Search

Since the yeast flora of Slovakian enology has not previously been investigated by culture-independent methods, this approach was applied to two most common cultivars Frankovka (red wine) and Veltlin (white wine), and complemented by cultivation. Model samples included grapes, initial must, middle fermenting must and must in the end-fermentation phase. The cultured isolates were characterized by length polymorphism of rDNA spacer two region using fluorescence PCR and capillary electrophoresis (f-ITS PCR), and some were identified by sequencing. The microbial DNA extracted directly from the samples without cultivation was analysed by f-ITS PCR, amplicons were cloned and sequenced. The use of universal fungal primers led to detection of both yeasts and filamentous fungi. The amplicon of highest intensity and present in all the samples corresponded to Hanseniaspora uvarum. Other species demonstrated by both approaches included Saccharomyces sp., Metschnikowia pulcherrima or M. chrysoperlae, Candida zemplinina, Cladosporium cladosporioides, Botryotinia fuckeliana, Pichia anomala, Candida railenensis, Cryptococcus magnus, Metschnikowia viticola or Candida kofuensis, Pichia kluyveri or Pichia fermentas, Pichia membranifaciens, Aureobasidium pullulans, Alternaria alternata, Erysiphe necator, Rhodotorula glutinis, Issatchenkia terricola and Debaryomyces hansenii. Endemism of Slovakian enological yeasts was suggested on the level of minor genetic variations of the known species and probably not accounting for novel species. The prevalence of H. uvarum over Saccharomyces sp. in the samples was indicated. This is the first culture-independent study of Slovakian enology and the first time f-ITS PCR profiling was used on wine-related microbial communities.

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682-C. boidinii and 625, 642, 647, and 941-P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10-30 degrees C) and (NH(4))(2)SO(4) (0.0-6.0 g l(-1)) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10 degrees C with 6.0 g l(-1) of (NH(4))(2)SO(4) added.

This article reports on the investigation on the bioactivity of eugenol, limonene, and citrus extract against three bacteria (Lactobacillus plantarum, Lactobacillus brevis, and Bacillus coagulans) and three yeasts (Saccharomyces bayanus, Pichia membranifaciens, and Rhodotorula bacarum), representing the spoilage microflora of fruit juices. The experiments were performed with laboratory media by using a microdilution method. Data were fitted using the Gompertz equation, and the kinetic parameters were used to evaluate the MIC and the dose-dependent effect (at suboptimal doses for each essential oil). Citrus extract was the most effective essential oil, and the results suggested the following susceptibility hierarchy, from the most sensitive microorganism to the most resistant one (values in parentheses represent MICs): S. bayanus (2 ppm) > R. bacarum (3 ppm) > P. membranifaciens (5 ppm) > B. coagulans (cells, 20 ppm) > L. brevis (40 ppm) > L. plantarum (>40 ppm).

The aims of this study were to isolate and characterize peptides present in chilli pepper seeds and evaluate their antifungal activities. An isolated peptide closer to 9 kDa showed high sequence homology to the antimicrobial peptide lipid transfer protein. The peptide fraction containing the LTP inhibited the growth of the fungi, Fusarium oxysporum, Colletotrium lindemunthianum, the yeasts, Saccharomyces cerevisiae, Pichia membranifaciens, Candida tropicalis, Candida albicans, inhibited glucose-stimulated acidification of the medium by yeast cells of S. cerevisiae and caused several morphological changes in P. membranifaciens.

Three plant growth-promoting yeasts and two rhizobacteria were tested for controlling tomato wilt caused by Fusarium oxysporum f. sp. lycopersici under greenhouse and field conditions. Under greenhouse and field conditions, all treatments were significantly reduced disease severity of tomato wilt relative to the infected control. The highest disease reductions in pots (75.0, 67.4%) and field (52.5, 42.4%) were achieved by Azospirillum brasilense and Bacillus subtilis compared to infected control. Under field condition all treatments produced the highest tomato yield compared to the control plants inoculated with the pathogen.

The molecular karyotyping analysis of 21 strains within the taxonomic complex Pichia membranifaciens allowed the sibling species P. membranifaciens and P. manshurica, as well as P. deserticola and P. punctispora, to be differentiated. Heterogeneity of the species P. membranifaciens at the variety level is discussed.

Pichia membranifaciensCYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 °C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibitBrettanomyces bruxellensiswhileSaccharomyces cerevisiaewas fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well asP. membranifaciensCYC 1086, was able to inhibitB. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.

Relationships among species assigned to the yeast genera Pichia, Issatchenkia and Williopsis, which are characterized by the ubiquinone CoQ-7 and inability to utilize methanol, were phylogenetically analyzed from nucleotide sequence divergence in the genes coding for large and small subunit rRNAs and for translation elongation factor-1alpha. From this analysis, the species separated into five clades. Species of Issatchenkia are members of the Pichia membranifaciens clade and are proposed for transfer to Pichia. Pichia dryadoides and Pichia quercuum are basal members of the genus Starmera. Williopsis species are dispersed among hat-spored taxa in each of the remaining three clades, which are proposed as the new genera Barnettozyma, Lindnera and Wickerhamomyces. Lineages previously classified as varieties of Pichia kluyveri, 'Issatchenkia'scutulata, Starmera amethionina and 'Williopsis'saturnus are elevated to species rank based on sequence comparisons.

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

The objective of this study was to investigate the effects of omija (Schiandra chinensis) and plum (Prunus mume) extracts on the fermentation and quality of Kimchi, especially with regard to extending its shelf-life. With the addition of omija and plum extracts, the final pH of Kimchi achieved levels of 4.18 (0.5% addition of omija extract: O-1), 4.36 (1.0% addition of omija extract: O-2), 4.33 (0.5% omija + 1.0% plum extract: O/P-1), and 4.33 (0.5% omija + 2.0% plum extract: O/P-2), respectively, which were higher than that of the Kimchi without extracts, 3.92, after 24 days of fermentation. The addition of the extracts caused a slight increase in titratable acidity and the growth of lactic acid bacteria as compared with the samples with no addition. Treatment O-2 showed a more profound inhibition effect on the growth of six strains, Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella koreensis, Leuconostoc citreum, Klebsiella pneumoniae and Pichia membranifaciens than the use of O/P-1 and O/P-2. Bitterness was increased with increase in the amount of omija extract. As for the evaluation of overall taste, the addition of the plum extract to omija extract induced a reduction in the bitterness of the Kimchi. Therefore, the addition of both plum and omija extracts appears to be an acceptable approach to enhance the quality of Kimchi without reducing acceptability.

The diversity of populations of yeast and lactic acid bacteria (LAB) in pig feeds fermented at 10, 15, or 20°C was characterized by rRNA gene sequencing of isolates. The feeds consisted of a cereal grain mix blended with wet wheat distillers' grains (WWDG feed), whey (W feed), or tap water (WAT feed). Fermentation proceeded for 5 days without disturbance, followed by 14 days of daily simulated feed outtakes, in which 80% of the contents were replaced with fresh feed mixtures. In WWDG feed, Pichia galeiformis became the dominant yeast species, independent of the fermentation temperature and feed change. The LAB population was dominated by Pediococcus pentosaceus at the start of the fermentation period. After 3 days, the Lactobacillus plantarum population started to increase in feeds at all temperatures. The diversity of LAB increased after the addition of fresh feed components. In W feed, Kluyveromyces marxianus dominated, but after the feed change, the population diversity increased. With increasing fermentation temperatures, there was a shift toward Pichia membranifaciens as the dominant species. L . plantarum was the most prevalent LAB in W feed. The WAT feed had a diverse microbial flora, and the yeast population changed throughout the whole fermentation period. Pichia anomala was the most prevalent yeast species, with increasing occurrence at higher fermentation temperatures. Pediococcus pentosaceus was the most prevalent LAB, but after the feed change, L. plantarum started to proliferate. The present study demonstrates that the species composition in fermented pig feed may vary considerably, even if viable cell counts indicate stable microbial populations.

Five new compounds, pichiafurans A-C (1-3) and pichiacins A and B (4 and 5), along with five known compounds (6-10), have been isolated from the yeast Pichia membranifaciens derived from a marine sponge Petrosia sp. Their structures were elucidated by 1D and 2D NMR and mass spectrometry techniques. Pichiafurans are rare examples of monofurano metabolites isolated from yeast.

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. Thispaper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckiiwith potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichiamanshurica and Pichiamembranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growthof all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 ° C. Pustulan andchitin - but not other cellwall polysaccharides - prevented sensitive yeast cells from being killed by TdKT, suggestingthat those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell deathafter 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanismsof action. Killertoxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool inwinemaking. Fil: Villalba, Maria Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina Fil: Saez, Julieta Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina. Universidad Nacional del Comahue. Facultad de Ciencias Agrarias; Argentina Fil: del Monaco, Silvana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina Fil: Lopes, Christian Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina. Universidad Nacional del Comahue. Facultad de Ciencias Agrarias; Argentina Fil: Sangorrin, Marcela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas. Universidad Nacional del Comahue. Instituto de Investigación y Desarrollo en Ingeniería de Procesos, Biotecnología y Energías Alternativas; Argentina. Universidad Nacional del Comahue. Facultad de Ingeniería; Argentina

Different types of antimicrobial peptides have been identified in seeds from different plant species. The aim of this study was to isolate and characterize peptides present in chilli pepper seeds (Capsicum annuum L.) and evaluate their toxic activities against some yeast species. Initially, proteins from seed flour were extracted in phosphate buffer, pH 5.4, for 3 h at 4 degrees C and the pellet obtained at 90% saturation with ammonium sulfate was heated at 80 degrees C for 15 min. The resulting suspension was clarified by centrifugation and the supernatant was extensively dialyzed against water; the peptide-rich extract was then named F/0-90. Cation-exchange chromatography was performed to separate low molecular mass proteins. One of the resulting fractions, named F3, enriched with basic proteins of 6-16 kDa, was submitted to reverse-phase chromatography in a C2/C18 column by HPLC, resulting in four fractions denominated RP1, RP2, RP3 and RP4. When these fractions were submitted to N-terminal sequencing, the comparative analysis in databanks revealed homology for two of these peptides, isolated from fractions RP3 and RP4, with sequences of proteinase inhibitors and 2S albumins, respectively. The F3 fraction, rich in peptides, inhibited the growth of yeasts Saccharomyces cerevisiae, Candida albicans, Candida parapsilosis, Candida tropicalis, Pichia membranifaciens, Kluyveromyces marxiannus and Candida guilliermondii. The RP3 and RP4 fractions showed high inhibitory activity against the growth of the yeast S. cerevisiae. The F3 fraction was also able to inhibit glucose-stimulated acidification of the medium by yeast cells of S. cerevisiae and to cause several morphological changes in different yeasts, such as cell wall disorganization, bud formation as well as the formation of pseudohyphae.

Summary PMKT is a channel-forming killer toxin secreted by Pichia membranifaciens. To identify novel genes that mediate cellular resistance to PMKT we screened a collection of 288 deletion mutants. We found 29 open reading frames (ORFs) that, when deleted, confer resistance to PMKT. In addition, the deletion of 15 ORFs was observed to increase protoplast resistance, in agreement with the initial assumption that a plasma membrane receptor for PMKT exists. Whole cells and protoplasts of a cwp2D mutant were found to be completely resistant to PMKT and were unable to bind PMKT, indicating that Cwp2p interacts with it. A protein with a molecular mass of 11.7 kDa was purified from PMKT-affinity columns. This protein was sequenced and identified as Cwp2p. Glycosylphosphatidylinositol (GPI) anchoring-defective mutants were much less sensitive to PMKT, as were wild-type protoplasts pretreated with phosphatidylinositolspecific phospholipase C to remove GPI-anchored proteins, indicating that the GPI-anchored precursor of Cwp2p is also necessary for PMKT activity. Carboxyfluorescein-entrapped liposomes containing a purified GFP–Cwp2p fusion protein in their membranes were much more sensitive to PMKT than protein-free liposomes. Cwp2p and its GPI-anchored precursor are proposed for the first time to be involved as PMKT secondary receptors.

The potential of biofilm formation of multifunctional starters Lactobacillus pentosus B281 and Pichia membranifaciens M3A during inoculated fermentation of Conservolea natural black olives according to Greek-style processing was investigated. Olives were directly brined in 8% (w/v) NaCl following three fermentation procedures namely, i) spontaneous fermentation, ii) inoculated fermentation with L. pentosus B281, and iii) co-inoculated fermentation with L. pentosus B281 and P. membranifaciens M3A. Lactic acid bacteria (LAB) and yeasts were monitored on olives by plate counting for a period of 153 days, whereas the survival of the inoculated strains was confirmed by Pulsed Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism (RFLP) analysis. Inoculated fermentation with L. pentosus B281 with/without the presence of the yeast resulted in higher acidification of the brine compared to the spontaneous process where no indigenous LAB could be enumerated. The population of LAB on olives ranged between 5.5 and 6.5 log CFU/g and it was maintained at higher levels compared to yeasts (3.5–4.5 log CFU/g) throughout the process. PFGE analysis revealed that L. pentosus B281 could successfully colonize the surface of black olives presenting high recovery rate (100%) at the end of fermentation in contrast to P. membranifaciens M3A that was successfully recovered (42%) only after 72 days of the process. The obtained results provide interesting perspectives for the production of natural black olives with functional properties.

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database ( http://www.yeast-id.com/ ). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to

Summary Yeasts, isolated from different sources, were identified and tested for inhibition using YMA-MB plates seeded with Botrytis cinerea strains. A total of 42 yeast strains of 20 different species were tested in vitro for antagonism against 18 pathogenic B. cinerea strains. Pichia membranifaciens , P. anomala and Debaryomyces hansenii displayed the most important inhibitory effect against Botrytis strains. In small-scale trials, post-harvest application of P. membranifaciens CYC 1106 to apple wounds inhibited B. cinerea CYC 20010. Purified killer toxin from P. membranifaciens CYC 1106 inhibited B. cinerea CYC 20010. Results indicated that certain yeasts, or their toxins such us P. membranifaciens CYC 1106 killer toxin, might have potential as novel agents to control B. cinerea .

The use ofPichia membranifaciensCYC 1106 killer toxin againstBotrytis cinereawas investigated. This strain exerted a broad-specificity killing action against other yeasts and fungi. At pH 4, optimal killer activity was observed at temperatures up to 20 °C. At 25 °C the toxic effect was reduced to 70 %. The killer activity was higher in acidic medium. Above about pH 4·5 activity decreased sharply and was barely noticeable at pH 6. The killer toxin protein fromP. membranifaciensCYC 1106 was purified to electrophoretic homogeneity. SDS-PAGE of the purified killer protein indicated an apparent molecular mass of 18 kDa. Killer toxin production was stimulated in the presence of non-ionic detergents. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a strain ofB. cinerea. The symptoms of infection and grey mould observed inVitis viniferaplants treated withB. cinereawere prevented in the presence of purifiedP. membranifacienskiller toxin. The results obtained suggest thatP. membranifaciensCYC 1106 killer toxin is of potential use in the biocontrol ofB. cinerea.

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (ΔΨm), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

Microbial transformation of xanthohumol using the culture broth of Pichia membranifaciens afforded three metabolites, (E)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":4',3']-2', 4-dihydroxy-6'-methoxychalcone, (2S)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":7,8]-4'-hydroxy-5-methoxyflavanone and (E)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":2',3']-4'-hydroxy-5-methoxychalcone.

A total of 293 strains of lactic acid bacteria and 78 yeast strains from original Bulgarian boza were isolated. The rod-shaped strains were identified as Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus maltaromicus, Lactobacillus fermentum, Lactobacillus parabuchneri, Lactobacillus paracasei subsp. paracasei and Weissella confusa. The strains of genus Leuconostoc were identified as Leuconostoc lactis, Leuconostoc amelibiosum, Leuconostoc mesenteroides subsp. dextranicum and Leuconostoc pseuclomesenteroides. The yeasts isolated belonged to the species Saccharomyces cerevisiae, Pichia membranifaciens, Dekkera bruxellensis and Nadsonici commutata. During the storage of boza the total number of the lactic acid bacteria increased slightly up to 48 h, while the n umber of yeasts increased considerably. The changes in pH and total acidity during the storage of boza were in accordance with the changes in viable count of lactic acid bacteria and their activity.

Microbial transformation of xanthohumol using the culture broth of Cunninghamella echinulata NRRL 3655 afforded (2S)-8-[4″-hydroxy-3″-methyl-(2″-Z)-butenyl]-4′,7-dihydroxy-5-methoxyflavanone (5) and (2S)-8-[5″-hydroxy-3″-methyl-(2″-E)-butenyl]-4′,7-dihydroxy-5-methoxyflavanone (6). Xanthohumol (1) and flavanone 6 as well as (E)-2″-(2′′′-hydroxyisopropyl)-dihydrofurano[2″,3″:4′,3′]-2′,4-dihydroxy-6′-methoxychalcone (2), (2S)-2″-(2′′′-hydroxyisopropyl)-dihydrofurano[2″,3″:7,8]-4′-hydroxy-5-methoxyflavanone (3) obtained with Pichia membranifaciens showed antimalarial activity against Plasmodium falciparum.

© 2015, Springer International Publishing Switzerland. This work examines the formation of poly-microbial communities adhered to the epidermis of natural green Gordal olives and the application of different methodologies for recovery and counting of the microorganisms embedded in olive biofilms. The fermentation process was physicochemical and microbiologically monitored for 90 days, at which, formation of true biofilms on the skin of fermented fruits was confirmed by scanning electron microscopy. Then, samples of olives were taken and treated with sonication, enzymes, mechanical homogenization with stomacher and ultrasonic bath for biofilm disaggregation. The use of the stomacher for 1 min was the most effective treatment to release the lactic acid bacteria (6.6 log10 cfu g−1), whereas sonication for 5 min was the most efficient method for quantification of yeasts (up to 3.5 log10 cfu g−1). Molecular identification of isolates obtained from natural Gordal olive biofilms revealed that Lactobacillus pentosus was the only species found among lactic acid bacteria, while Pichia membranifaciens was the dominant yeast species, with higher counts obtained for the bacteria., The research leading to these results has received funding from the Spanish Government (project OliFilm AGL-2013-48300-R: www.​olifilm.​science.​com.​es), and the Junta de Andalucía (through financial support to project P11-AGR-7051 and group AGR-125). A. Benítez and F.N. Arroyo-López wish to express thanks to the Spanish Government for their pre-doctoral fellowship and postdoctoral research contract (Ramón y Cajal), respectively. V. Romero-Gil would like also to thank the AgriFood Campus of International Excellence (ceiA3), Bank of Santander, Spanish Government and ‘Aloreña de Málaga’ Olive Manufacturing Association for her pre-doctoral fellowship (training program of Ph.D. in companies).

Pichia membranifaciens strain FY-101, isolated from grape skin, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mold disease of the grapevine. When grown together on solid as well as in liquid media, the yeast brings about the inhibition of Botrytis cinerea, which in turn loses its ability to produce the grey mold symptoms on the grapevine plantlets. The secretion of beta-1,3-glucanases by P. membranifaciens is one of the possible mechanisms related to this antagonism. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of this antagonism and the production of beta-1,3-glucanases by P. membranifaciens is given here.

Yeast population and dynamics associated to spontaneous fermentation of green table olives Negrinha de Freixo cv. were evaluated. Olives and brine samples were taken at different fermentation times, and yeast were enumerated by standard plate count and identified by sequencing of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA). Saccharomyces cerevisiae was the most frequent, followed by Candida tropicalis, Pichia membranifaciens and Candida boidini, representing together 94.8% of the total isolates. Galactomyces reessii was also identified for the first time in table olives. The highest species diversity was found between 44 and 54 days of fermentation, both in brine and olive pulp. Furthermore, high similarity was observed between brine and olive pulp microbiotas. In conclusion, these results give valuable information to table olive industrials in order to achieve more knowledge on the fermentation process of this important Protected Designation of Origin product. This work was supported by project SUB-IOC-TEC 11/12 “Table olives from the Northeast of Portugal: Contribution for their characterization and promotion”. info:eu-repo/semantics/publishedVersion

The host specificity of yeast-yeast interactions was investigated for two yeast types, represented by six pairs of Pichia membranifaciens clade yeasts (Pichia membranifaciens or Issatchenkia occidentalis) with apiculate yeasts (Kloeckera apis, Kloeckera africana, or Saccharomycodes ludwigii), commonly found in fruits. Competitive interactions between the two types were detected in both ripe tomato and guava fruit pulp. The differences in growth rates and carrying capacities depended on fruit type (host) and culture conditions (monocultures versus bicultures). These differences were probably due to nutrient composition of each fruit. Pichia membranifaciens did not show host dependent responses, but the apiculate yeasts and Issatchenkia occidentalis did. Depending on yeast strain and culture conditions (i.e., monoculture or biculture), carbon, nitrogen, and vitamins were investigated as potential limiting growth factors in guava fruit. Both singular and multiple limiting nutrients were implicated.

Based on the base composition of nuclear DNA and DNA/DNA hybridization, Pichia galeiformis IFO 10718T was reclassified as a synonym of Pichia manshurica, and Pichia scaptomyzae IFO 10731T was confirmed to be a synonym of Pichia membranifaciens. Comparison of 18S rRNA gene sequences indicated that IFO 10731T (P. scaptomyzae) is identical to P. membranifaciens IFO 10215T and IFO 10725, and IFO 10718T (P. galeiformis) is identical to P. manshurica IFO 10726T. These data were consistent with the view that P. scaptomyzae and P. membranifaciens should be conspecific, as should P. galeiformis and P. manshurica. Variation among 26S rRNA gene domain D1/D2 sequences from three P. membranifaciens strains indicated that this species encompasses a genetically heterogeneous population.

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4·5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3·5, the incidence of killer-sensitive interactions was reduced by 70% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.

Pichia membranifaciens exhibited a dissociative temperature profile (the temperature range of thermal death was distinct from the temperature range of growth) when incubation took place either in a commercial apple juice (AJ) or in a synthetic mineral medium with glucose and vitamins (MGV). In AJ the maximum temperature for growth (Tmax) was 38.6 degrees C, which decreased to 36 degrees C in the presence of either 1 mM sorbic or 1 mM benzoic acid. The minimum temperatures of thermal death (Tmind) were, respectively, 40 and 38 degrees C with either of the acids. The yeast could grow with up to 2 mM sorbic or 3 mM benzoic acid, at 25 degrees C, which is close to the optimum temperature for growth (Top). At temperatures slightly above Tmind, sorbic acid was an actual enhancer of death rather than benzoic, the latter conferring some protection. However, these effects were reversed at higher temperatures (above 43 degrees C), at which benzoic acid was the most operative, in contrast to sorbic which was highly protective of the yeast against thermal death. The addition of acetaldehyde to sulphur-dioxide-containing juice reduced the lag phase and increased the overall specific growth rates. Sporulated or stationary vegetative cultures were more heat-resistant than exponential cultures, particularly at temperatures above 45 degrees C.

The killer toxin fromPichia membranifaciensCYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1→6)-β-d-glucan of the cell wall of a sensitive yeast (Candida boidiniiIGC 3430). The (1→6)-β-d-glucan was purified from cell walls ofC. boidiniiby alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1→6)-β- and (1→3)-β-d-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and1H nuclear magnetic resonance) analyses of the fractions obtained showed that (1→6)-β-d-glucan was a receptor. Adsorption of most of the killer toxin to the (1→6)-β-d-glucan was complete within 2 min. Killer toxin adsorption to the linear (1→6)-β-d-glucan, pustulan, and a glucan fromPenicillium allahabadensewas observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1→6)-β-d-glucan as a receptor for this novel toxin.

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72-98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79-95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9-45.3 mol% for the P. membranifaciens group, 40.0-42.0 mol% for the P. manshurica group, and 27.2-44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.

Putative mechanisms of action associated with the biocontrol capacity of four yeast strains (Cryptoccocus albidus NPCC 1248, Pichia membranifaciens NPCC 1250, Cryptoccocus victoriae NPCC 1263 and NPCC 1259) against Penicillium expansum and Botrytis cinerea were studied by means of in vitro and in situ assays. C. albidus(YP), a commercial yeast was also evaluated for comparative purposes. The yeast strains exhibited a variety of different mechanisms including: wound colonization, germination inhibition, biofilm formation, secretion of killer toxins, competition for nutrient and secretion of hydrolytic enzymes (protease, chitinase and glucanase). The relationship between strains (and their associated antagonist mechanisms) and in situ antagonist activity was also evaluated. Results indicate that mechanisms such as production of hydrolytic enzymes, the ability for colonization of wounds, production of killer toxin and inhibition of germination are the most important for biocontrol activity. Our study indicate that multiple modes of action may explain why P. membranifaciens NPCC 1250 and C. victoriae NPCC 1263 provided excellent control of postharvest pears disease.

Pichia membranifaciens CYC 1086 secretes a unique 30kDa killer toxin (PMKT2) that inhibits a variety of spoilage yeasts and fungi of agronomical interest. The cytocidal effect of PMKT2 on Saccharomyces cerevisiae cells was studied. Metabolic events associated with the loss of S. cerevisiae viability caused by PMKT2 were qualitatively identical to those reported for K28 killer toxin activity, but different to those reported for PMKT. At higher doses, none of the cellular events accounting for the action of PMKT, the killer toxin secreted by P. membranifaciens CYC 1106, was observed for PMKT2. Potassium leakage, sodium influx and the decrease of intracellular pH were not among the primary effects of PMKT2. We report here that this protein is unable to form ion-permeable channels in liposome membranes, suggesting that channel formation is not the mechanism of cytotoxic action of PMKT2. Nevertheless, flow cytometry studies have revealed a cell cycle arrest at an early S-phase with an immature bud and pre-replicated 1n DNA content. By testing the sensitivity of cells arrested at different stages in the cell cycle, we hoped to identify the execution point for lethality more precisely. Cells arrested at the G1-phase by α-factor or arrested at G2-phase by the spindle poison methyl benzimidazol-2-yl-carbamate (MBC) were protected against the toxin. Cells released from the arrest in both cases were killed by PMKT2 at a similar rate. Nevertheless, cells released from MBC-arrest were able to grow for a short time, and then viability dropped rapidly. These findings suggest that cells released from G2-phase are initially able to divide, but die in the presence of PMKT2 after initiating the S-phase in a new cycle, adopting a terminal phenotype within that cycle. By contrast, low doses of PMKT and PMKT2 were able to generate the same cellular response. The evidence presented here shows that treating yeast with low doses of PMKT2 leads to the typical membranous, cytoplasmic, mitochondrial and nuclear markers of apoptosis, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release.

Benzoyl phenyl urea, a class of insect growth regulator's acts by inhibiting chitin synthesis. Carvacrol, a naturally occurring monoterpenoid is an effective antifungal agent. We have structurally modified carvacrol (2-methyl-5-1-methylethyl] phenol) by introducing benzoylphenyl urea linkage. Two series of benzoylcarvacryl thiourea (BCTU, 4a-f) and benzoylcarvacryl urea (BCU, 5a-f) derivatives were prepared and characterized by elemental analysis, IR, H-1 and C-13 NMR and Mass spectroscopy. Derivatives 4b, 4d, 4e, 4f and 5d, 5f showed comparable insecticidal activity with the standard BPU lufenuron against Dysdercus koenigii. BCTU derivatives 4c, 4e and BCU 5c showed good antifungal activity against phytopathogenic fungi viz. Magnaporthe grisae, Fusarium oxysporum, Dreschlera oryzae; food spoilage yeasts viz. Debaromyces hansenii, Pichia membranifaciens; and human pathogens viz. Candida albicans and Cryptococcus neoformans. Compounds 5d, 5e and 5f showed potent activity against human pathogens. Moderate and selective activity was observed for other compounds. All the synthesized compounds were non-haemolytic. These compounds have potential application in agriculture and medicine. (C) 2012 Elsevier Ltd. All rights reserved.

4 Páginas; 1 Figura, easts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future., This research line has received funding from the EU’s Seventh Framework Programme [FP7/2007-2013; under grant agreement n° 243471 (PROBIOLIVES)]. We also thank the Spanish Government for financial support to projects AGL2009-07436/ALI and AGL2010-15529/ALI, and Junta de Andalucía through financial support to group AGR-125. Thanks to JOLCA and ASEMESA for their invaluable collaboration. Joaquín Bautista-Gallego and Francisco N. Arroyo-López would also like to thank CSIC and Spanish Government for their JAE-PRE fellowship and Ramón y Cajal postdoctoral research contract, respectively.

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.

PMKT (Pichia membranifaciens killer toxin) reportedly has antimicrobial activity against yeasts and filamentous fungi. In previous research we posited that high PMKT concentrations pose a serious challenge for cell survival by disrupting plasma membrane electrochemical gradients, inducing a transcriptional response similar to that of certain stimuli such as hyperosmotic shock. This response was related to the HOG-pathway with Hog1p phosphorylation and a transitional increase in intracellular glycerol accumulation. Such a response was consistent with the notion that the effect induced by high PMKT concentrations lies in an alteration to the ionic homeostasis of the sensitive cell. By contrast, the evidence presented here shows that low PMKT doses lead to a cell death process in Saccharomyces cerevisiae accompanied by cytological and biochemical indicators of apoptotic programmed cell death, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release. Furthermore, dying cells progressed from an apoptotic state to a secondary necrotic state, and the rate at which this change occurred was proportional to the intensity of the stimulus. We have explored the global gene expression response of S. cerevisiae during that stimulus. The results obtained from DNA microarrays indicate that genes related with an oxidative stress response were induced in response to proapoptotic concentrations of PMKT, showing that the coordinated transcriptional response is not coincident with that obtained when ionophoric concentrations of PMKT are used. By contrast, cwp2Δ mutants showed no signs of apoptosis, indicating that the initial steps of the killer mechanism coincide when proapoptotic (low) or ionophoric (high) PMKT concentrations are used. Additionally, low dosages of PMKT promoted Hog1p phosphorylation and glycerol accumulation.

Publisher Summary This chapter studies the genus Pichia. In the determination of the asexual reproduction, it is seen that budding is multilateral on a narrow base. Cells are spherical, ovoid, or short elongate. Pseudohyphae may occur but true hyphae are not formed. In sexual reproduction it is found that asci are unconjugated or show conjugation between a parent cell and its bud or between independent cells. Some species are heterothallic, whereas others are homothallic. Asci may be persistent or deliquescent and form one to four, or rarely five to eight, ascospores. The chapter also discusses physiology/biochemistry and phylogenetic placement of the genus. The type species taken is Pichia membranifaciens. The key phenotypic characteristics of species assigned to the genus Pichia are presented. In the systematic discussion of the species, growth on 5% malt extract agar, growth on the surface of assimilation media, Dalmau plate culture on morphology agar, formation of ascospores, gene sequence accession numbers, type strain, origin of the strains studied, systematics, and ecology are determined.

In order to detect spoilage yeast species in wines showing off-odors, different yeast isolation protocols were evaluated. Independently of the isolation method, only Saccharomyces cerevisiae and Pichia manshurica were detected. The spoilage capacity of P. manshurica regional isolates was evaluated in red wine and the production of volatile phenols was evidenced. To evaluate the possible source of contamination, yeasts from both grapes and cellar surfaces were obtained. Hanseniaspora uvarum and Zygoascus hellenicus were detected in both sound and damaged grapes from sunny areas. The most frequent species in cellar surfaces was Candida boidinii, Pichia membranifaciens and P. manshurica were detected in filters. The intra-specific genetic characterization of the P. manshurica isolates by mtDNA-RFLP demonstrated that the same strain was detected in both wine and filter. Most P. membranifaciens isolates produced 4-EP (maximum level of 1.895 mg/L) and particularly high levels of 4-EG (maximum level of 10.260 mg/L) were produced by P. manshurica isolates in synthetic wine-like medium. In this work the capacity of P. manshurica and P. membranifaciens species to produce volatile phenols was shown for the first time.

Sequence analysis of D1/D2 region of 26S rDNA was used for identification of different yeasts isolated from food-borne material. Then, their aromas compounds were detected and analyzed by SPME-GC-MS, another biology information technology. 12 yeast strains were grouped in 7 species belonging to 5 genera as follows: Saccharomyces cerevisiae, Torulaspora delbrueckii, Kluyveromyces marxianus, Pichia cactophila, Clavispora lusitaniae, Pichia membranifaciens, and Pichia kudriavzevii. Among all yeast strains, most of volatiles were similar, but contents were affected by different strains. Strain A1 (Saccharomyces cerevisiae) produced relatively higher aromas compounds than S6 (Kluyveromyces marxianus). The result of GC-MS showed ethyl octanoate and phenylethyl acetate were high-impact flavors by strain A1 and S6, respectively. In conclusion, it is a reliable identification system to indentify food-borne yeasts at species level using 26S rDNA sequencing and phenotypic studies. SPME-GC/MS analysis highlights aroma compounds can be used for pre-screening potential edible yeast strains effectively.

Aims: To assess the yeast community structure and dynamics during Greek-style processing of natural black Conservolea olives in different brine solutions. Methods and Results: Black olives were subjected to spontaneous fermentation in 6% (w/v) NaCl brine solution or brine supplemented with (i) 0·5% (w/v) glucose, (ii) 0·2% (v/v) lactic acid and (iii) both glucose and lactic acid. Yeast species diversity was evaluated at the early (2 days), middle (17 days) and final (35 days) stages of fermentation by restriction fragment length polymorphism and sequence analyses of the 5·8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of isolates. Analysis revealed a relatively broad range of biodiversity composed of 10 genera and 17 species. In all treatments, yeasts were the main micro-organisms involved in fermentation together with lactic acid bacteria that coexisted throughout the processes. Metschnikowia pulcherrima was the dominant yeast species at the onset of fermentation, followed by Debaryomyces hansenii and Aureobasidium pullulans. Species heterogeneity changed as fermentations proceeded and Pichia membranifaciens along with Pichia anomala evolved as the main yeasts of olive elaboration, prevailing at 17 and 35 days of the process. Molecular techniques allowed for the identification of five yeast species, namely A. pullulans, Candida sp., Candida silvae, Cystofilobasidium capitatum and M. pulcherrima, which have not been reported previously in black olive fermentation. Conclusions: By using molecular techniques, a rich yeast community was identified from Conservolea black olive fermentations. Metschnikowia pulcherrima was reported for the first time to dominate in different brines at the onset of fermentation, whereas Pichia anomala and P. membranifaciens evolved during the course. The addition of glucose and/or lactic acid perturbed yeast succession and dominance during fermentation. Significance and Impact of the Study: Yeasts have an important role in black olive fermentation and contribute to the development of the organoleptic characteristics of the final product. At the same time, certain species can cause significant spoilage. The present study adds to a better knowledge of yeast communities residing in olive fermentations towards a well-controlled process with minimization of product’s losses.

Background: Absence or severe reduction in the amount of ergosterol in the fungal membrane and its replacement with other sterols have been described as potential antifungal resistance mechanisms in fungi. Aims and methods: The ergosterol content in a collection of 51 clinical yeast isolates, including susceptible and resistant strains to amphotericin B and azoles, was estimated by a simple chromatographic method (HPLC-UV). Results: A high content of ergosterol was detected for several strains of Candida glabrata, Candida tropicalis or Pichia membranifaciens. In contrast, strains of Cryptococcus neoformans and Dipodascus capitatus had the lowest ergosterol concentrations. No significant correlation was observed between antifungal susceptibility patterns and ergosterol content. Conclusions: We conclude from this study that ergosterol content on yeasts may not be associated with specific resistant patterns. © 2011 Published by Elsevier Espana, S.L. on behalf of Revista Iberoamericana de Micologia. Perfil de sensibilidad in vitro y contenido en ergosterol en levaduras de origen clinico

Yeasts were isolated from spontaneous fermentations of olives in brines. Ascomycetous species dominated the yeast flora (>90%) and among them Pichia membranae-faciens and related species. Some components of the olives were tested as substrates for growth. Killer activity was observed in approximately half of the isolates, and the wider spectra were displayed by strains of Pichia anomala.

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47 °C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42 °C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40 °C than at 30 °C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.

9 páginas, 4 figuras, 5 tablas.-- El Pdf del artículo es la copia de autor., Ripe olives account for ca. 30% of the world's table olive production. Fruits intended for this type of product are preserved in an aqueous solution (acidic water or brine) for several months, where they may undergo a spontaneous fermentation. Enterobacteriaceae and lactic acid bacteria were not detected in the present survey during storage. Thus, the work focused on studying the yeast microflora associated with the ripe olive storage of Manzanilla and Hojiblanca cultivars in acidified brines. A total of 90 yeast isolates were identified by means of molecular methods using RFLP analysis of the 5.8S-ITS rDNA region and sequencing of the D1/D2 domains of the 26S rDNA gene. The two most important species identified in both cultivars were Saccharomyces cerevisiae and Pichia galeiformis, which were present throughout the storage period, while Candida boidinii was detected during the later stages of the process. The species Pichia membranifaciens was detected only in the early stages of the Hojiblanca cultivar. The lipase assays performed with both extracellular and whole cell fractions of the yeast isolates showed that neither of the S. cerevisiae and P. galeiformis species had lipase activity, while the P. membranifaciens isolates showed a weak activity. On the contrary, all C. boidinii isolates gave a strong lipase activity. Change in olive fat acidity was markedly higher in the presence of the yeast population than in sterile storage, indicating that lipases produced by these microorganisms modify the characteristics of the fat in the fruit., This work was supported by the Spanish Government(project AGL-2006-03540/ALI, partially financed by European regional developmentfunds,ERDF),and Junta de Andalucía(through financial support to group AGR-125).J. Bautista-Gallego and F.N. Arroyo-López want to thank CSIC and the Spanish Government (MICINN) for their I3P fellowship and the ‘Juan de la Cierva’ postdoctoral research contract, respectively.

The physiological and kinetic capabilities of 233 Saccharomyces cerevisiae isolates, originating from traditional Brazilian cachaca fermentation, were evaluated under laboratory conditions to select flocculent and non-H2S producing strains to be employed in beverage production. Three flocculent S. cerevisiae strains were selected, two non-H2S producing and one H2S producing, and their kinetic performances were analysed during two large-scale fermentation experiments in a traditional cachaca distillery. One non-flocculent H2S-producing S. cerevisiae strain was also used for comparison with the flocculent strains. The results of mitochondrial DNA restriction analysis showed that the three flocculent starter S. cerevisiae strains, as well as the non-flocculent strain, remained in the process during the whole fermentation period, with cells numbering around 10(7) cfu/ml. All selected strains produced ethanol yields that were typically higher in the distillery than in the laboratory conditions, except for strain UFMGA-1240. The greatest diversity of non-Saccharomyces yeasts was observed prior to day 21 of cachaca fermentation; Pichia membranifaciens and Hanseniaspora guilliermondii were the most frequently isolated species. These yeasts were present in lower densities throughout the whole process. The cachaca produced by the selected strains contained concentrations of chemical compounds in accordance with current Brazilian legislation, and all cachacas scored well in sensory effective tests. In addition to the advantage of being flocculent, the strain UFMGA-1031 is non-H2S producing and also produces cachaca with good sensory acceptance. Therefore, this flocculent and non-H2S producing S. cerevisiae strain is highly suitable as a starter for production of high quality traditional cachaca.