Characterisation and improvement of whiskey yeast

La Grange-Nel, Karin (2003-03)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize, together with malted barley. In both processes, the enzymes from the barley are responsible for starch conversion and should always be derived entirely from the malted barley. No exogenous enzymes are allowed to be added to any mashing. The enzymes involved in the conversion process to fermentable sugars, are the aand p-amylases, limit dextrinase and p-glucosidase. Maize, on the other hand, contains no enzyme activity, therefore enzymes need to be added when producing whiskey from maize alone. In other whiskey-producing countries where maize is freely available and cheaper than barley, the use of exogenous enzymes are allowed in the mashing process and is crucial for the formation of fermentable sugars from complex carbohydrates. The cost of the enzymes, however, can push the production cost of whiskey to higher levels. Saccharomyces cerevisiae does not have any amylolytic activity, but is an excellent fermenter and produces favourable organoleptic notes, which makes it very suitable for producing potable spirit. Efforts have been made to genetically improve industrial strains, relying on classical genetic techniques followed by the selection of broad traits, such as ethanol tolerance, absence of off-flavours and carbohydrate/starch utilisation. No strain has thus far been selected for total starch degradation during the fermentation of whiskey mash. Over the last decade, considerable progress has been made in the development of genetically improved strains for the distilling, wine, brewing and baking industries. The expression of heterologous genes introduced a new dimension in approaches to the genetic improvement of industrial strains. It would therefore be cost-effective to use a yeast strain that can produce active and sufficient enzymes to ferment raw starch efficiently to alcohol without lowering the quality of the end product. No such strain has been developed to date, but the continuous improvement of starch-utilising strains has made this goal more achievable. Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria that are important for whiskey-specific strains. The selected strains were transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of its rapid fermentation rate. The genes were integrated into the genomes of the yeast strains and were stable after many generations. Assays showed that a significant increase in enzyme activity was induced in the whiskey strains, compared to the untransformed strains. The strains also showed good fermentation ability in whiskey fermentations, although optimum alcohol production was still not achieved.

AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s. gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~- arnitases, limiet dekstrinase en ~-glukosidase. Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme kan egter die produksiekoste van whiskey verhoog. Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol. Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie. Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie, maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel meer bereikbaar. Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53327
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