Thesis (MSc)--Stellenbosch University, 2003. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53327 |
| Date | 03 1900 |
| Creators | La Grange-Nel, Karin |
| Contributors | Van Rensburg, P., Lambrechts, M. G., Pretorius, I. S., Willemse, Q., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 71 pages : illustrations |
| Rights | Stellenbosch University |
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