1,720,967 research outputs found
Method development and validation for the efficient detection of super-attenuating (Over-Fermenting) yeast contaminants (Saccharomyces cerevisiae var. Diastaticus) in the brewery industry
Get PDFSaccharomyces cerevisiae var. diastaticus is the most dangerous spoilage yeast of fermented beverages in the brewery industry. In contrast to conventional brewing yeast, diastaticus yeast causes super-attenuation due to its ability to degrade residual dextrin and starch in beers, thereby causing degraded mouthfeel, over-carbonation, high alcohol levels, and package explosion. Usually, a diastaticus contamination can remain unnoticed several months after packaging. Diastaticus yeast is particularly difficult to detect with traditional microbiological analyses due to the common characteristics shared with brewing yeast. The objective of the present study was to optimize/develop the detection and growth control of diastaticus yeasts using three different assays: a) the modified Durham test, b) the dextrin agar test and, c) a novel test developed during this project, called the ‘attenuation test’. Strain DSM 70487 and Strain-Y were used as positive and negative controls, respectively. The attenuation test was the most reliable assay because all the investigated diastaticus strains were detected by monitoring the reduction in density (°P) associated with residual saccharides consumption in fully attenuated beer medium. Although the spoilage yeast strain TUM 1-B-8 demonstrated mild super-attenuating activity in the attenuation test, this strain did not show spoilage potential when assayed for growth on dextrin agar plates and did not demonstrate gas production potential in the Durham test. The positive control DSM 70487 showed spoilage potential in all assays, with the fastest detection time of 2 days recorded in the Durham test. Growth on dextrin agar at pH 5.2 and 6.2 revealed faster growth and more rapid detection at pH 5.2. The lowest detection limit (5x100 cells/ml) was noted in the agar tests. This research demonstrated clear variations in the super-attenuating strength of S. cerevisiae var. diastaticus yeast strains and highlighted the necessity to combine multiple assays for reliable detection of diastaticus activity in investigated samples.Saccharomyces cerevisiae var. diastaticus é uma estirpe da espécie utilizada na produção de cerveja, S. cerevisiae. S. cerevisiae var. diastaticus é a levedura de deterioração mais perigosa nas bebidas fermentadas obtidas na indústria cervejeira. As espécies do género Saccharomyces utilizadas no processamento de cerveja, como S. cerevisiae e S. Pastorianus degradam carboidratos numa determinada ordem: primeiro a glicose, posteriormente a frutose seguida da maltose e, finalmente, o trissacarídeo maltotriose, que dificilmente é consumido pela maioria das leveduras de cerveja. Em geral, estas leveduras são incapazes de utilizar oligossacarídeos (3-10 resíduos de monossacarídeos), e dextrinas e polissacarídeos (Ex.: amido). Os carboidratos não fermentados permanecem na cerveja e contribuem para o corpo e a sensação na boca do produto final. Contudo, S. cerevisiae var. diastaticus é uma levedura amilolítica que possui o gene STA1 o qual codifica a síntese da enzima glucoamilase extracelular. Esta enzima degrada as dextrinas e o amido presentes na cerveja acabada, levando à sua degradação, situação denominada superatenuação. A superatenuação caracteriza-se pela existência de fermentações secundárias de carboidratos residuais podendo causar a produção de dióxido de carbono, o aparecimento de sabores estranhos, o desenvolvimento de uma sensação desagradável na boca e a produção de níveis anormalmente altos de álcool. As contaminações por leveduras diastásicas são dificilmente detetadas, sendo os sinais de deterioração identificados vários meses após o embalamento, quando o produto já se encontra no mercado.
Na última década tem sido reportado um aumento no número de incidentes resultantes de contaminações por S. cerevisiae var. diastaticus os quais estão associados a perdas financeiras que atingem bilhões de euros/ano na Europa. Esta estirpe é difícil de detetar e identificar através de análises microbiológicas tradicionais devido às características comuns com a levedura de cerveja. Além disso, o gene STA1 pode estar presente em estirpes de levedura que não apresentam potencial de deterioração e superatenuação.
O objetivo do presente estudo foi otimizar/desenvolver e comparar métodos para detetar o crescimento e capacidade diastásica de várias estirpes de leveduras, utilizando três abordagens diferentes: a) o teste de Durham modificado, b) o teste em meio de cultura sólido de agar com dextrinas (com azul de bromofenol ou roxo de bromocresol), e c) um teste desenvolvido durante o presente projeto designado “teste de atenuação”. As estirpes DSM 70487 e Estirpe-Y foram utilizadas como controlos positivo e negativo, respetivamente. Foram também utilizadas misturas da estirpe diastásica TUM 1-B-8 com DSM 70487 (concentrações celulares variáveis de 1 x 108 a 5 x 100 células/ml) e com a estirpe de levedura lager (Estirpe-Y). Todos os ensaios foram realizados em duplicado. O teste de Durham e o “teste de atenuação” foram realizados no meio de cultura de “cerveja totalmente atenuado” com zinco (200 ppb) e sem zinco. A “cerveja totalmente atenuada” é um meio preparado pela reinoculação da cerveja embalada com uma levedura starter lager para garantir o consumo completo de qualquer açúcar fermentável residual. O teste de Durham permitiu avaliar a libertação de subprodutos gasosos (dióxido de carbono) em tubos Durham invertidos resultantes da fermentação de estirpes de levedura diastásicas que foram inoculadas em meio de “cerveja totalmente atenuado” e incubadas durante 30 dias à temperatura ambiente. O “teste de atenuação” consistiu em medições semanais do extrato aparente (EA) (°P) do meio de “cerveja totalmente atenuado” previamente inoculado e incubado à temperatura ambiente, durante 4 semanas. A presença das estirpes degradadoras provoca a diminuição do EA devido à hidrólise de sacarídeos residuais presentes no meio de cultura/cerveja. A capacidade de as leveduras utilizarem dextrina durante o seu crescimento foi estudada em meio de cultura sólido contendo dextrina, seguida de incubação aeróbia a 28 °C, durante 30 dias. Foi testado o meio de cultura sólido com os corantes azul de bromofenol (pH inicial 5,2) e roxo de bromocresol (pH inicial 6,2). A presença destes corantes indicadores de pH contribuiu para facilitar a deteção visual do crescimento das leveduras uma vez que uma alteração da cor do meio sólido resultava da sua acidificação devido ao crescimento das estirpes S. cerevisiae var. diastaticus previamente inoculadas.
A presença das estirpes investigadas foi confirmada no “teste de atenuação” após um período de duas semanas devido à redução dos valores do EA. Observaram-se reduções do EA entre a 2ª e a 3ª semanas de incubação no meio de ”cerveja totalmente atenuado”, suplementado com zinco, quando se utilizaram inóculos contendo 1x102 células/ml de DSM 70487. Apenas a estirpe utilizada como controlo positivo, DSM 70487 (1x108 células/ml), demonstrou potencial de deterioração no teste de Durham, tendo a primeira visualização do gás sido registada após 2 dias de incubação. Não houve diferenças nos resultados obtidos nos testes de Durham realizados com e sem zinco. Os ensaios realizados em meio sólido com dextrina (pH 5,2 e 6,2) revelaram que as estirpes superatenuantes cresceram mais rapidamente nos meios com valores de pH inicial de 5,2 pelo que a sua presença foi detetada mais rapidamente quando o pH inicial foi 5,2 em comparação com 6,2. Os testes realizados no meio de cultura sólido apresentaram um limite mínimo de deteção de 5x100 células/ml. A estirpe DSM 70487 apresentou um potencial de deterioração mais alto do que TUM 1-B-8 em todos os estudos realizados. A estirpe DSM 70487 revelou capacidade de superatenuação em todos os testes, enquanto a estirpe TUM 1-B-8 mostrou atividade superatenuante nos estudos de refermentação em meio de “cerveja totalmente atenuado”, mas não apresentou potencial de deterioração quando testada em meio sólido com dextrina e não foi detetada a produção de gás nos testes de Durham.
Com base nos resultados obtidos no presente estudo, o “teste de atenuação” permitiu obter melhores resultados para a deteção de estirpes de leveduras de degradação S. cerevisiae var. diastaticus quando comparado com os outros ensaios realizados. Este estudo permitiu também evidenciar a existência de variações na capacidade superatenuante das estirpes testadas e destacou a necessidade de combinar várias estratégias experimentais para a deteção destes contaminantes nas amostras a estudar durante o controlo de qualidade no ambiente de cervejaria
Het gebruik van modelsystemen om het werkingsmechanisme van amylasen te begrijpen - Een focus op anti-broodverouderende enzymen
No full textStarch is an important source of carbohydrates in the human diet. It consists mainly of two glucose polymers, the essentially linear amylose and the highly branched amylopectin. Starch polymers can be degraded by amylases from various sources. These enzymes are widely used in numerous applications, such as in syrup production, brewing, and detergent systems. One important application area is the bread making industry where they are used as bread crumb anti-firming agent. However, not every amylase is suitable as bread crumb anti-firming enzyme. Today, the action pattern of these enzymes and the bread crumb firming process are still not completely understood. Against this background, the present dissertation aimed to unravel the action pattern of amylases with different efficiency in bread crumb anti-firming applications.Amylopectin crystallisation seems to play a role in bread crumb firming. Amylases acting on the outer chains of amylopectin retard amylopectin crystallisation. Depending on their action pattern, they have a different efficiency. The extent of multiple attack action is an important parameter when studying the action pattern and is expressed as the degree of multiple attack (DMA). It is defined as the average number of linkages broken during the lifetime of an enzyme-substrate complex minus one (i.e. the initial random cleavage). Three amylases were characterised and their action pattern on starch polymers studied. The first one was maltogenic alfa-amylase from Bacillus stearothermophilus (BStA) with good bread crumb anti-firming properties. BStA releases maltose from starch polymers by means of exo-action. A modified maltotetraose forming alfa-amylase from Pseudomonas saccharophila (PSA) with increased thermostability was also studied. It forms maltotetraose by means of exo-action. However, not much is known about this enzyme. Next to these two enzymes, a beta-amylase from Clostridium thermosulfurogenes (CTB) with improved thermostability was included because of its purely exo-action pattern.In the first part of the work, the temperatures for optimal activity and stability and action patterns were determined. Optimal temperatures for activity were about 60, 60 and 60-70 °C for PSA, BStA and CTB, respectively. Temperature stability decreased in the following order: BStA > PSA >> CTB. PSA action caused a rapid decrease in amylose molecular weight (MW). However, in the beginning of hydrolysis, the decrease in amylose MW by BStA or CTB was very slow. This suggested that PSA shows more endo-action on amylose and/or has a lower DMA than BStA and CTB, respectively. This relative endo-activity remained constant for PSA when temperature increased, whereas it increased with temperature for BStA. In line with the action pattern noted for amylose, the decrease in amylopectin MW was more pronounced for PSA than for BStA and CTB, respectively. Under the experimental conditions, the MW of amylopectin decreased to a larger extent by action of BStA than by action of CTB, confirming that BStA performs endo-activity and degrades amylopectin to a larger extent than CTB. These observations were confirmed when studying the hydrolysis of maize starch. This showed that PSA decreases the MW of maize starch more rapidly than BStA or CTB. Additionally, the impact on viscosity development was studied. In general, addition of alfa-amylases to maize starch suspensions prior to running a heating cycle reduced the viscosity of the slurry. These effects were more pronounced for PSA than for BStA due to the higher endo-activity of PSA. Due to the exo-action and the slightly lower temperature stability of CTB, the impact on viscosity was limited.In a second part, the action patterns of PSA and BStA on the inner chains of amylopectin were compared with those of alfa-amylase from either porcine pancreas (PPA) or Bacillus subtilis (BSuA). These amylases were incubated with beta-limit dextrins (beta-LDs) to further investigate their endo-action. beta-LDs are amylopectin molecules from which the outer chains have been trimmed down to two or three glucose units. As a result, only endo-action is possible on this substrate, which can thus be used to study the action pattern on the inner chains of the amylopectin molecule. At least three different action patterns could be differentiated, suggesting a different specificity for the internal chains of amylopectin and/or different DMA. A first group of enzymes, such as BSuA, combined inner chain attack with a low DMA. The longer B-chains of beta-LDs were degraded first with low DMA, resulting in a rapid decrease in MW. A second type of action pattern (such as displayed by PSA and PPA) was characterised by inner chain attack and intermediate or high DMA. In this case, beta-LDs were swiftly degraded by cleavage of the long B-chains, but at a lower relative rate than displayed by the first group of enzymes. The third action pattern was characterised by a limited inner chain attack and a very high DMA. Presumably, BStA performed a limited number of endo attacks with a very high DMA. Finally, the addition of PSA and BStA to model bread systems was studied using an Electrical Resistance Oven (ERO). ERO baking allowed minimising the temperature differences in different parts of the dough/bread during baking, which is an advantage when studying the temperature dependent action of enzymes. The temperature-time evolution observed in the centre of a conventionally baked bread was simulated in ERO bread baking. Before studying the impact of amylases during bread making in ERO, the starch population and water migration in ERO breads (during storage) were compared with those of conventional breads. No differences were observed between the gelatinisation and retrogradation properties of the starch fraction of ERO and conventional breads. Differences in water migration during storage of ERO and conventional breads were studied with proton nuclear magnetic resonance (1H NMR). The proton populations in both fresh and stored EROand conventional breads differed due to the generally lower moisture contents in ERO than in conventional breads. In freshly baked ERO breads, less proton populations could be differentiated and they had lower mobility than those of conventionally baked breads.In a last part, the effect of amylases during bread making was studied from mixing to baking. The baking phase was studied by means of an ERO. Initially, very high amylase activity was noticed, caused by endogenously present amylases, which resulted in the release of a significant amount of maltose in the first stages of the baking process. Possibly, the impact of the presence of beta-amylases in wheat flour and dough is larger than previously assumed. The maltose concentration in bread was higher in amylase supplemented than in control breads. In contrast to what was expected, next to maltose, also maltotriose and maltotetraose were retrieved in the supernatant of the BStA supplemented bread crumb extracts. Additionally, there were only small changes in the chain length distribution of the residual starch fraction of PSA and BStA supplemented breads than in control breads. The most important changes were a decrease in levels of chains with a degree of polymerisation (DP) of 11-13, and concomitant an increase in levels of DP 6-9 chains. These changes were more pronounced for the starch fraction in BStA supplemented breads than in its PSA supplemented counterparts. Amylase activity was inactivated completely in control breads after 7 min baking (when a temperature of approximately 99 °C was reached), while there was still amylase activity in the PSA and BStA supplemented breads after 7 min baking, which corresponded to temperatures of approximately 95 and 99 °C, respectively.In conclusion, this thesis increased our understanding of the action patterns of amylases, and in particular those with interesting properties as bread crumb anti-firming agents. Moreover, it demonstrated how the amylases impact starch during bread making. Even if the enzymes only have a limited effect on starch chain length distributions, they have pronounced effects on starch retrogradation.status: Publishe
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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