Infrared spectra of microbial cells are highly specific, fingerprint-like signatures which can be used to differentiate microbial species and strains from each other. In this study, the potential applicability of Fourier transform infrared (FTIR) spectroscopy for the classification of yeast strains in terms of their biological taxonomy, their use in the production of wine, beer, and bread, and their sensitivity to killer yeast strains was investigated. Sample preparation, spectral data preprocessing methods and spectral classification techniques were also investigated. All yeast strains were grown on a single growth medium. The FTIR spectra were baseline corrected and the second derivative spectra were computed and employed in spectral analysis. The classification accuracy was improved when the principal component spectra (calculated from the second derivative spectra) were employed rather than the second derivative spectra or raw spectra alone. Artificial neural network (ANN) with 10 units in the input layer and 12 units in the hidden layer produced a robust prediction model for the identification of yeasts. Cluster analysis was employed for the classification of yeast strains in terms of their use in the production of wine, beer, and bread and in terms of their sensitivity to killer yeast strains. The optimum region for the classification in the former case was found to be between 1300 and 800 cm-1 in the infrared spectrum whereas the optimum region for the classification of yeast strains in terms of their sensitivity was between 900 and 800 cm-1 . The results of this work demonstrated that FTIR spectroscopy could be successfully employed for the classification and identification of yeast strains with minimal sample preparation.

The purpose of this study is twofold: (1) conduct an epidemiological survey of yeasts found in clinical material; and (2) suggest an identification scheme that would identify yeasts of medical importance in the shortest possible time.

This diploma thesis focuses on isolation and taxonomic classification of yeast species isolated during the red wine (Pinot noir) fermentation. Grapes were grown under organic and integrated farming in South Moravia wine region, Czech Republic. Processing was controlled – for inoculation was used strain Saccharomyces cerevisiae BS6. Polymerase chain reaction followed by restriction fragment lenght polymorphism of PCR-amplified fragments (PCR-RFLP) was used for yeast species identification. For DNA analysis we used coding region of 5.8S ITS rDNA which was amplified using ITS1-ITS4 primers. Amplicon was digested by three restriction endonucleases - HaeIII, HinfI and HhaI. Isolates were divided into eleven groups using UPGMA cluster analysis (software BioNumerics). We identified following yeast species: Candida valida, Candida vini, Issatchenkia occidentalis, Pichia fermentans, Saccharomyces cerevisiae and Zygosaccharomyces bailii. We were not able to identify some yeast species. Differences between organic and integrated farming were demonstrated with varying composition of yeast species.

No description available.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Wine production involves complex interactions between a variety of yeasts and bacteria. Conventional microbiological methods can be used to identify the different microorganisms present in wine, but prove to be time-consuming and certain microbial species may not grow on synthetic isolation media. The aim of this study was to evaluate the microbial population present in two South African red wines, Pinotage and Merlot, as well as five spoilt commercial South African wines by using a non-culturable approach, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE). The results from the non-culturable approach were compared to conventional platings. Unique PCR-based DGGE fingerprints were obtained for the Bacteria and yeasts present in the South African Pinotage and Merlot wines. Using yeast specific primers the Pinotage wine showed the presence of non-Saccharomyces yeasts at the beginning of the alcoholic fermentation, while Saccharomyces cerevisiae was present until the completion of the malo-lactic fermentation (MLF). This yeast was also identified during both the alcoholic fermentation and MLF of the Merlot wine using PCR-based DGGE and conventional plating. Using Bacteria specific primers, Lactobacillus plantarum and Lactobacillus sp. was identified in the Pinotage wine using PCR-based DGGE, while Lactobacillus brevis were isolated from Merlot wine using conventional platings. Although the presence of S. cerevisiae is expected during wine fermentation, the presence of this microbe in bottled wine could lead to spoilage. Four of the spoilt commercial wine samples (RW1, RW2, RoW1 and WW1) were found to be spoilt by S. cerevisiae, while a fifth wine sample (RW3) was found to be spoilt by an Acetobacter sp. using PCR-based DGGE. Members of the family Enterobacteriaceae were identified from all the wines using PCR-based DGGE, while Enterobacter sakazakii was identified from RW1 using PCR-based DGGE and conventional plating. The members of the family Enterobacteriaceae could possibly have contributed to the spoilage of the wine by producing undesirable secondary metabolites. PCR-based DGGE proved to be an alternative to conventional microbiological methods for the identification of the microbial species in South African red grape must and wine. This method also proved to be useful in the identification of spoilage microbes in spoilt commercial South African wines. / AFRIKAANSE OPSOMMING: Die produksie van rooi wyn behels komplekse interaksies tussen ‘n verskeidenheid van giste en bakterieë. Konvensionele mikrobiologiese metodes kan gebruik word om die verskillende mikro-organismes wat in rooi wyn teenwoordig is te identifiseer, maar dit blyk tydrowend te wees, terwyl sekere mikro-organismes nie groei op sintetiese media nie. Die doel van hierdie studie was om die mikrobiologiese populasie wat in twee Suid-Afrikaanse rooi wyne, Pinotage en Merlot, en vyf bederfde kommersiële wyne teenwoordig is, te evalueer met die gebruik van ‘n kultuur-onafhanklike benadering, polimerase ketting-reaksie (PKR)-gebaseerde denaturerende gradiënt jel elektroforese (DGJE). Die resultaat van die kultuur-onhafhanklike benadering was vergelyk met konvensionele uitplating tegnieke. Unieke, ongeëwenaarde PKR-gebaseerde DGGE vingerafdrukke was verkry van die Bakterieë en giste aanwesig in die Pinotage en Merlot wyne. Deur gebruik te maak van gis-spesifieke inleiers het die Pinotage wyn die teenwoordigheid van nie-Saccharomyces giste getoon, terwyl Saccharomyces cerevisiae teenwoordig was tot en met die afhandeling van die appel-melksuur gisting (AMG). Hierdie gis is ook geïsoleer gedurende beide die alkoholiese gisting en AMG van die Merlot wyn deur gebruik te maak van PKR-gebaseerde DGGE en konvensionele uitplating tegnieke. Met Bakterieë-spesifieke inleiers, was Lactobacillus plantarum en Lactobacillus sp. geïdentifiseer in die Pinotage wyn deur gebruik te maak van PKR-gebaseerde DGGE, terwyl Lactobacillus brevis geïsoleer is uit Merlot wyn deur gebruik te maak van konvensionele uitplatings. Alhoewel die teenwoordigheid van S. cerevisiae verwag word gedurende wynfermentasie, kan die teenwoordigheid van hierdie mikrobe in gebottelde wyn tot bederwing lei. Vier van die bedorwe kommersiële wynmonsters (RW1, RW2, RoW1 en WW1) was bederf deur S. cerevisiae, terwyl ‘n vyfde wynmonster (RW3) bederf was deur ‘n Acetobacter sp. deur die gebruik van PKR-gebaseerde DGGE. Van al die wyne is lede van die Enterobacteriaceae familie geïdentifiseer deur gebruik gemaak te maak van PKR-gebaseerde DGGE, terwyl Enterobacter sakazakii geïsoleer is van RW1 met konvensionele uitplating. Die lede van die familie Enterobacteriaceae kon moontlik bygedra het tot die bederwing van die wyn deur ongewenste sekondêre metaboliete te produseer. PKR-gebaseerde DGGE bewys ‘n alternatief tot die konvensionele mikrobiologiese metodes vir die identifikasie van die mikrobiese spesies in Suid-Afrikaanse rooi druif mos en wyn te wees. Hierdie metode het ook die bruikbaarheid in die identifikasie van mikrobes wat kommersiële Suid-Afrikaanse wyne bederf, bewys.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-05-11T14:03:23Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao Final.pdf: 3056948 bytes, checksum: c1f1bdb2193095261e3561f1c0508bd9 (MD5) / Made available in DSpace on 2016-05-11T14:03:23Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertacao Final.pdf: 3056948 bytes, checksum: c1f1bdb2193095261e3561f1c0508bd9 (MD5) Previous issue date: 2015-02-20 / CNPq / Piedra branca é uma infecção de etiologia fúngica caracterizada pela formação de nódulos restritos a porção extrafolicular dos pelos e cabelos, sendo ocasionada por leveduras do gênero Trichosporon. O diagnóstico é clínico e laboratorial micológico, sendo concluído com a identificação precisa do agente etiológico. Esta pesquisa teve como objetivo elaborar um modelo de infecção in vitro da piedra branca, analisar os aspectos morfológicos e ultraestruturais dos nódulos e seus agentes, e autenticar taxonomicamente os isolados por meio de uma abordagem de identificação polifásica. A infecção dos cabelos foi induzida utilizando 18 isolados do gênero Trichosporon, mantidos na Coleção de Culturas Micoteca URM. Para confirmação taxonômica dos isolados, foi adotada métodos empregados na taxonomia clássica, Espectroscopia de Massa (MALDI-TOF MS) e sequenciamento das regiões ITS (ITS1–5.8s-ITS2), IGS1 (Intergenic spacer) e domínios D1/D2 do rDNA. Foi verificado que 15 (83,33%) dos isolados de levedura foram identificados por meio da Espectrometria de Massa MALDI-TOF MS. Os métodos moleculares foram considerados os mais precisos, possibilitando a identificação dos isolados de levedura como: T. asahii (8), T. faecale (3), T. montevideense (5), T. mycotoxinivorans (1) e Hyphopichia burtonii (1). Os nódulos típicos foram formados por 12 isolados, seis formaram nódulo atípico (Quatro de T. asahii e dois de T. montevideense) e dois não formaram nódulo (um de T. montevideense e um de T. faecale). Os constituintes químicos detectados nos nódulos foram carbono, oxigênio, sódio, cálcio, manganês e magnésio. A consistência friável dos nódulos está relacionada com as baixas concentrações de cálcio e enxofre, conferindo a facilidade de rompimento do nódulo. Neste modelo, foi verificado que temperaturas elevadas e umidade excessiva não estão relacionadas com o desenvolvimento desta tricopatia. / White piedra is a fungal infection caused by Trichosporon species. The clinical condition of this mycosis is the production of white nodules adhered to the hair shaft. The diagnosis take in account the clinical feature and mycological laboratory results, which is finalized with the precise etiologic agent identification. This research aimed to develop an in vitro infection model of white piedra, analyzing the morphological and ultrastructural aspects of nodules and aetiological agents, and to identify the isolates by a polyphase approach. The infection of the hair was induced using 18 isolates of the genus Trichosporon, from the Culture Collection Micoteca URM. To taxonomic confirmation of the Trichosporon isolates, were adopted methods used in the classical taxonomy, Mass Spectroscopy (MALDI-TOF MS) and sequencing of the ITS (ITS1-5.8s-ITS2), IGS1 (Intergenic spacer) and D1/D2 domains of rDNA regions. A total of 15 (83.33%) yeast isolates were identified by mass spectrometry MALDI-TOF MS. Molecular tols were considered more accurate, allowing the identification of all yeast isolates as: T. asahii (8), T. faecale (3), T. montevideense (5), T. mycotoxinivorans (1) and Hyphopichia burtonii (1). Typical nodules were formed by 12 isolates, 6 isolates formed atypical nodules (T. asahii (4) and T. montevideense (2)) and 2 isolates did not formed nodules (T. montevideense (1) and T. faecale (1)). The chemical constituents detected in the nodules were carbon, oxygen, sodium, calcium, manganese and magnesium. The softh consistency of the nodules is associated with the low concentrations of calcium and sulfur, that providing ease disruption of the nodule. In this in vitro infection model, it was observed that the temperatures and humidity were not related to the development of this tricopatia.

Thesis (MSc Food Sc )--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Kepi is a refreshing, fermented dairy beverage that differs from other fermented milk products in that it is produced with a mixed microbial community which is confined to discrete grains. These grains can be recovered as a solid matrix at the end of the fermentation and then be reutilised as a starter to ferment the next batch of milk. The grain microbial community consists of a symbiotic association of yeasts and lactic acid bacteria, but the overall composition of the grains has not been completely elucidated. The microbes in the grains are embedded in a protein-polysaccharide Kefiran matrix, which appears essential for grain formation. The mechanism of grain formation is still not fully understood and it thus remains undecided which organism is really responsible for the production of this proteinpolysaccharide matrix. The aim of this study was to isolate, characterise and identify the microbes present in Kefiran from mass cultured South African grains and then to evaluate grain formation with these purified cultures isolated from Kefiran strings using a mass cultivation process. Sixteen strains of lactic acid bacteria and one yeast strain were isolated from Kefiran strings produced during the mass cultivation of South African Kepi grains. API technology, numerical clustering and DNA sequence comparisons were used to identify the purified isolates. The isolates were grouped into seven clusters by numerical clustering and clustering distance from selected reference and marker strains. The heterofermentative lactobacilli were identified as Lactobacillus parakefiri and Lb. kefiri and the homofermentative strains as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus and Lb. bavaricus. One isolate was found to be a member of the genus Lactobacillus, but was not positively identified to species level. Cultures isolated from Kefiran were evaluated for ability to grain formation by adding 1 x 109 cfu.ml:' bacteria and 1 x 108 cfu.ml' yeast to double pasteurised, full cream milk during the mass cultivation process. It was found that the control and all the cultures in double pasteurised milk showed grain accumulation indicating that other microbes were present in pasteurised and double pasteurised milk which had an influence on the grain forming ability. The cultures isolated from pasteurised and double pasteurised milk included members of the species Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliermondii, Chryseobacterium meningosepticum, Pseudomonas putida and four isolates of the Bacillus cereus group. It was found that these rod-shaped "milk isolates" resulted in grain accumulation when inoculated into UHT milk and it was concluded that the "milk isolates" did contribute to grain formation. These isolates were then combined with the Kefiran cultures and this resulted in grains very similar to the traditional Kepi grains. These grains were made from Lb. gallinarum in double pasteurised milk as well with a combination of Lb. gallinarum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica and Pseudomonas putida in URT milk. The grains were firm, elastic and did not dissolve in water but kept their structure and were retained when sieved. An acceptable Kepi beverage was produced from these grains. From these typically traditional grain characteristics it was concluded that, even though the microbial compositions were probably not the same, the general appearance was similar to traditional grains and that it is thus possible to produce grains from pure single strain Kefiran cultures and "milk isolates". Furthermore, it was possible to produce a Kepilike beverage from these grains, which included similar characteristics as the traditional Kepi beverage. / AFRIKAANSE OPSOMMING: Kepi is "n verfrissende, gefermenteerde suiweldrankie wat van ander gefermenteerde produkte verskil in die opsig dat dit vervaardig word deur Kepi korrels in melk te inkubeer. Die Kepi korrels kan aan die einde van die fermentasie herwin word en weer gebruik word om die volgende lot melk te fermenteer. Die korrels bestaan uit "n simbiotiese samestelling van giste en melksuurbakterieë, maar die presiese samestelling van die korrels is steeds onbekend. Die mikro-organismes is vasgevang in "n proteïen-polisakkaried Kefiran matriks en die Kefiran word as essensieel beskou vir korrelvorming. Die meganisme van korrelvorming bly steeds onbekend en daar is nog nie tot "n gevolgtrekking gekom oor watter organisme die Kefiran produseerder is nie. Die doel van die studie was om die mikro-organismes in Kefiran te isoleer en te identifiseer deur Suid-Afrikaanse Kepi korrels te massa kweek. Hierdie mikroorganismes was dan verder geëvalueer ten opsigte van korrel vorming. Sestien melksuurbakterieë isolate en een gis isolaat is geïsoleer vanuit die Kefiran. API tegnologie, numeriese groepering en DNA volgorde vergelykings was gebruik om die isolate te identifiseer. Die isolate is in sewe groepe verdeel volgens numeriese groepering. Die afstand van verwysings en merker organismes is ook in ag geneem. Die heterofermentatiewe organismes is geïdentifiseer as Lactobacillus parakefiri en Lb. kefiri en die heterofermentatiewe organismes as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus en Lb. bavaricus. Een isolaat kon nie geïdentifiseer word tot op spesie vlak nie, maar is verwant aan die genus Lactobacillus. Hierdie geïsoleerde Kefiran kulture is geëvalueer ten op sigte van korrelvorming, deur 1 x 109 kve.ml' van die bakterieë en 1 x 108 kve.ml' van die gis by dubbel gepasteuriseerde volroom melk te voeg tydens die massakwekings proses. Die kontrole wat geen bygevoegde kulture bevat nie, sowel as die wat wel bygevoegde kulture bevat, het korrel vorming getoon. Laasgenoemde toon dat daar organismes teenwoordig is in gepasteuriseerde en dubbel gepasteuriseerde melk wat "n rol kan speel tydens korrelvorming. Die kulture wat geïsoleer is vanuit gepasteuriseerde en dubbel gepasteuriseerde melk, sluit in: Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliennondii, Chryseobacterium menigosepticum, Pseudomonas putida en vier isolate van die Bacillus cereus groep. Hierdie organismes wat uit melk geïsoleer is, het korrelvorming getoon in UHT melk en die gevolgtrekking kan gemaak word dat die "melk organismes" wel "n rol speel tydens korrel vorming. Hierdie "melk isolate" in kombinasie met die Kefiran kulture het korrels tot gevolg gehad wat baie dieselfde was as tradisionele Kepi korrels. Laasgenoemde korrels is gemaak deur Lb. gallina rum in dubbel gepasteuriseerde melk, sowel as deur "n kombinasie van Lb. gallina rum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica en Pseudomonas putida in UHT melk. Die korrels was stewig, elasties, het nie opgelos in water nie en het hulle struktuur behou wanneer gesif. Wanneer hierdie tipiese tradisionele korrels se eienskappe in ag geneem word, kan die gevolgtrekking gemaak word dat alhoewel die mikrobiese samestelling van die korrels nie dieselfde is as die tradisionele korrel nie, is die algemene voorkoms en eienskappe dieselfde en dat dit wel moontlik is om korrels te produseer deur isolate geïsoleer vanuit Kefiran en melk. Verder was dit moontlik om "n drankie te vervaardig met die korrels wat baie dieselfde is as tradisionele Kepi.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The chemical profile of a fermented product depends upon the raw basic material and the fermentative microbiota. The yeast microbiota is responsible for fermentation and contributes to fermented product aroma by several mechanisms. The study of this microbiota is relevant because the microorganisms utilize the constituents of the raw basic material and transform them into aroma or flavour impacting components. Autochthonous yeasts belonging to the blueberry microbiota of two different cultivars, Florida M and Climax, were investigated by analyzing the fermentative capacity, the hydrogen sulfide (H2S) production, the film-forming ability, killer feature, sensitivity and neutrality to killer factor. Three methods employed to distinguish autochthonous yeast species were used: MALDI-TOF MS, PCR and PCR-RFLP of ITS region of rRNA gene and, if necessary, genetic sequencing of the D1/D2 26S rRNA region. The species Hanseniaspora uvarum, Hanseniaspora opuntiae, Candida sorboxylosa, Metschnikovia kunwiensis, Candida bentonensis, Candida oleophila/Candida railenesis and Candida quercitrusa were found on the surface of Florida M. Hanseniaspora uvarum, Issatchenkia terricola, Candida sorboxylosa, Candida asiatica, Candida oleophila/railenensis, Issatchenkia hanoiensis and Kazachstania intestinalis were of berries of Climax variety. Only three species were found in both varieties: H. uvarum, C. sorboxylosa and C. oleophila/railenensis. The species H. uvarum was the predominant in both cultivars, representing 70.4% and 78% of yeasts isolated from the surface of the Florida M and Climax, respectively. The killer feature was not detected in any strain isolated from Florida M. From strains isolated from Climax, only one strain C. asiatica 133 MCMCF/14 was killer against the 26B. Moreover, the K. intestinalis 91 MCMCF/14 was the only sensitive when tested against both the patterns killer S. cerevisiae K1 (Lallemand), EMBRAPA 1B, EMBRAPA 91B and the killer yeast C. asiatica 133 MCMCF/14. All strains showed low fermentative capacity and all of them were non-Saccharomyces yeasts. All yeasts isolated from Florida M produced H2S. Only four strains from Climax did not produce H2S. The highest evolution of H2S was observed with the genus Issatchenkia and with the majority strains of C. sorboxylosa. The species C. oleophila/C. railenensis showed low production of this parameter. The fermentation process of fermented products from blueberries of both Florida and Climax can be severely affected by this kind of autochthonous yeasts. / O perfil químico de um produto fermentado depende da matéria prima de base e da microbiota que participam do processo fermentativo. No presente estudo, leveduras pertencentes à microbiota do mirtilo foram investigadas, isolando-se estes micro-organismos da superfície de duas diferentes cultivares de mirtilo, Florida M e Climax. Foram avaliadas a capacidade fermentativa, produção de sulfeto de hidrogênio (H2S), formação de filme, característica killer, sensibilidade e neutralidade a este fator. Três metodologias para identificação foram empregadas: espectrometria de massas MALDI-TOF, análise da região ITS do gene rRNA por PCR-RFLP e, quando necessário, sequenciamento genético da região D1/D2 do 26S do gene rRNA. A cultivar Florida M apresentou em sua superfície as espécies Hanseniaspora uvarum, Hanseniaspora opuntiae, Candida sorboxylosa, Metschnikovia kunwiensis, Candida bentonensis, Candida oleophila/Candida railenensis e Candida quercitrusa. Na cultivar Climax, foram encontradas as espécies Hanseniaspora uvarum, Issatchenkia terricola, Candida sorboxylosa, Candida asiatica, Candida oleophila/Candida railenesis, Issatchenkia hanoiensis e Kazachstania intestinalis. Apenas três espécies foram comuns as duas cultivares, sendo elas H. uvarum, C. sorboxylosa, Candida oleophila/Candida railenesis. H. uvarum foi a espécie predominante em ambas cultivares, representando 70,4% e 78% do total de leveduras isoladas da cv. Florida M e Climax, respectivamente. A característica killer não foi detectada em nenhuma linhagem isolada da cv. Florida M. Na cv. Climax, apenas a linhagem C. asiatica 133 MCMCF/14 se comportou como killer. A linhagem K. intestinalis 91 MCMCF/14 foi a única que demonstrou sensibilidade em relação a todas as linhagens killer padrão K1 (Lallemand), EMBRAPA 1B, EMBRAPA 91B. Esta mesma linhagem se mostrou sensível também à linhagem isolada da cultivar Climax C. asiatica 133 MCMCF/14. Todas as linhagens isoladas de ambas cultivares apresentaram baixa capacidade fermentativa e todas elas foram identificadas como sendo não-Saccharomyces. Houve de moderada a alta produção de H2S em 21 e 34% nas linhagens isoladas das cultivares Florida M e Climax, respectivamente. A produção máxima de H2S se destacou principalmente em linhagens das espécies I. terricola e C. sorboxylosa. A maioria das linhagens de leveduras da espécie C. oleophila/C. railenensis apresentaram baixa produção deste gás. O processo de produção de fermentados de mirtilo tanto da cultivar Florida M como da cultivar Climax pode ser severamente afetado por este tipo de leveduras autóctones.