Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes

Flow cytometry was applied to assess the antimicrobial activity of oregano, thyme and cinnamon essential oils against Listeria monocytogenes ATCC19114, using combined staining with propidium iodide (PI) for membrane damage evaluation and carboxyfluorescein diacetate (cFDA) for esterase activity detection. The antimicrobial activity of the essential oils was also tested at different NaCl concentrations.Significant differences were observed between plate count results and flow cytometric data, which suggested the presence of a sublethally stressed subpopulation, not able to form colonies on agar plates.Following treatments, flow cytometric assessment clearly discriminated three different subpopulations: viable, dead and injured cells. Cinnamon essential oil exerted a different impact on the cellular subpopulations, with a lower overall activity and a large percentage of cells having minimally damaged membranes. On the contrary, membrane disintegration seemed to be the primary inactivation mechanism of oregano and thyme essential oils.The antimicrobial activity of the essential oils increased with NaCl concentration increase, but higher NaCl concentrations were necessary following treatments with cinnamon essential oil.Our findings suggest differences in the mode of action of cinnamon essential oil against L. monocytogenes, in comparison with thyme and oregano essential oils.     

Antibacterial activity of essential oils from plants of the genus Origanum

In this study, three plant species, members of the family of Lamiaceae and the genus Origanum, namely, Origanum vulgare subsp. hirtum, Origanum onites L., and Origanum marjorana L. were studied for their chemical composition and antibacterial activity. Essential oils of these plants were received by means of micro-steam distillation and their components were analyzed by gas chromatography and mass spectrometry (GC–EIMS). The major components identified in all three species are carvacrol and thymol. The oils were assayed as potential food control antimicrobial agents. In vitro studies showed that the essential oils showed strong antimicrobial activity against 5 bacterial and 1 yeast strains.     

Antifungal activity of thyme, summer savory and clove essential oils against Aspergillus flavus in liquid medium and tomato paste

Antifungal activity of essential oils of thyme, summer savory and clove were evaluated in culture medium and tomato paste. Aspergillus flavus were inoculated in Sabouraud Dextrose Broth and tomato paste and then 0, 50, 200, 350 and 500 ppm of essential oils were added to each sample and then kept at 25 ± 0.5 °C for 2 months. Results showed that all essential oils could inhibit the growth of A. flavus and the thyme oil and summer savory, showed the strongest inhibition at 350 ppm and 500 ppm, respectively. Taste panel evaluations were carried out in a tomato ketchup base, and the percent of inhibition of each essential oil in tomato paste was lower than culture medium. Taste panel was carried out and sample with 500 ppm thyme oil was accepted by panelists.     

Antimicrobial activity of nanoemulsions containing essential oils and high methoxyl pectin during long-term storage

The antimicrobial activity against Escherichia coli and Listeria innocua of nanoemulsions containing oregano, thyme, lemongrass or mandarin essential oils and high methoxyl pectin was assessed during a long-term storage period (56 days). On one hand, a higher antimicrobial activity was detected against E. coli compared to L. innocua regardless the EO type. Transmission Electron Microscopy (TEM) images showed a significant damage in the E. coli cells for both the cytoplasm and cytoplasmic membrane, led to cell death. The antimicrobial activity of the nanoemulsions was found to be strongly related to the EO type rather than to their droplet size. The lemongrass-pectin nanoemulsion had the smallest droplet size (11 ± 1 nm) and higher antimicrobial activity reaching 5.9 log reductions of the E. coli population. Nevertheless, the freshly made oregano, thyme and mandarin EO-pectin nanoemulsion led to 2.2, 2.1 or 1.9 E. coli log-reductions, respectively. However, the antimicrobial activity decreased significantly during storage regardless the EO type, which was related to the loss of volatile compounds over time according to our results. The current work provides valuable information in order to make progress in the use of nanoemulsions containing EOs as decontaminating agents in food products.     

Antibacterial activity of two Phlomis essential oils against food pathogens

Phlomis species from the Lamiaceae family are widely distributed in Turkey. In this study, the essential oils of Phlomis russeliana (Sims.) Bentham and Phlomis grandiflora H.S. Thompson var. grandiflora collected from North and Southern parts of Turkey, were obtained by hydrodistillation of the aerial parts. The essential oils were subsequently analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS). The major constituents of P. russeliana essential oil were identified as sesquiterpenes β-caryophyllene (23%), germacrene-D (15%), and caryophyllene oxide (8%). Analysis of P. grandiflora var. grandiflora oil also showed oxygenated sesquiterpenes such as β-eudesmol (42%) and α-eudesmol (16%) as major constituents.Furthermore, essential oils were tested in vitro against common food borne bacteria such as Aeromonas hydrophila, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, and the anaerobic pathogen Clostridium perfringens using the micro-broth dilution assay. When compared with antimicrobial standards weak to moderate (125 to >1000 μg/ml) minimum inhibitory concentrations (MIC) were observed. The results show that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.     

Antibacterial activity of Oregano,Rosmarinus and Thymus essential oils against Staphylococcus aureus and Listeria monocytogenes in beef meatballs

Antimicrobial activity of five essential oils (EOs) was investigated up to 72 h against foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enteritidis, Campylobacter jejuni) through disk diffusion and determination of Minimum Inhibitory Concentrations. The most active EOs were Thymus vulgaris and Origanum vulgare, followed by Cinnamomum zeylanicum, Rosmarinus officinalis, and Salvia officinalis. The antimicrobial activity of O. vulgare, Rosmarinus officinalis and T. vulgaris was investigated against five enterotoxin producers of S. aureus and five L. monocytogenes strains, for different amounts of time (up to 14 days), at 4 °C, in meatballs. Concentrations of 2% and 1% restricted the growth of both the pathogens but, as a result of panel tests, altered the meat flavor. The cooked meatballs containing 0.5% of EO were acceptable in terms of taste, and the oils were able to suppress concentrations of <10² CFU/g of the pathogens, revealing the potential use of R. officinalis, T. vulgaris and O. vulgare as food preservatives at this concentration.     

Effects of exposure to biocides on susceptibility to essential oils and chemical preservatives in bacteria from organic foods

A collection of 38 biocide-adapted strains with significant increases in their tolerance to biocides after step-wise exposure to these compounds were screened for sensitivity to essential oils and chemical preservatives. Several strains adapted to quaternary ammonium compounds (benzalkonium chloride, hexadecylpyridinium chloride or cetrimide) showed a generalized increase in the sensitivity to preservatives. Similar results were found among hexachlorophene- or chlorhexidine-adapted strains. Moreover, tolerance to hexadecylpyridinium chloride showed a very strong positive correlation with 4-hydroxybenzoic acid, thyme oil and sodium nitrite increased sensitivity, as well as a strong correlation with clove oil, potassium sorbate and potassium nitrate increased sensitivity. On the contrary, an increase in tolerance to preservatives was detected among triclosan-adapted strains. Results from this study suggest that exposure of bacteria from foods to biocides is not always associated with co-selection for other antimicrobial resistances, especially against essential oils or chemical preservatives used in the food industry.     

Evaluation of the antimicrobial and antioxidant activities of essential oils, extracts and their main components from oregano from Madeira Island, Portugal

Origanum vulgare subsp. virens growing wild in Madeira Island, Portugal was studied within the ongoing investigations on polymorphic Lamiaceae species. The antimicrobial activity of the essential oils and n-hexane extracts of origanum was determined against 10 strains of bacteria and yeasts, found as human pathogenic or food spoilage microorganisms. The essential oils, n-hexane extracts and isolated compounds showed moderately activity, compared to standard antibiotics, inhibiting all tested bacteria except Pseudomonas aeruginosa. The most sensitive microorganism was Mycobacterium smegmatis, reaching MIC = 25 μg mL⁻¹. The results obtained suggest a potential application of these oils in preventing the human pathogenic and food spoilage due to microorganism’s growth. The essential oils and n-hexane extracts have greater RSC than polar extracts, probably due to the high contents in thymol, which demonstrated the highest activity in the DPPH assay. All studied origanum samples showed a large content in non-esterified 1-hexacosanol, C₂₆H₅₄O, accumulated mainly in bracts and flowers.     

Efficacy of Fir and Qysoom essential oils,alone and in combination,in controlling Listeria monocytogenes in vitro and in RTE meat products model

Listeria monocytogenes, frequently associated with ready-to-eat meat products (RTE-MP), is the causal agent of listeriosis, the virulent foodborne disease. Accordingly, this work aimed to study the effectiveness of essential oils (EOs) of different plants to control growth of L. monocytogenes in RTE-MP model. EOs antilisteric activities were screened by disk diffusion method. Then, efficacy of EOs (1% v/w) with strong inhibition activities were further examined in meat luncheon model, against 2 levels of L. monocytogenes strains cocktail (3 and 6 log CFU/g) coupled with storage at 4 °C for 14 days. The EOs of Fir and Qysoom showed to have the highest significant (p < 0.05) antilisteric activity. In the food model, L. monocytogenes populations in control samples increased by 4 log cycles after 14 days of storage at 4 °C. At the end of storage, for samples with low contamination; Fir, Qysoom, and EOs mixture had approximately 6.37, 6.04, and 5.53 log CFU/g of L. monocytogenes respectively, compared to 6.90 log of control. Whereas in the samples with high contamination level, populations reached to 8.43, 8.88 and 6.75 log CFU/g for Fir, Qysoom, and EOs mixture respectively, compared to 9.90 log of the control. The application of 1% EOs (v/w) to RTE-MP surfaces significantly showed to reduce (p < 0.05) the L. monocytogenes populations growth rate as compared to control in the 2 levels treatments after 14 days of storage at 4 °C. Accordingly, our results suggest that these EOs could be used as natural bio-preservatives in many food products produced in Jordan and worldwide, particularly in RTE-MP.     

Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms

Bacterial biofilms pose health risks in clinical environments, food industry and drinking water systems. Here, we investigated in vitro antibiofilm activities of essential oils (EO) and plant extracts of peppermint (Mentha × piperita L.), coriander (Coriandrum sativum L.), and anise (Pimpinella anisum L.). Minimum inhibitory concentration assay (MIC) was carried out using two-fold serial dilution method and MTT assay against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Biofilm growth and development were assessed using crystal violet (CV) and XTT reduction assays. Antibacterial activity was observed for almost all plant extracts and all EOs against both bacterial strains with stronger activity against S. aureus. All EOs (at MIC value of 0.8 to 0.63 μl/ml) and 8 out of 14 plant extracts (at MIC value of 2–4 mg/ml) inhibited bacteria cell attachment of both bacteria. CV and XTT reduction assay for the plant extracts and EOs with inhibition of bacteria attachment by at least 50%, demonstrated that coriander EO had the highest antibiofilm activity against biofilm formed by both tested bacteria (S. aureus and E. coli) at lowest MIC value 0.8 μl/ml and 1.6 μl/ml, respectively, indicating further investigations due to the oil's high antibiofilm activity potential.     

Influence of emulsifier type on the antifungal activity of cinnamon leaf,lemon and bergamot oil nanoemulsions against Aspergillus niger

Only exiguous data are currently available on the antifungal properties of essential oil (EO) nanoemulsions against spore-forming microorganisms. The aim of this work is to develop physically stable nanoemulsion-based delivery systems for different EOs (cinnamon leaf, lemon, and bergamot), to exploit their antifungal properties against Aspergillus niger. The inhibition of mycelial radial growth and spore germination were used as indicators of antifungal activity of the nanoemulsions, which were prepared at 3 wt% EO, using non-ionic Tween 80 (T80) or anionic whey protein isolate (WPI) (1 wt%) as emulsifiers, and sunflower oil (1 wt%) as ripening inhibitor. The nanoemulsions were physically stable over seven days of accelerated aging at 35 °C.The minimal inhibitory concentration of free cinnamon leaf and of both citrus EOs were 0.35 and 5.50 μg/g, respectively. The encapsulation of cinnamon leaf EO in nanoemulsions significantly enhanced the inhibiting effect against A. niger mycelial growth and spore germination, with respect to the free EO. In contrast, for citrus EOs, the encapsulation in nanoemulsions generally decreased the antifungal activity, likely because of the nanoemulsion acting as a hydrophobic sink for the main constituents of citrus EOs. The emulsifier played a fundamental role in the resulting antifungal activity, with WPI-based nanoemulsions being more effective in inhibiting the mycelial growth and the spore germination of A. niger than T80-based ones. The antifungal action was correlated to the morphological alterations observed in A. niger, such as the loss of cytoplasm in fungal hyphae and hyphal tip. The results of this study show the importance of nanoemulsions design in the development of efficient and stable natural antifungal agents for food applications.     

Antibacterial activity of essential oils and their main components enhanced by atmospheric RF plasma

The antibacterial activity of low concentration clove oil, sweet basil oil, and lime oil (5–20 μl ml⁻¹) and their main components (eugenol, β-ocimene, and d-limonene) at the same concentration was enhanced by atmospheric RF plasma at 20W and 40W for 10 min to effectively control the growth of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus on chicken egg. Number of colony germination after plasma treatment was evaluated. Results showed that growth of E. coli, S. thyphimurium, and S. aureus on eggshell mixed with clove oil (10 μl ml⁻¹) or its main component (eugenol at 5 μl ml⁻¹) was completely inhibited after using plasma treatment at 40W. Without the plasma treatment, all essential oils and all their main components at concentrations of 5–20 μl ml⁻¹ could not inhibit bacterial growth. Thus, this study has demonstrated the good potential of using atmospheric RF plasma treatment to enhance the antibacterial activity of essential oil at a relatively low concentration.     

Efficacy of the combined application of oregano and rosemary essential oils for the control of Escherichia coli,Listeria monocytogenes and Salmonella Enteritidis in leafy vegetables

This study assessed the effect of the combined application of essential oils (EOs) from Origanum vulgare L. – oregano (OVEO) and Rosmarinus officinalis L. – rosemary (ROEO), alone or in combination at subinhibitory concentrations, against three pathogenic bacteria that are associated with fresh leafy vegetables: Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli) and Salmonella enterica Serovar Enteritidis (S. Enteritidis). The inhibitory effects were evaluated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI) and assessing the viable cell counts in vegetable broth and artificially infected vegetables over time. Still, the effects of the EOs on native spoilage native flora were assessed. The MIC of OVEO was 0.6 μL/mL against the test strains either in single and mixed inoculum. The MIC of ROEO was 5 μL/mL against L. monocytogenes and E. coli and 10 μL/mL against S. Enteritidis in single inocula, whereas it was 10 μL/mL against the mixed inoculum. The FICI of the combined EOs was 0.5 against the mixed bacterial inoculum, which suggested a synergic interaction. The incorporation of OVEO and ROEO alone (MIC) or combined at different subinhibitory concentrations in vegetable broth resulted in a decrease in the viable cell counts of all test strains over 24 h. Similarly, the EOs alone or in the tested combinations reduced the viable cell counts of all test strains in experimentally infected fresh vegetables, besides to decrease the counts of spoiling native flora (mesophilic bacteria, enterobacteria and fungi). These findings reinforce the rational for the use of OVEO and ROEO in combination at subinhibitory concentrations to guarantee the safety and extend the shelf life of fresh vegetables.     

Antifungal, aflatoxin inhibition and antioxidant activity of Callistemon lanceolatus (Sm.) Sweet essential oil and its major component 1,8-cineole against fungal isolates from chickpea seeds

The present investigation reports the extent of molds and aflatoxin contamination to Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea seeds. The study also examines the chemical composition of Callistemon lanceolatus (Sm.) Sweet essential oil and its antifungal, antiaflatoxin and antioxidant activity. During standardization of chemical profile, a total of 8 compounds constituting 0.862 mg/mL of oil composition were analyzed by GC-MS analysis where 1,8-cineole was recorded as a major component (0.56 mg/mL). The antifungal activity of EO and 1,8-cineole was evaluated by contact assay on Czapek’s dox agar. The EO (0.227-0.908 mg/mL) and 1,8-cineole (0.918 mg/mL) showed remarkable antifungal effect against all the fungal isolates of chickpea. Their minimal inhibitory (MIC) and fungicidal (MFC) concentrations for Aspergillus flavus were lower than those of the prevalent systemic fungicide Nystatin. Aflatoxin B₁ (AFB₁) production by NKD-208 isolates of A. flavus was strongly inhibited even at the lower fungistatic concentration of EO and 1,8-cineole.There was no adverse effect of EO treatment on chickpea seed germination suggesting its non-phytotoxic nature. Based on the findings of present investigation, C. lanceolatus essential oil may be recommended as botanical preservative for the enhancement of shelf life of food items in- view of the adverse effect of synthetic preservatives and its strong antifungal, aflatoxin inhibition and antioxidant activity.     

Behaviour of food-borne pathogens on dry cured sausage manufactured with herbs and spices essential oils and their sensorial acceptability

Dry cured sausages such as Portuguese chouriço de vinho are made generally with roughly minced pork and fat and seasoned with a large variety of herbs and spices. It is a particular sausage because meat is marinated in wine with other seasonings during 24–48 h before stuffing. Nowadays, health concerns of consumers have left food processors with less flexibility to choose preservative substances and methods. Thus, the objective of this study was to assess the antimicrobial effect of essential oils (EOs) of herbs and spices traditionally used in seasoning dry cured sausage chouriço against Salmonella spp., Listeria monocytogenes and Staphylococcus aureus. First, antibacterial activity of 14 EOs was screened by disk diffusion assay. Those EOs that displayed antimicrobial activity were further characterized by GC–MS and added as natural antimicrobial substances to the manufacture of chouriço at two concentrations (0.005% and 0.05%). Samples were analyzed 24 h after the preparation of the batter and after 3, 8, 15 and 21 days.After 15 days of drying, Salmonella spp. and L. monocytogenes counts decreased by ca. 2 log cfu/g in all samples. At 3 days, Salmonella spp. was already undetectable in chouriço made with 0.05% of garlic and oregano EOs. Both Salmonella spp. and L. monocytogenes were not detected after 8 and 15 days of drying respectively. S. aureus was still presented after 21 days of drying although its counts were not high enough to a potential enterotoxins presence. Utilization of EOs in manufacture of dry cured sausage resulted in an interesting strategy to improve its safety against Salmonella spp, L. monocytogenes and S. aureus, but with sensory limitations, that does not allow its use in high concentrations, that are those more interesting for pathogen inhibition.However, results with good potential to be applied by the industry were found, once the level of addition of 0.005% contributed for significant reductions on the pathogen's counts and for a shorter period to achieve the not detectable level, allowing the industry to shorten the drying period and, thereafter increasing yield production.     

Antimicrobial effects of modified chitosan based coating containing nanoemulsion of essential oils,modified atmosphere packaging and gamma irradiation against Escherichia coli O157:H7 and Salmonella Typhimurium on green beans

The antibacterial activity of modified chitosan-based coatings containing nanoemulsion of essential oils (EOs), gamma irradiation, modified atmosphere packaging (MAP), alone or in combinations, against Escherichia coli O157:H7 and Salmonella Typhimurium was evaluated on inoculated green bean samples. Firstly, four different nanoemulsions, made of carvacrol, mandarin, bergamot and lemon Eos, respectively, were compared in terms of minimum inhibitory concentration (MIC) against the two bacteria evaluated in vitro using the micro-broth dilution method. Carvacrol nanoemulsion resulted to be the most effective antibacterial agent and was therefore selected to be incorporated into modified chitosan (MC) to form a bioactive coating. Secondly, the radiosensitivity of E. coli and S. Typhimurium to gamma irradiation was evaluated on inoculated green beans after coating deposition and MAP. Results showed that, without MAP, MC-based coating containing carvacrol nanoemulsion significantly increased the radiosensitization of E. coli and S. Typhimurium by 1.32-fold and 1.30-fold, respectively. Remarkably, the use of bioactive coating under MAP caused a synergistic effect with an increase in radiosensitivity by 1.80-fold and 1.89-fold for E. coli and S. Typhimurium, respectively. Thirdly, the antibacterial effects of the antimicrobial coating, gamma irradiation, MAP alone and their combinations were evaluated against these two bacteria during a 13-days storage of green beans at 4 °C. Bioactive coating deposition or gamma irradiation treatment resulted effective in controlling the growth of the two bacteria during the entire shelf-life. Moreover, it was also found that the combined treatment of antimicrobial coating, gamma irradiation and MAP caused the reduction of microbial population to undetectable levels during the whole storage period for E. coli and from day 7 to the end of storage for S. Typhimurium. The obtained results can be interested to food companies aiming to ensure the food safety with a prolonged shelf life.     

Chemical composition,antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.)

Fennel (Foeniculum vulgare Mill.) is widely cultivated and used as a culinary spice. In this work, the chemical composition of the essential oil obtained by hydrodistillation of fennel seeds was analyzed by gas chromatography-mass spectrometry (GC–MS), and 28 components were identified. Trans-anethole (68.53%) and estragole (10.42%) were found to be the major components. The antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericide concentration (MBC) of essential oil against several food-borne pathogens were evaluated. The results showed that the gram positive and gram negative strains of bacteria had different sensitivities to essential oil of fennel seeds, the essential oil exhibited antibacterial activity against Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae and Escherichia coli according to the results of MIC and MBC. Among these bacteria, S. dysenteriae was the most sensitive to essential oil, showing the lowest MIC and MBC values of 0.125 and 0.25 mg/mL respectively. In addition, kill-time assay also showed that the essential oil had a significant effect on the growth rate of surviving S. dysenteriae. We concluded that the mechanism of action of the essential oil against S. dysenteriae might be described as essential oil acting on membrane integrity according to the results of the leakage of electrolytes, the losses of contents (proteins, reducing sugars and 260 nm absorbing materials) assays and electron microscopy observation.     

Essential oil composition and biological/pharmacological properties of Salmea scandens (L.) DC

Salmea scandens (L.) DC is an indigenous edible plant whose stem bark is traditionally used as food by people of Oaxaca, México. Proximate analysis of the edible stem bark revealed abundant amounts of fiber (43.67%) and protein (9.27%). GC and GC-MS analyses demonstrated that the essential oil from leaves contained high levels of germacrene D (47.1%) and elemol (15.3%), whereas that of the stem bark contained the alkylamides N-Isobutyl-(2E,4E,8Z,10E/Z)-dodecatetraenamide isomers (39.7%). Levels of these compounds in the essential oil from both organs were in similar concentrations in all seasons except winter. The HPLC purified N-Isobutyl-(2E,4E,8Z,10E/Z)-dodecatetraenamide isomers produced a non-competitive inhibition on porcine pancreatic lipase. The enzymatic assays with these compounds revealed a modification on V_max (0.0431–0.0533 mM min⁻¹) whereas the K_m value (0.880–0.881 mM) was not significantly changed. Essential oil from the stem bark showed a high anti-microbial activity against some phytopathogenic microorganisms. The MIC's in μg mL⁻¹ for Pseudomonas syringae pathovars were tabaci 56.1, tomato 91.2 and phaseolitica 196.4, for Clavibacter michiganensis 35.8 and Erwinia carotovora 48.1. The fungi Fusarium oxysporum and Phytophthora infestans had MIC's in μg mL⁻¹ of 3.3 and 2.4 respectively. Same essential oil was highly effective against larvae from Aedes aegypti (LC₅₀ = 0.3 μg mL⁻¹) and Anopheles albimanis (LC₅₀ = 2.5 μg mL⁻¹).     

Evaluation of Zataria multiflora Boiss. essential oil activity against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes by propidium monoazide quantitative PCR in vegetables

Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157:H7 and dead bacteria killed by cumin, clove, oregano and cinnamon EOs. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Zataria multiflora EOs on E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population treated with Z. multiflora EO. Inactivation was obtained at EO concentrations of 0.02, 0.035, 0.045 for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed in 30 min while it took 1 h 30 min for the gram negative pathogens. As a conclusion Z. multiflora EO has potential as natural food additive or biopreservative since it was able to irreversibly inactivate the three pathogens tested, at lower concentrations than other EOs and short exposition times. In addition, the PMA-qPCR approach proved efficient to selectively detect live pathogenic bacteria in vegetables following inactivation with Z. multiflora EO.     

Synergistic action between fractions of essential oils from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against Penicillium expansum

Penicillium expansum is a mould that causes the rotting of several fruits and vegetables, especially apples onto which it also synthesizes some dangerous mycotoxins. The degree of synergism between fractions of essential from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris was evaluated against two mycotoxin producing strains of P. expansum. The antifungal activity determined by dilution method and expressed as a Number of Decimal Reduction of the colony forming units per ml (NDR cfu) showed that the essential oils extracted from O. gratissimum was significantly (P < 0.05) more active against P. expansum than those extracted from C. citratus and T. vulgaris. Fractions enriched with oxygenated terpenes were significantly (P < 0.05) more active than their respective essential oils, whereas most of the fractions enriched with terpene hydrocarbons, were significantly (P < 0.05) less active. The fungicidal activity of mixtures of fractions from the same essential oils or from two different essential oils showed that there exist synergistic, additive and antagonistic effects between fractions of the three essential oils tested against both fungal strains. The synergistic effects observed could be exploited in order to maximize the antimicrobial activity of essential oils and to minimize the concentrations of essential oil required to produce a given antimicrobial effect without any alteration of the food test.