Recovery of heat-stressed yeasts in media containing plant oleoresins

C onner , D.E. & B euchat , L.R. 1985. Recovery of heat-stressed yeasts in media containing plant oleoresins. Journal of Applied Bacteriology 59 , 49–55.
Oleoresins from seven plants (allspice, cinnamon, clove, garlic, onion, oregano, and thyme) were tested for their effects on eight genera of sublethally heated food-spoilage and industrially important yeasts ( Candida lipolytica, Debaryomyces han-senii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata ). All heat-stressed yeasts had increased sensitivity to oleoresins in recovery media. Heated cells were most sensitive to cinnamon oleoresin, a lethal effect being observed to concentrations as low as 5 μg/ml. Results indicate that all eight yeasts underwent metabolic and/or structural injury as a result of exposure to elevated temperature. Whilst changes in sensitivity to oleoresins were not the same for all yeasts, it is concluded that the oleoresins may have a pronounced synergistic or additive effect on thermal inactivation of yeasts in foods. Furthermore, carryover of oleoresins in low dilutions of food to enumeration media could adversely influence the recovery of heat-stressed cells, thus resulting in apparent low populations.     

Effectiveness of cell-adsorbed bacteriocin produced by Lactobacillus curvatus CWBI-B28 and selected essential oils to control Listeria monocytogenes in pork meat during cold storage

Aims:  To study the effectiveness of a combination of cell-adsorbed bacteriocin (CAB; a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments) of a Lactobacillus curvatus strain with oregano or savory essential oil to control Listeria monocytogenes in pork meat at 4°C.
Methods and Results:  The antimicrobial activity of the CAB and six different essential oils was tested by the well diffusion assay against L. monocytogenes M, Escherichia coli 10536 and Salmonella serotype Typhi CWBI-H1. The anti- Listeria activity of the CAB and oregano or savory essential oils was also investigated in pork meat. The results of the well diffusion assay showed that CAB was only inhibitory to L. monocytogenes while savory and oregano essential oils were the most active against the three indicator bacteria. In pork meat, Listeria counts have declined from c. 10² CFU g⁻¹ to below the detectable limit during the first week of storage in samples treated with CAB or oregano essential oil and in those treated with CAB combined with oregano or savory essential oil. However, the counts of L. monocytogenes have increased after the third week of storage in all samples with the exception of those treated with the combination of CAB and oregano essential oil. The combination of CAB with savory essential oil resulted in a 2-week delay of the growth rebound compared with samples treated with CAB alone.
Conclusions:  Addition of oregano or savory essential oil exhibited a synergistic effect with CAB to control L. monocytogenes in pork meat during storage at 4°C.
Significance and Impact of the Study:  The combination of CAB with oregano or savory essential oil may be effectively used in meat industry to enhance the safety and stability of meat products.     

Inhibition of oxidation of unsaturated fatty acid methyl esters by essential oils

The essential oils from 16 various spice plants were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids methyl esters isolated from linseed oil. The content of methyl oleate, methyl linoleate, and methyl linolenoate after 1, 2, and 4 months of autooxidation were used as criteria to estimate the antioxidant efficiencies of essential oils. In 4 months, 92% of the methyl linolenoate and 79% of the methyl linoleate were oxidized in a control sample of a model system. It was found that the most effective antioxidants were essential oils from clove bud, cinnamon leaves, and oregano. They inhibited autooxidation of methyl linolenoate by 76–85%. The antioxidant properties of these essential oils were due to phenols— eugenol, carvacrol, and thymol. Essential oil from coriander did not contain phenols, but it inhibited methyl linolenoate oxidation by 38%. Essential oils from thyme, savory, mace, lemon, and tea tree inhibited methyl linolenoate oxidation by 17–24%. The other essential oils had no antioxidant properties.     

In vitro activity of essential oils extracted from plants used as spices against fluconazole-resistant and fluconazole-susceptible Candida spp

In the present study, the antifungal activity of selected essential oils obtained from plants used as spices was evaluated against both fluconazole-resistant and fluconazole-susceptible Candida spp. The Candida species studied were Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata, and Candida krusei. For comparison purposes, they were arranged in groups as C. albicans, C. dubliniensis, and Candida non-albicans. The essential oils were obtained from Cinnamomum zeylanicum Breyn, Lippia graveolens HBK, Ocimum basilicum L., Origanum vulgare L., Rosmarinus officinalis L., Salvia officinalis L., Thymus vulgaris L., and Zingiber officinale. The susceptibility tests were based on the M27-A2 methodology. The chemical composition of the essential oils was obtained by gas chromatography-mass spectroscopy and by retention indices. The results showed that cinnamon, Mexican oregano, oregano, thyme, and ginger essential oils have different levels of antifungal activity. Oregano and ginger essential oils were found to be the most and the least efficient, respectively. The main finding was that the susceptibilities of fluconazole-resistant C. albicans, C. dubliniensis, and Candida non-albicans to Mexican oregano, oregano, thyme, and ginger essential oils were higher than those of the fluconazole-susceptible yeasts (P<0.05). In contrast, fluconazole-resistant C. albicans and Candida non-albicans were less susceptible to cinnamon essential oil than their fluconazole-susceptible counterparts (P<0.05). A relationship between the yeasts' susceptibilities and the chemical composition of the essential oils studied was apparent when these 2 parameters were compared. Finally, basil, rosemary, and sage essential oils did not show antifungal activity against Candida isolates at the tested concentrations.     

Inhibitory effect of oregano and thyme essential oils on moulds and foodborne bacteria

The essential oil of oregano ('origanum oil'; thymol type oil from Origanum vulgare) inhibited completely the mycelial growth of Aspergillus niger and A. flaous at 400 μg/ml, while A. ochraceus was inhibited at 600 μg/ml. At 700 μg/ml, thyme oil inhibited the mycelial growth of A. flavus and A. niger but not that of A. ochraceus . Fungal spore germination was inhibited by 600 μg/ml of origanum oil and (with the exception of A. ochraceus) by 700 μg/ml of thyme oil. Under aerobic conditions, the essential oils of oregano (250 μg/ml) and thyme (350 μg/ml) inhibited to some extent the growth of Staphylococcus aureus and Salmonella typhimurium. Pseudomonas aeruginosa was not affected by either oregano or thyme oil at concentrations up to 500 μg/ml. The origanum oil was very effective against Campylohacter jejuni and Clostridiurn sporogenes and thyme oil was very effective against C. jejuni. The antagonistic effect of the two oils on Staph. aureus and Salm. typhimuriutn was greatly enhanced when those organisms were incubated in atmospheres of low oxygen tensions     

Antiradical properties of oregano, thyme, and savory essential oils

In model reactions with the stable free 2,2-diphenyl-1-picrylhydrazyl radical, the antiradical properties of essential oils of thyme (Thymus vulgare), oregano (Origanum vulgare), and savory (Satureja hortensis) that are similar in the qualitative composition, but differ in the quantitative content of the main components, were studied and compared with the properties of synthetic antioxidant ionol. The reaction rates of components of essential oils with the radical were almost identical for all essential oils and were twice the reaction rate of ionol. The antiradical efficiency values were close to each other for all essential oils and by an order of magnitude smaller than for ionol.     

Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7

AIMS: To quantify the antibacterial properties of five essential oils (EO) on a non-toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures. METHODS AND RESULTS: Five EOs known to exhibit antibacterial properties were screened by disc diffusion assay and the most active were selected for further study in microdilution colorimetric assays. Oregano (Origanum vulgare) and thyme (Thymus vulgaris; light and red varieties) EO had the strongest bacteriostatic and bactericidal properties, followed by bay (Pimenta racemosa) and clove bud (Eugenia caryophyllata synonym: Syzygium aromaticum) EO. Oregano oil was colicidal at 625 microl l(-1) at 10, 20 and 37 degrees C. The addition of 0.05% (w/v) agar as stabilizer reinforced the antibacterial properties, particularly at 10 degrees C, whereas 0.25% (w/v) lecithin reduced antibacterial activity. Scanning electron micrographs showed extensive morphological changes to treated cells. CONCLUSIONS: Oregano and thyme EO possess significant in vitro colicidal and colistatic properties, which are exhibited in a broad temperature range and substantially improved by the addition of agar as stabilizer. Bay and clove bud EO are less active. Lecithin diminished antibacterial properties. The bactericidal concentration of oregano EO irreversibly damaged E. coli O157:H7 cells within 1 min. SIGNIFICANCE AND IMPACT OF THE STUDY: Oregano and light thyme EO, particularly when enhanced by agar stabilizer, may be effective in reducing the number or preventing the growth of E. coli O157:H7 in foods.     

Efficacy of essential oils and biopesticides on Phytophthora infestans suppression in laboratory and growth chamber studies

Control of late blight by alternative products is important for reduction of fungicide inputs and potato production costs. The efficacy of essential oils and biopesticides for inhibition of growth of Phytophthora infestans on media and suppression of late blight on potato plants in growth chambers was examined. Growth of pathogen isolates of diverse genotypes was evaluated on Rye B media amended with essential oils (lavender, thyme, thyme borneal, and oregano) and the biopesticide Serenade (Bacillus subtilis strain QST 713). Over 90% inhibition of pathogen growth was achieved with oregano and Serenade amendments. The protective foliar application of Serenade, an aerated compost tea (ACT), Effective Microorganism mix (EM), and oregano, resulted in disease suppression of 5-40% relative to the untreated control. ACT had no significant suppressive effects (0-15% reduction), EM resulted in mild suppression (15-30% reduction), and oregano and Serenade consistently resulted in moderate disease reduction (20-40%). No oil or biological treatment produced disease control comparable to the chemical control chlorothalonil, which resulted in disease reductions of 80-98%. Both oregano and Serenade resulted in some phytotoxicity at high doses. These results suggest that the natural products and biological amendments tested are not sufficient for effective late blight control by themselves; however, when used in combination with other established disease control practices, these approaches may contribute to improved, integrated, and more sustainable management options for late blight.     

Efficacy of some medicinal plant extracts and essential oils against Colorado potato beetle,Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

The Colorado potato beetle (CPB), Leptinotarsa decemlineata Say is one of the most important defoliator pests of potato in the world and it often causes extremely large potato yield losses. Potatoes are the preferred hosts for the pest, but it may feed and reproduce on a number of other plants in the Solanaceae family. Public concern related to pesticides and their residues in and on the foods had prompted a rise of consumer interest in organically produced foods. There have been growing efforts to detect and introduce suitable plant compounds that they have insecticidal properties. However, discovering of plant extracts for possible use in control of this pest requires more studying about plant extracts and compounds. Since resistance of CPB to common chemical insecticides is well documented and potato is one of the most prominent nutritious food products for many people in many countries, we examined the effect of essential oils (EOs) of European pennyroyal, lavander, mint, oregano and savory and methanolic extracts of fumitory, licorice and oregano on the pest. These plants were selected because they have medicinal properties and they are safe to human and environment. Adult CPBs were exposed to mentioned plant extracts and essential oils. LC₅₀ values for EOs of lavander and European pennyroyal were 4154 and 3561 ppm, respectively. The results demonstrated that essential oil of European pennyroyal (Mentha longifolia) would be suitable compound to control the pest, but essential oil of mint (Mentha spicata) was not effective against the pest. Also it is notable that at all treatments, the amount of adult feeding was very low.     

Inhibitory effect of Thymus vulgaris and Origanum vulgare essential oils on virulence factors of phytopathogenic Pseudomonas syringae strains

• Pseudomonas syringae is a phytopathogenic bacterium that causes lesions in leaves during the colonisation process. The damage is associated with production of many virulence factors, such as biofilm and phytotoxins. The essential oils of Thymus vulgaris (thyme) and Origanum vulgare (oregano) have been demonstrated to inhibit P. syringae. The aim of this study was to investigate the effects of T. vulgaris and O. vulgare essential oils on production of virulence factors of phytopathogenic P. syringae strains, including anti‐biofilm and anti‐toxins activities.
• The broth microdilution method was used for determination of MIC and biofilm inhibition assays. Coronatine, syringomycin and tabtoxin were pheno‐ and genotypically evaluated.
• Both oils showed good inhibitory activity against P. syringae, with MIC values from 1.43 to 11.5 mg·ml^?1 for thyme and 5.8 to 11.6 mg·ml^?1 for oregano. Biofilm formation, production of coronatine, syringomycin and tabtoxin were inhibited by thyme and oregano essential oil in most strains.
• The results presented here are promising, demonstrating the bactericidal activity and reduction of virulence factor production after treatment with thyme and oregano oil, providing insight into how they exert their antibacterial activity. These natural products could be considered in the future for the control of diseases caused by P. syringae.

     

Antimicrobial Activities of the Essential Oils of Various Plants against Tomato Late Blight Disease Agent Phytophthora infestans

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 μg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4–2.0 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 μg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 μg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.     

Essential oils: effects of application rate and modality on potential for combating northern fowl mite infestations

The northern fowl mite (NFM), Ornithonyssus sylviarum (Mesostigmata: Macronyssidae), is the primary blood‐feeding ectoparasite found on poultry in the U.S.A. Three experiments were conducted in vitro to test the acaricidal properties of cade, garlic, lavender, lemongrass, pine and thyme essential oils against NFM, and to evaluate whether these effects are altered by adjusting oil application rates and application modality (direct vs. vapour contact). Applied at the rate of 0.21 mg/cm², the essential oils of cade, thyme, lemongrass and garlic resulted in higher NFM mortality at 24 h post‐application than lavender and pine oils, and the untreated and ethanol‐treated controls. Cade and thyme were the most consistent and fast‐acting of the essential oils in terms of toxicity to NFM. Cade applied at 0.21 mg/cm² and 0.11 mg/cm² and thyme applied at 0.21 mg/cm² were effective in eliminating NFM within 2 h through direct contact. The modality of application did not affect the efficacy of cade and thyme essential oils. The results suggest that essential oils may be utilized as alternatives to chemical pesticides and could be used as fumigants for the control of NFM.     

Inhibitory activity of spices and essential oils on psychrotrophic bacteria

This study was designed to evaluate "in vitro" the inhibitory effects of spices and essential oils on the growth of psycrotrophic food-borne bacteria: Aeromonas hydrophila, Listeria monocytogenes and Yersinia enterocolitica. The sensitivity to nine spices and their oils (chilli, cinnamon, cloves, ginger, nutmeg, oregano, rosemary, sage, thyme) was studied. Antibacterial activity was evaluated on liquid and solid medium. Spices: 1% concentration of each spice was added separately to Triptic Soy Broth and then inoculated to contain 10(8)/ml organism and held to 4 degrees C for 7 days. Populations of test organism were determined on Triptic Soy Agar. Oils: Inhibition of growth was tested by using the paper disc agar diffusion method (at 35, 20 and 4 degrees C) and measuring their inhibition zone. MIC was determined by the broth microdilution method. Some culinary spices produce antibacterial activity: inhibition of growth ranged from complete (cinnamon and cloves against A. hydrophila) to no inhibition. Antibacterial inhibition zone ranged from 8 mm to 45 mm: thyme essential oil showed the greatest inhibition against A. hydrophila.     

Fumigant toxicity of plant essential oils against Camptomyia corticalis (Diptera: Cecidomyiidae)

The toxicity of 98 plant essential oils against third instars of cecidomyiid gall midge Camptomyia corticalis (Loew) (Diptera: Cecidomyiidae) was examined using a vapor-phase mortality bioassay. Results were compared with that of a conventional insecticide dichlorvos. Based on 24-h LC50 values, all essential oils were less toxic than dichlorvos (LC50, 0.027 mg/cm3). The LC50 of caraway (Carum carvi L.) seed, armoise (Artemisia vulgaris L.), clary sage (Salvia sclarea L.), oregano (Origanum vulgare L.), lemongrass [Cymbopogon citratus (DC.) Stapf], niaouli (Melaleuca viridiflora Gaertner), spearmint (Mentha spicata L.), cassia especial (Cinnamomum cassia Nees ex Blume), Dalmatian sage (Salvia offcinalis L.), red thyme (Thymus vulgaris L.), bay [Pimenta racemosa (P. Mill.) J.W. Moore], garlic (Allium sativum L.), and pennyroyal (Mentha pulegium L.) oils is between 0.55 and 0.60 mg/cm3. The LC50 of cassia (C. cassia, pure and redistilled), white thyme (T. vulgaris), star anise (Illicium verum Hook.f.), peppermint (Mentha X piperita L.), wintergreen (Gaultheria procumbens L.), cinnamon (Cinnamomum zeylanicum Blume) bark, sweet marjoram (Origanum majorana L.), Roman chamomile [Chamaemelum nobile (L.) All.], eucalyptus (Eucalyptus globulus Labill.), rosemary (Rosmarinus officinalis L.),Virginian cedarwood (Juniperus virginiana L.), pimento berry [Pimenta dioica (L.) Merr.], summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia Mill.), and coriander (Coriandrum sativum L.) oils is between 0.61 and 0.99 mg/cm3. All other essential oils tested exhibited low toxicity to the cecidomyiid larvae (LC50, >0.99 mg/cm3). Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the active essential oils as potential larvicides for the control of C. corticalis populations as fumigants with contact action.     

The Effect of Some Natural Essential Oils Against Bovine Mastitis Caused by <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Isolates In Vitro

The aim of the study was to evaluate the effect of essential oils obtained from Thymus vulgaris L., Origanum vulgare L., Origanum majerana L., Mentha × piperita L. and Allium ursinum L. against Prototheca zopfii strains that cause inflammation of the udder (mastitis) in cows. The study was conducted on ten strains derived from milk samples. The microdilution method was used to determine the sensitivity of P. zopfii strains to the studied essential oils, and the disk diffusion method was used to determine the sensitivity to antifungal chemotherapeutics. The plates were incubated for 48 h at 37 °C under aerobic conditions. All strains of algae were sensitive to the essential oils marjoram, thyme and oregano and resistant to mint and garlic oils. MIC values ranged from 0.25 to 1 μl/ml. Marjoram oil demonstrated the greatest activity, and oregano oil the weakest. Among the antifungal agents tested, 90% of strains showed sensitivity to nystatin. One of the tested strains (71/IV) was resistant to all investigated antifungal agents. The tested essential oils are known to have anti-algae activity and can be used as natural agents for prophylaxis in animals, particularly in mastitis-affected cows.     

Effect of two plant extracts and Lactobacillus fermentum on colonization of gastrointestinal tract by Salmonella enterica var. Düsseldorf in chicks

According to published data, it is well known that essential oils from plants possess antimicrobial activity against a wide range of pathogens, including Salmonella strains. The influence of the administration of essential oils from oregano (Origanum vulgare) and thyme (Thymus vulgaris) as well as Lactobacillus fermentum on crop, caecum, liver and spleen colonization by Salmonella enterica var. Düsseldorf in chicks was investigated in this study. For the experiment, one hundred 1-day old chicks were used, and they were divided into four groups: (i) untreated (C); (ii) treated with L. fermentum (L); (iii) treated with a mixture of oregano and thyme essential oils (OT); and (iv) treated with a combination of L. fermentum and a mixture of oregano and thyme essential oils (LOT). Essential oils from oregano and thyme were mixed with commercial poultry diet, which was offered ad libitum to chicks in appertaining groups. L. fermentum was added daily to drinking water. After 3 days all the chicks were challenged orally with S. enterica var. Düsseldorf. The crops, ceca, spleens and livers of the birds were examined for S. enterica var. Düsseldorf colonization 5 days after the challenge. Our results showed that a combined administration of L. fermentum and essential oils (oregano and thyme) in group with combined application of essential oils and lactobacillus strain reduced the percentage of colonized crops and ceca when compared to the control group without any treatment. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Antimicrobial activity of Pelargonium essential oils added to a quiche filling as a model food system

Eight essential oils obtained by steam distillation from the scented leaves of Pelargonium species and cultivars were added at 250, 500 and 1000 ppm to a quiche filling, inoculated with either Saccharomyces ludwigii or Zygosaccharomyces bailii (at 10⁸ cfu g⁻¹), Salmonella enteriditis or Listeria innocua (at 10⁹ cfu g⁻¹). The quiche fillings were then kept at 25 °C for 24 h and the residual number of micro-organisms determined using the pour plate technique. There was an effective antimicrobial activity by the Pelargonium essential oils at 250 ppm, comparable with that of commercial thyme oil, an excellent antimicrobial agent, against Saccharomyces ludwigii and Zygosaccharomyces bailii , and a lesser inhibition compared with commercial thyme against Salm. enteriditis. There was a greater diversity of activity against L. innocua, which was in some cases more effective than commercial thyme oil. At 500 ppm, there was a greatly increased inhibition of microbial growth using the Pelargonium essential oils, which was comparable with that of commercial thyme, clove, geranium and coriander oils. As there is no evidence for the toxicity of any of these novel Pelargonium oils, and their odour does not make the delicately flavoured quiche filling unpalatable, there is a strong potential for their use in food processing.     

Plating medium pH as a Factor in Apparent Survival of Sublethally Stressed Yeasts

Sublethally stressed cells of 9 of 10 species of yeast were recovered at maximum levels when potato dextrose agar was adjusted to approximately pH 8. The optimum for candida utilis was at approximately pH 10. At pH 3.5, as commonly employed with media selective for yeasts and molds, recovery of heat-stressed organisms ranged from essentially the same as at optimum pH to levels of 1% or less of the maximum count. The extent to which this may be of practical significance in assessing the microbiological quality of food products remains to be determined.     

Effects of selected plant essential oils on the growth and development of mouse preimplantation embryos in vivo

Plant essential oils (EOs) have been reported to have health benefit properties and their preventive and therapeutic use in animals is expected to increase in the future. We evaluated the influence of five essential oils obtained from plant species which are known to have positive antimicrobial, antioxidative and anti-inflammatory effects--sage EO from Salvia officinalis L. (Lamiaceae), oregano EO from Origanum vulgare L. (Lamiaceae), thyme EO from Thymus vulgaris L. (Lamiaceae), clove EO from Syzygium aromaticum L. (Myrtaceae) and cinnamon EO from Cinnamomum zeylanicum Blume (Lauraceae) on the growth and development of mouse preimplantation embryos in vivo. Essential oils were added to commercial diet at concentrations of 0.25% for sage EO, thyme EO, clove EO, cinnamon EO and 0.1% for oregano EO, and fed to ICR female mice for 2 weeks ad libitum. Females were then mated with males of the same strain. Embryos obtained on Day 4 of pregnancy at the blastocyst stage were stained by morphological triple staining (Hoechst, PI, Calcein-AM) and evaluated using fluorescent microscopy. The effects of essential oils were estimated by the viability of embryos, number of nuclei and distribution of embryos according to nucleus number. Cinnamon EO significantly decreased the number of nuclei and the distribution of embryos according to nucleus number was significantly altered. Sage EO negatively influenced the distribution of embryos according to nucleus number. Clove and oregano EOs induced a significantly increased rate of cell death. Only thyme EO had no detectable effects on embryo development. In conclusion, none of the essential oils had any positive effect on embryo development, but some of them reduced the number of cells and increased the incidence of cell death.     

Survival of yeasts inoculated in flavoured extra virgin olive oil

The survival of four strains of yeast belonging to the speciesSaccharomyces cerevisiae, Candida wickerhamii, Candida boidinii andWilliopsis californica was studied in extra virgin olive oil flavoured with garlic, lemon, oregano and red chilli pepper. The ingredients used in the doses of 1%, 5% and 10% profoundly modified the habitat of the extra virgin olive oil, reducing drastically, in 90 days of storage, the survival of the yeasts by 20–50%, in the following decreasing order: lemon, garlic, oregano and red chilli pepper. Among the yeasts studied,W. californica strain 1639 was found to be one of the most sensitive, whileS. cerevisiae strain 1525 was one of the most tolerant regarding the ingredients present in the flavoured olive oil. The observations carried out with a scanning electron microscope (SEM) highlighted the presence of frequent lesions on the cellular wall ofC. wickerhamii 1532,C. boidinii 1638 andS. cerevisiae 1525 and only in a few rare cases inW. californica 1639. Nevertheless, since the survival ofW. californica 1639 in the flavoured olive oil was compromised to a greater extent in respect to the other species, it is plausible to deduct that the damage to the cellular wall represents only one of the causes responsible for the death of the yeasts in the flavoured olive oil.