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Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:40:52Z No. of bitstreams: 1 S1516-89132007000400019.pdf: 95830 bytes, checksum: b08d1b51984e2bdeb3f3f2bbe5274cb5 (MD5) Made available in DSpace on 2013-08-22T18:40:52Z (GMT). No. of bitstreams: 1 S1516-89132007000400019.pdf: 95830 bytes, checksum: b08d1b51984e2bdeb3f3f2bbe5274cb5 (MD5) Previous issue date: 2007-07-01 Made available in DSpace on 2013-09-30T19:27:04Z (GMT). No. of bitstreams: 2 S1516-89132007000400019.pdf: 95830 bytes, checksum: b08d1b51984e2bdeb3f3f2bbe5274cb5 (MD5) S1516-89132007000400019.pdf.txt: 35466 bytes, checksum: 785445d4ad13703dbb9399735b094e1f (MD5) Previous issue date: 2007-07-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:07:32Z No. of bitstreams: 2 S1516-89132007000400019.pdf: 95830 bytes, checksum: b08d1b51984e2bdeb3f3f2bbe5274cb5 (MD5) S1516-89132007000400019.pdf.txt: 35466 bytes, checksum: 785445d4ad13703dbb9399735b094e1f (MD5) Made available in DSpace on 2014-05-20T15:07:32Z (GMT). No. of bitstreams: 2 S1516-89132007000400019.pdf: 95830 bytes, checksum: b08d1b51984e2bdeb3f3f2bbe5274cb5 (MD5) S1516-89132007000400019.pdf.txt: 35466 bytes, checksum: 785445d4ad13703dbb9399735b094e1f (MD5) Previous issue date: 2007-07-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Considerando o antagonismo e degradação de patulina detectados em Pichia membranifaciens e Sporobolomyces roseus no estudo preliminar, este trabalho avaliou o efeito antagônico de Pichia ohmeri 158 no desenvolvimento de Penicillium expansum e a degradação de patulina in vitro. O sobrenadante do cultivo de P. ohmeri 158 inibiu 66,17% do desenvolvimento micelial, indicando antibiose relacionada ao fator killer. A concentração inicial de patulina (223 µg) na presença de células íntegras de P. ohmeri foi reduzida em mais de 83% após dois dias de incubação a 25ºC e superior a 99% após 5 dias, com níveis indetectáveis no 15º dia. O decréscimo do pH 4,0 inicial para pH 3,3 sugeriu que a eliminação de patulina é um processo ativo e uma conseqüência do metabolismo da levedura. Os resultados obtidos concluem que P. ohmeri 158 é uma alternativa promissora na inibição do desenvolvimento de P. expansum e na degradação de patulina. Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 µg/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17% micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 µg in the presence of P. ohmeri 158 cells was decreased over 83% of the original concentration, when incubated at 25ºC/2 days and > 99% after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation. State University of Londrina Department of Biochemistry and Biotechnology State University of Ponta Grossa Department of Food Engineering UNESP Department of Food Engineering and Technology UNESP UNESP Department of Food Engineering and Technology UNESP

Four yeast strains (TM1-01T, TM1-07, TM3-47 and TM3-49) were isolated by membrane filtration from estuarine water collected from a mangrove forest in Phang-Nga province, southern Thailand. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that the sequences of the four strains were identical. The closest recognized species in terms of pairwise sequence similarity was Pichia deserticola, but the level of nucleotide substitution (4.8 %) was sufficient to justify the description of a separate species. Phylogenetic analysis demonstrated that the four strains occupy a basal position with respect to Pichia membranifaciens and close relatives. The four strains showed identical phenotypic characteristics, including proliferation by multilateral budding, absence of ascospores, arthrospores and ballistospores and negative reactions for Diazonium blue B and urease. The major ubiquinone was Q-7. On the basis of the above findings, these four strains were assigned to a single novel species of the genus Candida, for which the name Candida thaimueangensis sp. nov. is proposed. The type strain is TM1-01T (=CBS 10360T=NBRC 101967T=BCC 21229T).

The antimicrobial action of selected short synthetic peptides against wine spoilage yeasts such as Cryptococcus albidus, Dekkera bruxellensis, Pichia membranifaciens, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces bisporus has been examined. Peptides analyzed include nine sequence-related antifungal hexapeptides (PAFs) previously developed by a combinatorial approach, and two representative lactoferricin B (LfcinB)-derived peptides. Different peptides had distinct activity profiles. In vitro assays identified the peptides PAF26, PAF36, and LfcinB(17-31), as having growth inhibitory properties towards several of the yeasts at low micromolar concentrations. Z. bailii and Z. bisporus were the most sensitive yeasts. In addition to their fungistatic activity, the three peptides showed fungicidal properties towards Z. bailii, Z. bisporus, and S. cerevisiae in laboratory growth medium. Remarkably, only LfcinB(17-31) against Z. bisporus had inhibitory and fungicidal properties in wine at the concentrations assayed, showing that the antimicrobial action of each peptide is dependent on both the food matrix and the target micro-organism. Lack of fungicidal activity of peptides against Z. bailii in wine is related to the presence of salt ions other than divalent cations. On the contrary, fungicidal activity of LfcinB(17-31) towards Z. bisporus was not significantly affected by wine salts. Our data identify a bioactive peptide from natural origin with potential use against the food spoilage yeast Z. bisporus, and indicate that the application of antimicrobial peptides in wine preservation deserves further investigation.

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.

M. COMBINA, L. MERCADO, P. BORGO, A. ELIA, V. JOFRE´ , A. GANGA, C. MARTINEZ AND C. CATANIA. 2005. Aims: The aims of this work were to evaluate different pre-isolation treatments applied to complete yeastextraction from grapes and to identify the yeast microflora associated to Malbec grapes from two vineyards locatedin Mendoza, Argentina.Methods and Results: The pre-isolation treatments evaluated were shaking, jet streaming with pressurized waterand grape blending. The overall results clearly indicated that when a more vigorous and disruptive pre-isolationtreatment was applied; larger numbers of yeast species were recovered. The yeast population on healthy and ripeMalbec grapes was in the order of 10 5 –10 6 CFU g )1 . Eight different yeast species were isolated from berries,including Kloeckera apiculata, Metschnikowia pulcherrima, Pichia membranifaciens, Saccharomycodes ludwigii, Candidaspecies (Candida stellata and Candida raghi), Issatchenkia orientalis and Rhodotorula spp.Conclusions: Grape blending gave the highest yeast counts. Rainfall near grape harvest time quantitatively andqualitatively modifies the yeast microflora. The yeast species identified on ripe grapes from the Mendoza region,partially match those previously documented in different parts of the world related. S. ludwigii has not beenpreviously reported in grapes.Significance and Impact of the Study: The report is on yeast microbiota in grapes from Mendoza, Argentina.Saccharomycodes ludwigii was found in high percentage (17%), this species has not been described before on grapessurface. The importance of pre-isolation steps to the recovery of high number of yeasts was shown. Influence ofclimatic conditions near harvest time on microflora was confirmed.Keywords: Argentina, grape berry, Malbec, pre-isolation, yeast.

Aims: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. Methods and Results: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1·5–2 mol l−1 NaCl. Although the presence of H2O2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity. Conclusions: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. Significance and Impact of the Study: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.

In this experiment a differential culture medium is used for the rapid and efficient detection and enumeration of Z. bailii in the presence of other yeast species encountered in wines, such as S.cerevisiae and P. membranifaciens.

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.

Enzymatic activity of potential interest in wine-making was studied for 182 non-Saccharomyces yeasts isolated from musts before and at the onset of fermentation at wine cellars operating under the La Mancha Appellation of Origin in Spain. Tests were carried out on plates containing differential substrates appropriate for each case (casein, gelatin, polygalacturonic acid, and arbutin) to determine whether each of the isolates exhibited proteolytic, polygalacturonase, and beta-glucosidase activities. Nearly 80% of the wild yeasts possessed one or more enzymes of biotechnological interest. Once the enzymatic activities of the isolates had been established, 69 of the isolates that exhibited pronounced enzymatic activity and 11 randomly selected isolates that were devoid of any activity were typed using PCR/RFLP, which gave 13 different molecular profiles. The isolates for each of the profiles were then identified by classical methods. The enzyme beta-glucosidase was linked to the species Metschnikowia pulcherrima, and polygalacturonase activity was common in most of the species identified. Proteolytic activity was observed in Pichia membranifaciens and in Metschnikowia pulcherrima. Typing revealed the possibility of intraspecific differences in Pichia membranifaciens, because six different molecular profiles with one or more shared restriction bands were recorded for that species.

Microflora in wound sites of preharvest maize (including bacteria, yeasts, and filamentous fungi) may play a role in attracting insects to maize plants and may also interact with growth and mycotoxin production by filamentous fungi. As little data are available about the yeasts occurring on maize from the U.S. corn belt, samples of milled maize from experimental plantings at the University of Illinois River Valley Sand Field were analyzed. Yeast counts showed slight yearly fluctuation and varied between 3.60 and 5.88 (log cfu/g maize). The majority of the yeasts were Candida guilliermondii (approximately 55%), Candida zeylanoides (24 %), Candida shehatae (11%), and Debaryomyces hansenii (3%). Also present were Trichosporon cutaneum, Cryptococcus albidus var. aerius, and Pichia membranifaciens. The occurrence of killer yeasts was also evaluated. Killer yeasts were detected in maize for the first time and were identified as Trichosporon cutaneum and Candida zeylanoides. These were able to kill some representative yeasts isolated from maize, including Candida guilliermondii, Candida shehatae, and Cryptococcus albidus var. aerius. Other maize yeasts (Candida zeylanoides, Debaryomyces hansenii, Pichia membranifaciens) were not affected. The majority of yeasts found on maize were unable to ferment its major sugars, i.e., sucrose and maltose. Some (e.g., Candida zeylanoides) were not even able to assimilate these sugars. The importance of these properties in relation to insect attraction to preharvest ears of maize is discussed.Key words: corn, maize, yeast, killer.

Yeasts had mean counts of above 106 CFU/g in the fecal pellets of small mammals from tropical forest fragments. Most of the 55 species isolated were fermentative ascomycetes, with the most frequent being Debaryomyces hansenii, Pichia membranifaciens and Issatchenkia orientalis, whereas Rhodotorula mucilaginosa was the most frequent yeast of basidiomycetous affinity.Leveduras em pelotas fecais de pequenos mamíferos provenientes de fragmentos de Mata Atlântica tiveram contagem média acima de 106 UFC/g. A maioria das 55 espécies isoladas foi de ascomicetos fermentativos, com as mais freqüentes sendo Debaryomyces hansenii, Pichia membranifaciens e Issatchenkia orientalis, enquanto Rhodotorula mucilaginosa foi a levedura mais freqüente de afinidade basidiomicética.

Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non-Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non-Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima, and Pichia membranifaciens) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae, to assess the impact of the non-Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae. Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae. We report here, for the first time, that non-Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris, aspartic acid was assimilated very slowly by M. pulcherrima, and glutamine was not assimilated by P. membranifaciens. By contrast, cysteine appeared to be a preferred nitrogen source for all non-Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non-Saccharomyces yeasts may account for some of the interactions observed here, such as poorer performances of S. cerevisiae and volatile profile changes.