INSTITUTUL NAIONAL DE CERCETARE DEZVOLTARE PENTRU BIORESURSE ALIMENTARE
INSTITUTUL NAȚIONAL DE CERCETARE DEZVOLTARE PENTRU BIORESURSE ALIMENTARE - IBA BUCUREȘTIwww. bioresurse. ro
PLANT ESSENTIAL OILS AS ACTIVE ANTIMICROBIAL AGENTS MICROBIOLOGY – ELISA LABORATORY Alina A. Dobre, Mirela Cucu, Ioana Vatuiu, Nastasia Belc www. bioresurse. ro
INTRODUCTION DEFINITION: ü Essential oils are mixtures of natural volatile compounds deriving from plant secondary metabolism, mainly monoterpenes, sesquiterpenes, and their oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols and oxides); ü The biological activity of essential oils depends on their chemical composition, which is determined by genotype and influenced by environmental and agronomic conditions; ü 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. www. bioresurse. ro
CHEMICAL NATURE OF ESSENTIAL OILS (1) ü Essential oils were proved to be good sources of bioactive compounds, with antioxidative and antimicrobial properties Solvent extraction Hydrodiffusion Hydrodistillatio n Steam distilation www. bioresurse. ro
CHEMICAL NATURE OF ESSENTIAL OILS (2) Terpene hydrocarbones: - monoterpene hydrocarbons - sesquiterpenes Oxygenated compounds: - phenols - alcohols (monoterpene alcohols, sesquiterpene alcohols) - aldehydes - ketones - esters - lactones - coumarins - ethers - oxides www. bioresurse. ro
CHEMICAL NATURE OF ESSENTIAL OILS (3) ü GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC/MS) TESTING OF ESSENTIAL OILS – RESEARCH RESULTS ü Essential oils: ü ü 3 high purity essential oils Purchased from Sigma Aldrich, Germany Natural origin Extraction method: steam distillation Thymus vulgaris Thymus capitatus Eugenia caryophyllata ü AGILENT GC- 6890 equiped with a MS system MS – 5973), 7673 autosampler and a capillary column HP-5 MS (lenght 30 m, diameter 0. 25 mm and film thickness 0. 25µm) and a flame ionization detector which was operated in EI mode at 70 e. V ü Helium was employed as a carrier gas at a flow rate of 1. 0 ml/min and a nominal pressure of 7. 64 psi. ü The oven temperature programmed was 50 – 2800 C at a rate of 40 C/min. ü 3. 0 µl of essential oil in chlorophorm was injected in the capillary column using a 10 µl volume syringe. ü Identification of the main components of essential oils was carried out using NIST spectra bank and Kovats indexes. www. bioresurse. ro
CHEMICAL NATURE OF ESSENTIAL OILS (4) 16 compounds of Lamiaceae family (thyme and oregano) Monoterpene hydrocarbons (19. 03%) Oxygenated monoterpene s (3. 66%) Phenolic monoterpeno ids (72. 84%) GC-MS profile of oregano essential oil ü 26 compounds were found, from which 17 chemical compound were identified, which represent about 99. 16% of the total detected constituents ü The major constituents of oregano essential oil were phenols like carvacrol (60. 77%), tymol (12. 07%) and p – cymene (14. 85%) www. bioresurse. ro Sesquiterpene hydrocarbons (3. 63%)
CHEMICAL NATURE OF ESSENTIAL OILS (5) α- pinen (2. 23%) linalol (4. 65 %) γ- terpinen (7. 17%) thymol (32, 63%) 16 chemicals - 93. 97% p – cymene (25. 44%) carvacrol (12, 87%) GC-MS profile of thyme essential oil ü The high concentration of phenolic compounds suggests that tested thyme essential oils belongs to tymol chemotype. www. bioresurse. ro
CHEMICAL NATURE OF ESSENTIAL OILS (6) 6 compounds were detected and identified - 99. 98% eugenol (53. 94%) isoeugenol (18. 57%) β – caryophyllen (8. 66%) GC-MS profile of clove bud essential oil ü 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 www. bioresurse. ro eugenil acetate (6. 62%)
IN VITRO ANTIMICROBIAL ACTIVITY TESTING (1) ü The CLSI (Clinical and Laboratory Standards Institute) method for antimicrobial susceptibility testing has been modified for testing essential oils AGAR DISC DIFFUSION METHOD PCA was inoculated with 100 µl of bacterial inoculum (106 CFU/m. L) sterile filter discs (6 mm) impregnated with 10 µl of stock solutions of EOs spred over the plates using a sterile rod display 370 C for 24 h Mean diameter of inhibition halo - clearly visible inhibition zone - was measured in mm www. bioresurse. ro
IN VITRO ANTIMICROBIAL ACTIVITY TESTING (1) ü Results Antibacterial activity of oregano EO Antibacterial activity of thyme EO Ø Direct contact method Ø higher antibacterial activity against E. coli (42 mm, 39, 3 mm) and B. cereus (45, 2 mm, 35, 5 mm) Ø 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 www. bioresurse. ro
IN VITRO ANTIMICROBIAL ACTIVITY TESTING (2) Ø Agar dilution method ü 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). ü 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. ü Inoculated plates were incubated at 370 C for 24 h and the MIC was determined. ü 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. ü The effect was bacteriostatic if the resumption of bacterial growth occurred and bactericidal in the contrary case. www. bioresurse. ro MIC – the lowest concentration of oil inhibiting the visible growth of each bacterial strain on the agar plate
IN VITRO ANTIMICROBIAL ACTIVITY TESTING (2) ü Results ü Bacterial growth inhibition was influenced by the essential oils concentration and their chemical composition ü The essential oil with the lowest MIC and MBC was oregano oil with values that vary from 200 ppm to 400 ppm ü The concentration of 400 ppm inhibited all the test strains, but only E. coli was more sensible presenting growth inhibition at 200 ppm ü Essential oil of thyme presentd a MIC of 400 ppm for all the bacterial strains tested and clove bud oil of 600 ppm Bacterial strain Clove bud oil White thyme oil Oregano oil MIC MBC S. aureus 600 1000 400 600 400 B. cereus 600 1000 400 800 400 E. coli 600 800 400 200 400 S. enteritidis 600 800 400 400 ü 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 ü E. coli, and S. enteritidis were more sensitive than S. aureus and B. cereus in the medium containing thyme and clove essential oil www. bioresurse. ro
IN VITRO ANTIMICROBIAL ACTIVITY TESTING (3) ü These evaluation techniques could be used as a preliminary, qualitative steps. ü Can determine the sensitivity of many microorganisms to EOs and select the oils with the strongest antimicrobial activity. ü 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. ü The antimicrobial activity of EOs is strictly connected to their chemical composition. ü Antimicrobial proprieties of the EOs from many plants are of great interest in both academia and the food, cosmetic and pharmaceutical industries. www. bioresurse. ro
MODE OF ANTIMICROBIAL ACTION (1) Influence of essential oils on bacteria morphology ü the Transmission Electron Microscopy (MET) technique - Philips EM 208 S was used ü 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 www. bioresurse. ro
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 www. bioresurse. ro
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 www. bioresurse. ro
MODE OF ANTIMICROBIAL ACTION (4) ü 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 ü The hydrophobicity that is typical of EOs is responsible for the disruption of bacterial structures that leads to increased permeability due to an inability to separate the EOs from the bacterial cell membrane ü Toxic effects on membrane structure and function are generally used to explain the antimicrobial activity of Eos ü This could explain why EOs are generally most effective against Gram-positive microorganisms. The external capsule of some Gram-negative bacteria limits or prevents the penetration of EOs into the microbial cell. www. bioresurse. ro
CONCLUSIONS ü 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. ü However, there are certain limitations, such as strong organoleptic flavor, low water solubility and low stability. ü In vitro studies in this research showed that the EOs inhibited bacterial growth but their effectiveness varied. ü The antimicrobial activity of EOs is strictly connected to their chemical composition. ü Antimicrobial proprieties of the EOs from many plants are of great interest in both academia and the food, cosmetic and pharmaceutical industries. ü Their possible use as natural additives emerged from growing tendency to replace synthetic preservatives by natural ones. www. bioresurse. ro
Contact: alina. dobre@bioresurse. ro www. bioresurse. ro
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