PLANT ESSENTIAL OILS AS ACTIVE ANTIMICROBIAL AGENTS

2. PLANT ESSENTIAL OILS AS ACTIVE ANTIMICROBIAL AGENTS MICROBIOLOGY – ELISA LABORATORY Alina A. Dobre, Mirela Cucu, Ioana Vatuiu, Nastasia Belc

3. INTRODUCTION • DEFINITION: • Essentialoils are mixtures of natural volatile compoundsderivingfromplantsecondary metabolism, mainlymonoterpenes, sesquiterpenes, and theiroxygenatedderivatives (alcohols, aldehydes, esters, ethers, ketones, phenols and oxides); • The biological activity of essential oils depends on their chemical composition, which is determined by genotype and influenced by environmental and agronomic conditions; • The use of essential oils and their chemical compounds, categorized as aromas • by the European Union [1] [2] and as GRAS (Generally Recognized as Safe) by • the US Food and Drug Administration, in food preservation is a attractive • opinion for “green” food products.

4. Chemicalnature of essentialoils (1) • Essential oils were proved to be good sources of bioactive compounds, with antioxidative and antimicrobial properties

5. Chemicalnature of essentialoils (2) • Terpene hydrocarbones: • - monoterpene hydrocarbons • - sesquiterpenes • Oxygenated compounds: • - phenols • - alcohols (monoterpene alcohols, sesquiterpene alcohols) • - aldehydes • - ketones • - esters • - lactones • - coumarins • - ethers • - oxides

6. Chemicalnature of essentialoils (3) • Gas chromatography mass spectrometry (GC/MS) testing of essential oils – researchresults • Essential oils: • 3 high purity essential oils • Purchased from Sigma Aldrich, Germany • Natural origin • Extraction method: steam distillation • AGILENT GC- 6890 equipedwith a MS system MS – 5973), 7673 autosampler and a capillarycolumn HP-5MS (lenght 30 m, diameter 0.25 mm and film thickness 0.25µm) and a flame ionization detector whichwasoperated in EI mode at 70 eV • Helium wasemployed as a carrier gas at a flow rate of 1.0 ml/min and a nominal pressure of 7.64 psi. • The oventemperatureprogrammedwas 50 – 2800C at a rate of 40C/min. • 3.0 µl of essential oil in chlorophorm was injected in the capillary column using a 10 µl volume syringe. • Identification of the main components of essential oils was carried out using NIST spectra bank and Kovats indexes. Thymuscapitatus Thymusvulgaris Eugenia caryophyllata

7. Chemicalnature of essentialoils (4) GC-MS profile of oregano essential oil • 26 compounds were found, from which 17 chemical compound were identified, which represent about 99.16% of the total detected constituents • The major constituents of oregano essential oil were phenols like carvacrol (60.77%), tymol (12.07%) and p – cymene (14.85%)

8. Chemicalnature of essentialoils (5) GC-MS profile of thyme essential oil • The high concentration of phenolic compounds suggests that tested thyme essential oils belongs to tymol chemotype.

9. Chemicalnature of essentialoils (6) GC-MS profile of clove bud essential oil • The main constituents of clove essential oil are phenylpropanoides such as eugenol and isoeugenol, which are produced by plants as defense mechanisms against animals and microorganisms and to attract pollinating insects

10. IN VITRO ANTIMICROBIAL ACTIVITY TESTING (1) Bacillus cereus ATCC 11778 Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Salmonella enteritidis ATCC 14028 • The CLSI (Clinical and Laboratory Standards Institute) method forantimicrobial susceptibility testing hasbeen modified for testing essential oils AGAR DISC DIFFUSION METHOD sterile filter discs (6 mm) impregnated with 10 µl of stock solutions of EOs PCA was inoculated with 100 µl of bacterial inoculum (106 CFU/mL) spred over the plates using a sterile rod display 370C for 24h Mean diameter of inhibition halo - clearly visible inhibition zone - was measured in mm

11. IN VITRO ANTIMICROBIAL ACTIVITY TESTING (1) • Results Antibacterial activity of oregano EO Antibacterial activity of thyme EO • Direct contact method • higher antibacterial activity against E. coli(42 mm, 39,3 mm) and B. cereus(45,2 mm, 35,5 mm) • Gram-positive bacteria, B. cereus and S. aureus, as a mean sensitivity against all essential oils tested, were more sensitive than the Gram-negative bacteria E. coli and S. enteritidis

12. IN VITRO ANTIMICROBIAL ACTIVITY TESTING (2) • Agar dilution method • different dilutions of each essential oil were made in melted PCA medium with 10% DMSO depending on their density, obtaining seven concentrations from 100 ppm to 15000 ppm (mg/L). • Work concentrations of essential oils obtained in agar dilution were placed in sterile Petri dishes (aprox 15 ml) and left to dry at room temperature for 30 minutes prior to spot inoculation with 10 µl impregnated filter disks (6 mm in diameter) of each bacterial culture of 106 ufc/ml. • Inoculated plates were incubated at 370C for 24 h and the MIC was determined. • Minimum bactericidal/bacteriostatic concentration was determined by transferring impregnated disks coming from Petri dish were the bacterial growth inhibition by essential oils was total during incubation period, on a PCA medium without essential oil. • The effect was bacteriostatic if the resumption of bacterial growth occurred and bactericidal in the contrary case. MIC – thelowest concentration of oil inhibiting the visible growth of each bacterial strain on the agar plate

13. IN VITRO ANTIMICROBIAL ACTIVITY TESTING (2) • Results • Bacterial growth inhibition was influenced by the essential oils concentration and their chemical composition • The essential oil with the lowest MIC and MBC was oregano oil with values that vary from 200 ppm to 400 ppm • The concentration of 400 ppm inhibited all the test strains, but only E. coli was more sensible presenting growth inhibition at 200 ppm • Essential oil of thyme presentd a MIC of 400 ppm for all the bacterial strains tested and clove bud oil of 600 ppm • In the case of MBC, oregano oil inhibited all the bacterial strains at 400 ppm, the concentration at which no growth accured on normal culture medium • E. coli, and S. enteritidiswere more sensitive than S. aureus and B. cereus in the medium containing thyme and clove essential oil

14. IN VITRO ANTIMICROBIAL ACTIVITY TESTING (3) • These evaluation techniques could be used as a preliminary, qualitative steps. • Can determine the sensitivity of many microorganisms to EOs and select the oils with the strongest antimicrobial activity. • The esential oils of oregano and thyme (Lamiaceae family) apperead to be equally effective against both Gram positive and Gram negative bacteria at MIC concentration but in the case of MBC concentration, essential oils were more active with respect to Gram positive bacteria, exerting greated inhibition. • The antimicrobial activity of EOs is strictly connected to their chemical composition. • Antimicrobial proprieties of the EOs from many plants are of great interest in both academia and the food, cosmetic and pharmaceutical industries.

15. MODE OF ANTIMICROBIAL ACTION (1) • Influence of essential oils on bacteria morphology • the Transmission Electron Microscopy (MET) technique - Philips EM 208 S was used • Bacterial inoculum was treated with sub-inhibitory concentrations of essential oils for three hours Electronic images of B. cereus cells untreated with EOs 1 - integral cell wall; 2-cell cytoplasmic membrane bound to the cell wall; 3 fine-grained cytoplasm; 4 - cross section Electronic images of E. coli cells untreated with EOs 1 - integral cell wall; 2-well peritrichi located all over the cell surface

16. MODE OF ANTIMICROBIAL ACTION (2) Electronic images of E. coli cells treated with oregano oil 800 ppm 1-cell wall degradation; 2 coagulated cytoplasm; 3- destruction of the scourges; 4 - the internal membrane shows retraction Electronic images of B. cereus cells treated with oregano oil 800 ppm 1-degradation of septal division; 2. partially disintegrated cytoplasm; 3. Partially disintegrated cell wall; 4 - the cytoplasmic membrane was coagulated

17. MODE OF ANTIMICROBIAL ACTION (3) Electronic images of E. coli cells treated with white thyme oil 800 ppm 1 - Coagulation and stiffness of the internal membrane; 2 - alteration of external membrane integrity; 3,4-thinning of cytoplasmic material in cell mass and cell membrane internal membrane aggregation Electronic images of B. cereus cells treated with white thyme oil 800 ppm 1-coagulated cell mass; 2 cells unaffected; 3. The cell wall destroyed; 4 - the cytoplasmic membrane is receded

18. MODE OF ANTIMICROBIAL ACTION (4) • According to the MET analysis, the site of action of essential oils on bacterial cells was the cell wall and cytoplasmic membrane, acting by destroying their integrity • The hydrophobicitythatistypical of EOsisresponsible for thedisruption of bacterialstructuresthatleadstoincreasedpermeabilitydueto an inabilityto separate theEOsfromthebacterialcell membrane • Toxic effects on membrane structure and function are generallyusedtoexplaintheantimicrobialactivity of Eos • ThiscouldexplainwhyEOs are generallymosteffectiveagainst Gram-positivemicroorganisms. The external capsule of some Gram-negative bacteria limits or preventsthepenetration of EOsintothemicrobialcell.

19. CONCLUSIONS • Essential oils possess important volatile compounds with diverse bioactivities including antimicrobial potential. EOs have been used in drugs, food, and cosmetics due to these properties.  • However, there are certain limitations, such as strong organoleptic flavor, low water solubility and low stability. • In vitro studies in this research showed that the EOs inhibited bacterial growth but their effectiveness varied. • The antimicrobial activity of EOs is strictly connected to their chemical composition. • Antimicrobial proprieties of the EOs from many plants are of great interest in both academia and the food, cosmetic and pharmaceutical industries. • Their possible use as natural additives emerged from growing tendency to replace synthetic preservatives by natural ones.

20. Contact: alina.dobre@bioresurse.ro