Useful cryophilic killer strains of Saccharomyces cerevisiae were bred by back-crossing KL-88, which is a wild killer sake yeast, with a repetitive parent WL-7and is one of the best cryophilic wine yeasts. The hybrids maybe aid in producing good wines in pure cultures at relatively low temperature (13~15°C).A haploid of KL-88 was crossed with haploids of WL-7to yield first-generation hybrids. Haploids possessing both killing activity and SO2 tolerance were selected from the hybrids and back-crossed with a haploid of WL-7 to yield second-generation hybrids. Several strains of killer hybrids were comparable to WL-7 in fermentation ability at 15°C, SO2 tolerance, TTCstain, and growth on jS-alanine mediumat 35°C. One strain (2HYL-2) aided in producing wine as good as with WL-7, in fermentation at 15°C on pilot-plant scale.The hybrid killed only Saccharomyces yeasts in grape must. In the last-stage of fermenting Koshu grape must with the killer hybrid (2HYL-2), no sensitive yeasts were detected, because they were killed by the killer yeast. Moreover, most of the neutral yeasts, such as Kloeckera and Torulopsis, that were abundant in grape juice before the addition of the starter yeast, disappeared in the last-stage.On the other hand, many sensitive pseudo-film-forming yeasts of the genus Saccharomyces were detected in the last-stage of fermenting must with WL-7strain. These results show the possibility of fermenting musts with only a single strain"of cryophilic killer yeasts at a relatively low temperature without contaminating other yeast strains.Because grape musts are usually not pas-tolerance by back-crossing KL-88 strain, a teurized before fermentation in wine making, wild killer sake yeast, with OC-2 strain, one of many wild yeasts from natural grapes are the excellent wine yeasts, for repetitive parent brought to fermenting musts. Ohara and age. One ofthehybrids, 2HY-1strain, successNonomura1} reported that many yeast strains fully killed spontaneous wild yeasts of other than the yeast strain inoculated were Saccharomyces in grape musts containing SO2; participating in the fermentation ofmusts in a consequently, no film formation occurred in Japanese winery. Undesirable yeasts in the the resultant wine. must can harm the wine (off odor, excessive However, the hybrid was slow in fermenvolatile acid, incomplete fermentation, tur-tation at low temperature, around 15°C. It is bidity, etc.).2>3) Furthermore, bottled wine can highly desirable that fermentation proceeds begin fermentation again or film can form on smoothly at relatively low temperature to prothe surface of table wine during storage if duce wine of good quality, especially in white processing has been faulty, e.g., lack of SO2 or wine. Because Koshu grapes, a typical variety improper topping. Such phenomenawere for white wine in Japan, are usually harvested mainly caused by the growth of pseudo-film-late in cooler weather, the fermentation is apt forming yeasts, belonging to Saccharomyces to be delayed. Accordingly, cryophilic killer oviformis, S. ...
Useful cryophilic killer strains of Saccharomyces cerevisiae were bred by back-crossing KL-88, which is a wild killer sake yeast, with a repetitive parent WL-7 and is one of the best cryophilic wine yeasts. The hybrids maybe aid in producing good wines in pure cultures at relatively low temperature (13 ~ 15°C).A haploid of KL-88 was crossed with haploids of WL-7 to yield first-generation hybrids. Haploids posses'sing both killing activity and S02 tolerance were selected from the hybrids and back-crossed with a haploid of WL-7 to yield second-generation hybrids. Several strains of killer hybrids were comparable to WL-7 in fermentation abilIty at 15°C, S02 tolerance, TTC stain, and growth on p-alanine medium at 35°C. One strain (2HYL-2) aided in producing wine as good as with WL-7, in fermentation at 15°C on pilot-plant scale.The hybrid killed only Saccharomyces yeasts in grape must. In the last-stage of fermenting' Koshii grape must with the killer hybrid (2HYL-2), no sensitive yeasts were detected, because they were killed by the killer yeast. Moreover, most of the neutral yeasts, such as Kloeckera and Torulopsis, that were abundant in grape juice before the addition of the starter yeast; disappeared in the last-stage. On the other hand, many sensitive pseudo-film-forming yeasts of the genus Saccharomyces were detected in the last-stage of fermenting must with WL-7 strain. These results show the possibility of fermenting musts with only a single strain' of cryophilic killer yeasts at a relatively low temperature without contaminating other yeast strains.
RESUMO -A motivação para as pesquisas na área do etanol de primeira e segunda geração se deve, em grande medida, a uma responsabilidade compartilhada e diferenciada do meio ambiente. Nesse panorama científico, este trabalho avaliou o uso de sete extratos enzimáticos (E1 a E7) obtidos a partir de três meios (M1, M2 e M3) da fermentação em estado sólido na produção de etanol de bagaço de cana explodido. Cotejou-se a produção de etanol pelos extratos enzimáticos E1 a E7 para tempos de 24 h e 48 h de fermentação de 250 g/L de bagaço de cana explodido, sendo observado que a utilização de diferentes combinações de meios de FES na geração de extratos enzimáticos representa uma estratégia salutar para auferir menores tempos e maiores concentrações de etanol de segunda geração. Os melhores resultados obtidos nesses ensaios foram 21.5 g/L de etanol após 24 h de fermentação. INTRODUÇÃOSegundo Trigueiro et al. (2008) construímos um modelo de mundo civilizatório depredador e consumista que, caso seja universalizado, seria necessário três planetas semelhantes ao nosso. Neste milênio, o etanol consagra-se como alternativa para minorar, sobretudo, a problemática ambiental e energética mundial, adensadas pela nociva poluição, escassez e alta dos preços de combustíveis fósseis.O alto custo das enzimas celulases (um dos grandes entraves para a consolidação do etanol de segunda geração) torna a fermentação em estado sólido (FES) uma alternativa promissora para a produção enzimática, sendo o bagaço da cana-de-açúcar o principal substrato microbiano utilizado nessa técnica. Na FES o micro-organismo cresce sobre substratos sólidos na ausência livre de água para a produção de extratos enzimáticos (Lopes, 2013).No cenário atual, a precípua e a potencial aplicação do complexo enzimático obtido da FES é na hidrólise da biomassa lignocelulósica. Desta forma, a premente e fundamental pesquisa científica/tecnológica nos âmbitos acadêmico e industrial devem ser os pilares para a viabilidade econômica e técnica da produção do etanol de segunda geração (Zúñiga, 2010).Nessa incessante busca por novas rotas alternativas, o presente trabalho avaliou extratos enzimáticos de três diferentes meios de FES e de suas combinações selecionadas na produção de etanol.
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