Sensitivity of heat-stressed yeasts to essential oils of plants.

Eight strains of yeasts (Candida lipolytica, Debaryomyces hansenii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata) were examined for changes in sensitivity to eight essential oils of plants (allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme) after being sublethally heat stressed. With the exception of garlic oil for all test yeasts, onion oil for S. cerevisiae, and oregano oil for R. rubra, the essential oils at concentrations of up to 200 ppm in recovery media did not interfere with colony formation by unheated cells. However, some oils, at concentrations as low as 25 ppm in recovery media, reduced populations of sublethally heat-stressed cells compared to populations recovered in media containing no test oils. This demonstrates that the yeasts were either metabolically or structurally damaged as a result of being exposed to elevated temperatures and that essential oils prohibited repair of injury. The size (diameter) of colonies produced on oil-supplemented recovery agar by heat-stressed cells was reduced compared to that observed on unsupplemented agar. Pigment production by heated R. rubra was inhibited by oils of oregano, savory, and thyme, but enhanced by garlic and onion oils. The influence of essential oils on survival of yeasts in thermally processed foods and in the enumeration of stressed cells in these foods should not be minimized.     

Survival of Candida parapsilosis yeast in olive oil

Candida parapsilosis is a human opportunistic pathogen yeast isolated from different habitats like animals, man, pickled cucumber, fruit juices, and water. Recent studies have demonstrated that C. parapsilosis can survive in olive oil for very long periods even exceeding 24 months. The survival of two strains of C. parapsilosis named DAPES 1890 and 1892, previously isolated from extra virgin olive oil, was influenced by the state of hydration of the cells and the polyphenols concentration of olive oil. When the cells of the two strains of C. parapsilosis were inoculated under a liophilized form into olive oil containing 45–312 mg/kg of total polyphenols, their survival in some olive oil samples reached approximately 18 months. However, if the above-mentioned inoculum was rehydrated with 1 % of distilled water, then the survival of both yeast strains in some samples of oil exceeded 24 months. The two yeast strains, recovered from the olive oil samples after 24 months of storage, showed, under SEM, spherical shapes with and without buds according to whether the inoculum was made up of rehydrated or lyophilized cells. The survival of all the C. parapsilosis strains was also negatively influenced by the polyphenols concentration of the olive oil samples inoculated both with lyophilized and rehydrated yeast cells. In the oily habitat, the polyphenols sorption to the C. parapsilosis yeast surface was observed, and during storage the polyphenols reacted with the yeast cell walls according to their concentration in the inoculated olive oil.     

Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations

The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of “sticky” material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing.     

Antifungal activity of olive leaf (Olea Europaea L.) extracts from the Trilye Region of Turkey

Antimicrobial properties of olive leaf extract on some yeast were examined in this study. Fresh olive leaf extracts were prepared using various solvents (water, ethanol, acetone, ethyl acetate) in Soxhlet apparatus. Antimicrobial effects of these extacts were tested againstSaccharomyces cerevisiae ATCC 9763,Schizosaccharomyces pombe, Saccharomyces uvarum, Candida oleophila, Metschnikowia fructicola andKloeckera apiculata. The antifungal activities of these extracts were tested by the disc diffusion assay, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). All extracts showed various degrees of antifungal effects with 10–28 μg/ml MIC, 20–48 μg/ml MFC and 1.5–9.3 mm inhibitory zone values against yeasts utilised, except water. The results indicated that the tested yeasts were sensitive to acetone and ethyl acetate extracts. It was determined thatSaccharomyces cerevisiae ATCC 9763 was the most resistant among the yeasts.     

The impact of different plant extracts on biological parameters of Housefly [Musca domestica (Diptera: Muscidae)]: Implications for management

Housefly is a significant domestic pest, which causes nuisance. The use of insecticides is discouraged to manage housefly; therefore, alternative management strategies are inevitable. The current study investigated the impact of different plant extracts, i.e., Moringa oleifera (moringa), Allium sativum (garlic) and Piper nigrum (black pepper) on biological parameters of house fly. Two different concentrations (i.e., 25 and 50%) of the extracts were blended in larval diet made through mixing of wheat bran, yeast and dried milk powder. The results indicated significant differences for larval duration. Maximum larval duration was recorded for garlic followed by black pepper and moringa, respectively. In case of pupal duration, non-significant differences were observed among plant extracts. Increase rate of oviposition was noticed with moringa at 25% concentration, while decreased oviposition rate was noted for garlic with 50% concentration. Egg hatching percentage remained non-significant for the botanical extracts. The highest survival was observed with moringa, while garlic resulted in the lowest survival. The highest repellency was noticed for garlic followed by black pepper, whereas moringa resulted in the lowest repellency after 30, 60 and 90 min. Prolonged developmental time was observed for bot concentrations of garlic, whereas moringa noted the shortest developmental time. Thus moringa was found to be a promoter of housefly development. Minimum adult emergence was found with both concentrations of garlic followed by 50% concentration of black pepper. The 50% concentration of black pepper promoted the population of adult males, while both concentrations of moringa and 25% concentration of black pepper encouraged the population of female adults. Study outcomes depicted that tested botanical extracts had significant potential for disturbing biological parameters of housefly. The garlic extracts can potentially be used to manage housefly. However, further investigations on the larval and adult mortality are needed.     

Development and Evaluation of a Model Predicting the Survival of Escherichia coli O157:H7 NCTC 12900 in Homemade Eggplant Salad at Various Temperatures, pHs, and Oregano Essential Oil Concentrations

Homemade eggplant salad, a traditional Greek appetizer, was inoculated with Escherichia coli O157:H7 NCTC 12900 supplemented with different concentrations of oregano essential oil (0.0, 0.7, 1.4, and 2.1% [vol/wt]) and stored at different temperatures (0, 5, 10, and 15°C). The product's pH was adjusted to 4.0, 4.5, or 5.0 with lemon juice. For each combination of the environmental factors, the bacterial counts were modeled, using the Baranyi model, as a function of time to estimate the kinetic parameters of the pathogen. A reduction of more than 1 log unit in E. coli O157:H7 counts was observed in all cases, and the death rate depended on the pH, the storage temperature, and the essential oil concentration. Separate quadratic models were developed with natural logarithms of the shoulder period and death rate as estimated by the growth model, as a function of temperature, pH, and oregano essential oil concentrations. These were further used to predict the population of E. coli O157:H7 NCTC 12900 from other inoculated eggplant salads at random conditions of temperature, pH, and oregano oil concentration. The predicted values were compared with viable-count measurements for validation.     

Isolation of auxotrophic mutants of the methylotrophic yeastCandida boidinii and determination of its ploidy

Auxotrophic mutations in the methylotrophic yeast strainCandida boidinii 11Bh were induced by different mutagens and their combinations (nitrosoguanidine, UV light, HNO₂+UV). Majority of the mutants obtained carried defects in histidine, arginine, proline and/or adenine biosynthetic pathways. His^- mutants were distributed into four complementation groups using the protoplasts fusion technique. Ploidy determination ofCandida boidinii 11Bh was performed by measuring its DNA content and by following its survival after chemical mutagens treatment. The DNA content of this strain was found to be similar to that of aSaccharomyces cerevisiae diploid strain. Also the kinetics of survival ofCandida boidinii cells indicate thatCandida boidinii 11Bh is a diploid.     

Extra virgin olive oil rich in polyphenols modulates VEGF-induced angiogenic responses by preventing NADPH oxidase activity and expression

Previous studies have shown the antiinflammatory, antioxidant and antiangiogenic properties by pure olive oil polyphenols; however, the effects of olive oil phenolic fraction on the inflammatory angiogenesis are unknown. In this study, we investigated the effects of the phenolic fraction (olive oil polyphenolic extract, OOPE) from extra virgin olive oil and related circulating metabolites on the VEGF-induced angiogenic responses and NADPH oxidase activity and expression in human cultured endothelial cells. We found that OOPE (1–10 μg/ml), at concentrations achievable nutritionally, significantly reduced, in a concentration-dependent manner, the VEGF-induced cell migration, invasiveness and tube-like structure formation through the inhibition of MMP-2 and MMP-9. OOPE significantly (P<0.05) reduced VEGF-induced intracellular reactive oxygen species by modulating NADPH oxidase activity, p47phox membrane translocation and the expression of Nox2 and Nox4. Moreover, the treatment of endothelial cells with serum obtained 4 h after acute intake of extra virgin olive oil, with high polyphenol content, decreased VEGF-induced NADPH oxidase activity and Nox4 expression, as well as, MMP-9 expression, as compared with fasting control serum. Overall, native polyphenols and serum metabolites of extra virgin olive oil rich in polyphenols are able to lower the VEGF-induced angiogenic responses by preventing endothelial NADPH oxidase activity and decreasing the expression of selective NADPH oxidase subunits. Our results provide an alternative mechanism by which the consumption of olive oil rich in polyphenols may account for a reduction of oxidative stress inflammatory-related sequelae associated with chronic degenerative diseases.     

Ornamental pepper as banker plants for establishment of Amblyseius swirskii (Acari: Phytoseiidae) for biological control of multiple pests in greenhouse vegetable production

Silverleaf whitefly, Bemisia tabaci biotype B (Gennadius) (Hemiptera: Aleyrodidae), western flower thrips, Frankliniella occidentalis (Pergande), and chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae), are key pests of vegetable crops in the US. The present study established ornamental peppers as banker plants supporting Amblyseius swirskii (Acari: Phytoseiidae) against the three pests. Specifically, this study (a) evaluated survival and population buildup of A. swirskii on three ornamental pepper varieties, Masquerade (MA), Red Missile (RM), and Explosive Ember (EE) in both laboratory and greenhouses and (b) determined the predation of A. swirskii reared on ornamental pepper plants to the targeted pests under greenhouse conditions. The results showed that the three pepper varieties were excellent banker plants and able to support at least ∼1000 of all stages of A. swirskii per plant in greenhouse conditions and allow them to complete their life cycle. A. swirskii dispersed or released from the banker plants to target plants, resulting in significant suppression of the three pests, i.e., after 14 d post-release, a significantly lower average of 2.75 B. tabaci and 13.4 all stages of thrips (chilli thrips and western flower thrips) were found per bean plant, respectively, compared to 379.5 B. tabaci and 235.4 all stages of thrips per plant in the control. Furthermore, our experiment observed that the sweet pepper seedlings closed to banker plants were healthy, whereas those without banker plants were heavily infested by chilli thrips; their growth seriously stunted or died. This is the first report of ornamental pepper as banker plants supporting A. swirskii against three notorious pests. This established banker plant system could be a new addition to the integrated pest management programs for sustainable control of these three pests in greenhouse vegetables.     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

Development of Transparent Curcumin Loaded Microemulsions by Phase Inversion Temperature (PIT) Method: Effect of Lipid Type and Physical State on Curcumin Stability

In this study, curcumin loaded transparent microemulsions obtained using the phase inversion temperature (PIT) method were developed. Different lipids (sunflower, peanut, castor, and extra virgin olive oil, tristearin, and tripalmitin) were tested as curcumin carrier in microemulsions. The obtained systems were analyzed for transparency, particle size, lipid crystal polymorphism, and curcumin stability at 20 °C up to 120 days. It was found that the maximum lipid content allowing transparent microemulsions (mean particle diameter of around 25 nm) to be obtained was greatly affected by the lipid characteristics. By using oils rich in long chain fatty acids, such as sunflower, peanut, and extra virgin olive oil, transparent microemulsions can be obtained with oil fractions up to 7.5 % (w/w). On the contrary, when fat containing crystals (e.g. tripalmitin or tristearin) was used, the maximum lipid loading capacity was reduced to 5 % (w/w). Castor oil, rich in polar groups, did not permit the formation of transparent microemulsions at any tested concentration (from 1 to 9 % w/w). The oil type also affected curcumin stability: curcumin degradation rate was lower in tristearin containing microemulsions than in those containing extra virgin olive oil. This result was attributed to the protective effect of solid lipid particles into lipid droplets.     

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.     

Role of yeasts in the qualitative structuring of extra virgin olive oil

This review sought to describe the role played by some components of the microbiota of extra virgin olive oil (EVOO), particularly yeasts, in structuring the physicochemical and sensorial quality of freshly produced olive oil. Yeasts can survive during the entire storage period of the product. To date, approximately 25 yeast species isolated from oil produced in more than six countries have been identified, eight of which are classified as new species. Some yeast species improve the health qualities of oil, whereas many others improve the chemical composition and sensory characteristics based on β-glucosidase and esterase enzymes, which are involved in the hydrolysis of the bitter glucoside known as oleuropein. However, some species, which are typically favoured by the high water content in the oily matrix, such as lipase-producing yeasts, can worsen the initial chemical characteristics of EVOO oil during storage. Some physical treatments that are compatible with the EVOO production specification affect the biotic component of the oil by reducing the concentration of yeasts. The possibility of minimizing the invasive action on the biotic component of the oil by appropriately selecting the physical treatment for each oil is discussed.     

Effect of formaldehyde on the level of cytochrome c in methylotrophic yeasts

The effect of formaldehyde on the cytochrome system of the methylotrophic yeasts Candida tropicalis, Candida boidinii, Candida methylica, Hansenula capsulata, Hansenula polymorpha, Pichia pastoris and nonmethylotrophic yeasts Saccharomyces cerevisiae was studied. In the investigated concentration range of 0.58 – 2.5 mol · 1^?1, formaldehyde decreases the level of the reduced cytochrome c in the above-mentioned microorganisms. The kinetics of the change in the reduced cytochrome c content has two phases: a phase of slow decrease and a phase of complete disappearance of the reduced cytochrome c in the cells. The duration of the first phase shortens with increasing concentrations of formaldehyde.     

Characterization of yeasts isolated from spontaneously fermenting fresh olive effluents

Summary Spontaneous fermentation of sugars in fresh olive oil extraction effluents was caused by yeasts tentatively identified asCandida wickerhamii, C. molischiana andSaccharomyces cerevisiae. These strains may serve to convert olive oil waste effluents into useful products.

---

Caractérisation de levures isolées d'effluents d'extraction d'huile d'olive en fermentation spontanée

Résumé La fermentation spontanée des sucres dans des effluents d'extraction d'huile d'olive est due à des souches de levures tentativement identifiées commeCandida wickerhamii, C. molischiana etSaccharomyces cerevisiae. Ces souches peuvent servir à la conversion d'effluents résiduaires d'extraction d'huile d'olive en produits utiles.

     

Assessment of Helicobacter pylori eradication by virgin olive oil

Background: A recent study conducted by Medina et al. disclosed that virgin olive oil has a bactericidal effect in vitro against Helicobacter pylori because of its contents of certain phenolic compounds with dialdehydic structures. We carried out two clinical trials to evaluate the effect of virgin olive oil on H. pylori‐infected individuals. Materials and Methods: Two different pilot studies were performed with 60 H. pylori‐infected adults. In the first study, thirty subjects who tested positive for H. pylori received 30 g of washed virgin olive oil for 14 days, and after 1 month, the patients took 30 g of unwashed virgin olive oil for another 14 days. In a second study, a group of 30 subjects received 30 g of a different virgin olive oil for 14 days. Helicobacter pylori‐infection status was checked by the urea breath test. Results: Helicobacter pylori was eradicated in 8 of 30 individuals when microorganism status was checked after 4–6 weeks from the first clinical intervention although 12 of 30 individuals did not show H. pylori infection at 24–72 hour of the last oil dose. Eradication rates were 27 and 40% by intention to treat and per protocol, respectively. Moreover, only 3 of 30 individuals were H. pylori negative after 4–6 weeks from the second clinical intervention but 5 of 30 were negative at 24–72 hour of the last oil dose. Eradication rates were 10 and 11% by intention to treat and per protocol, respectively. It must also be noted that 13 subjects withdrew from the studies because of taste and nausea drawbacks. Conclusions: The administration of virgin olive oil showed moderate effectiveness in eradicating H. pylori. Further studies are needed to confirm these findings, especially with longer periods, different administration conditions, and several types of olive oils.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

Ectopic expression of an osmotin gene leads to enhanced salt tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annum</Emphasis> L.)

Plants, when exposed to abiotic or biotic stress, produce several pathogenesis-related proteins to counteract the effects of stress. Osmotin is one of the important pathogenesis-related proteins induced during several stress conditions. We have developed improved salt stress tolerant transgenic chilli pepper plants (Capsicum annum L. var. Aiswarya 2103) by ectopic expression of the Nicotiana tabaccum osmotin gene using Agrobacterium tumefaciens EHA105 as a vector. Four-week-old chilli pepper leaves were used as an explant and A. tumefaciens EHA105 harboring pBINASCOSM plasmid that contains osmotin gene under the control of CaMV 35S promoter and npt II as a selectable marker was used in co-cultivation. Transgene integration and expression were analyzed using molecular, immunochemical, and biochemical assays. PCR and Southern blot analysis confirmed that osmotin gene has been successfully integrated into the genome of chilli pepper plants. The osmotin gene was stably segregated and expressed in T₂ generation transgenic chilli pepper plants, and it was confirmed by Western blot analysis. Biochemical assays of these putative transgenic plants revealed enhanced levels of chlorophyll, proline, glycinebetaine, APX, SOD, DHAR, MDHAR, GR, and relative water content. Yield potential of the putative transgenic chilli pepper plants was evaluated under salinity stress conditions in a green house. The putative transgenic chilli pepper plants overexpressing the osmotin gene were morphologically similar to wild-type plants and produced 3.32 kg chilli pepper fruits per plant at 300 mM NaCl concentration.