Method development and validation for the efficient detection of super-attenuating (Over-Fermenting) yeast contaminants (Saccharomyces cerevisiae var. Diastaticus) in the brewery industry

Saccharomyces 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

Adsorption of phenol/tyrosol from aqueous solutions on macro-reticular aromatic and macro-porous polystyrene cross-linked with divinylbenzene polymeric resins

The current work aims at separating by adsorption of low-molecular-weight organic compounds in a nanofiltration concentrate of the olive mill wastewaters. The experimental investigations on adsorption of phenol/tyrosol in single and binary systems were conducted in batch mode by using the commercially available macroporous resins FPX66 and MN202. The structures of such resins were examined by FTIR before and after adsorption. The operating parameters affecting the adsorption process such as resin dosage, contact time, pH, and initial concentration of phenol/tyrosol were investigated. Fast phenol and tyrosol uptakes were observed for both resins. It can be attributed to their physical properties, for instance high specific area and microporous area. The adsorption selectivity of phenol is larger than tyrosol when using FPX66 resin, but smaller if MN202 resin is used. Acidic pH appeared to be always favourable for the adsorption. A synergetic effect between solutes was observed since adsorption of phenol and tyrosol in the binary systems was faster than the individual sorption of each solute. Five isotherms namely Langmuir, Freundlich, DubininRadushkevich, Temkin and Redlich-Peterson were selected to fit the obtained equilibrium experimental data. Finally, desorption of the examined compounds with ethanol (EtOH) allowed a maximum around 85 % of phenol, and equal to 94 % of tyrosol on FPX66 and MN202 resins

Adsorption of phenol/tyrosol from aqueous solutions on macro-reticular aromatic and macro-porous polystyrene cross-linked with divinylbenzene polymeric resins

The current work aims at separating by adsorption of low-molecular-weight organic compounds in a nanofiltration concentrate of the olive mill wastewaters. The experimental investigations on adsorption of phenol/tyrosol in single and binary systems were conducted in batch mode by using the commercially available macroporous resins FPX66 and MN202. The structures of such resins were examined by FTIR before and after adsorption. The operating parameters affecting the adsorption process such as resin dosage, contact time, pH, and initial concentration of phenol/tyrosol were investigated. Fast phenol and tyrosol uptakes were observed for both resins. It can be attributed to their physical properties, for instance high specific area and microporous area. The adsorption selectivity of phenol is larger than tyrosol when using FPX66 resin, but smaller if MN202 resin is used. Acidic pH appeared to be always favourable for the adsorption. A synergetic effect between solutes was observed since adsorption of phenol and tyrosol in the binary systems was faster than the individual sorption of each solute. Five isotherms namely Langmuir, Freundlich, DubininRadushkevich, Temkin and Redlich-Peterson were selected to fit the obtained equilibrium experimental data. Finally, desorption of the examined compounds with ethanol (EtOH) allowed a maximum around 85 % of phenol, and equal to 94 % of tyrosol on FPX66 and MN202 resins

Non-discrimination et droits des peuples minoritaires

Placés dans une situation d’infériorité numérique au sein des États, les peuples minoritaires ont fréquemment subi des persécutions ou des tentatives d’élimination orchestrées par une majorité de la population dont l’avantage du nombre lui a souvent permis de s’arroger l’essentiel du pouvoir politique, économique et social. Depuis quelques décennies, le principe de non-discrimination consacré notamment par le droit supranational a permis aux membres des peuples minoritaires de bénéficier, au sein des États qui les abritent, des droits analogues à ceux de leurs concitoyens. Par ailleurs, il n’est pas incongru que les peuples minoritaires puissent se voir octroyer des prérogatives particulières leur permettant de défendre leur identité culturelle ou linguistique. Ces prérogatives peuvent être reconnues grâce à une politique de discrimination positive. Le droit international encourage en effet, dans certaines circonstances, les États à mettre en œuvre un traitement différencié au profit des minorités dont le retard économique et/ou social est souvent lié aux mauvais traitements dont ils ont été l’objet dans le passé. Toutefois, les peuples minoritaires ont un devoir de loyauté vis-à-vis de leur pays d’établissement et doivent, par exemple, éviter tout comportement qui serait de nature à en compromettre l’intégrité territoriale. Les États sont en effet hantés par les velléités sécessionnistes d’un peuple minoritaire qui peut se révéler majoritaire sur une portion du territoire national ; d’autant plus que, sauf cas exceptionnel, la sécession n’est qu’une simple situation de fait que le droit international ne condamne pas expressément.As a numeric minority in States, minority peoples have frequently suffered persecution or attempts to remove them orchestrated by a majority of the population, whose advantage in number often allowed them to assume the bulk of political, economic and social power. In recent decades, the principle of non-discrimination, which is enshrined in supranational law, has enabled minority peoples to enjoy the same rights as their fellow citizens in the States in which they live. Furthermore, it is not incongruous for minority peoples to be granted specific rights that allow them to defend their cultural or linguistic identity. These rights may be recognised through a policy of positive discrimination. Indeed, in some circumstances, international law encourages States to implement differential treatment in favour of minorities, whose economic and/or social disadvantage is often linked to the poor treatment that they received in the past. However, minority peoples do have a duty of loyalty to the country in which they have settled and must, for example, avoid any behaviour that could undermine its territorial integrity. States are indeed haunted by the secessionist desires of a minority people which could become a majority in a portion of the national territory, particularly since, in all but exceptional cases, secession is nothing more than a simple de facto situation, which international law does not condemn specifically

COMPARISON OF ELECTROCOAGULATION AND CHEMICAL COAGULATION IN THE TREATMENT OF ARTISANAL TANNERY EFFLUENTS

In this study, the treatment of the effluents of an artisanal tannery by electrocoagulation with iron electrodes was carried out. During electrolytic treatment, a current intensity of 0.4 A was used, and the electrolysis time was varied from 0 to 40 minutes. The performance of electrocoagulation was compared with that of chemical coagulation with ferric sulfate. The results obtained showed that highest removals after electrocoagulation were 71.15, 98.26, and 86.59% for the COD, colour and turbidity respectively. Chemical coagulation reduced the COD up to 83.17%, while colour and turbidity removals were 99.81 and 98.62% respectively. Although chemical coagulation leads to highest percentage removals after treatment, electrocoagulation results in near neutral pH values and also decrease conductivity weakly. By contrast, chemical coagulation increases the acidity of the effluent and its conductivity. Hence with close to neutral pH and weak conductivity values obtained for effluents treated by electrocoagulation, it is inferred that such treated effluents can be recycled. http://dx.doi.org/10.4314/njt.v35i1.2

Impact De L’exploitation Artisanale De La Sabliere De Toutsang Sur La Structure Des Communautes De Macroinvertebres Du Cours D’eau Doulahang A Dschang (Ouest Cameroun)

The impact of wastewaters from the Toutsang sandpit (Dschang), both on water parameters and on benthic macro-invertebrate community structure of the Doulahang stream was studied from March 2015 to February 2016 on a monthly basis in three sampling stations named TS1, TS2 and TS3. The physico-chemical analysis were carried out according to standard protocols while the benthic macro-fauna was harvested using a haze net of 30 cm side and 400 μm mesh size. The analysis of physico-chemical parameters revealed that the introduction of wastewater from the sandpit at the TS2 station led to an increase of the temperature, conductivity, total dissolved solids, salinity, suspended matter, turbidity and colour. The wildlife inventory identified 63 taxa divided into 4 phyla, 6 classes, 13 orders and 31 families. The lowest values of the taxonomic richness (18 taxa, against 46 taxa and 47 taxa respectively to stations TS1 and TS3), the Shannon diversity index (2.48 bits / ind.) and the Pielou's evenness (0.62) were observed at station TS2 which received wastewaters from the sandpit. Similarly, the low values of the richness and abundance of the pollutionsensitive groups (Ephemeroptera-Trichoptera-Odonata) were recorded in this station. Significant negative correlations were found between some metrics (total taxonomic richness, taxonomic richness of the Diptera, Richness of Ephemeroptera-Trichoptera-Odonata, Taxonomic richness of insects, Relative abundance of Ephemeroptera-Trichoptera-Odonata, Shannon Weaver diversity index, Pielou's evenness) descriptors of the structure of benthic macro-fauna communities and physico-chemical parameters indicative of physical pollution such as turbidity, suspended matter and colour

Ecotoxicological Effects of Imidacloprid and Lambda-Cyhalothrin (Insecticide) on Tadpoles of the African Common Toad, Amietophrynus Regularis (Reuss, 1833) (Amphibia: Bufonidae)

Agriculture is the main activity carried out in Cameroon and pesticide use is believed to be a common rule for its success. Most of the farms are situated close to water bodies, thus constituting a potential risk to non-target aquatic organisms. Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. The aim of this study was to assess the effects of an insecticide used by local farmers in Buea on amphibians. Parastar 40WP® which is constituted of imidacloprid + lambda-cyhalothrin a commonly used insecticide was applied on tadpoles of the African common toad Amietophrynus regularis (Reuss, 1833) in a static renewal experiment. The acute toxicity test was carried out after a range finding test from which seven test concentrations were selected. These concentrations were 0.0025mg/l, 0.005mg/l, 0.01mg/l, 0.05mg/l, 0.1mg/l, 0.5mg/l and 0.2mg/l. Signs of toxicity such as hyperactive symptoms, loss of balance, motionlessness and death were recorded. A varying degree of mortality (dose-dependent) was noticed during the test. On the contrary, no such toxicity signs and mortality occurred in the control, indicating that they were caused by the test substance. The 24h LC50 was 3.66mg/l, which is less than the recommended application dose (125mg/l). Products constituted of imidacloprid and lambda-cyhalothrin should therefore be handled with care and far from water bodies because of their potential to cause harm to non-target aquatic biota