Jagos Perovic


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Yeast Management


“Give man a beer, waste an hour. Teach a man to
brew, and waste a lifetime.” -Bill Owen. Beer is seen as an everyday beverage
all around the world, but people do not know how much goes into a good beer
bottle. This excellent quote by Bill Owen shows that beer making is an art,
perfected by years of experience. The basic idea of making beer is to extract
the sugars out of grains so that the yeast can convert it into carbon dioxide
and alcohol. Yeast does a lot of work and is very important, as it causes fermentation.
That is why proper yeast management is the key to developing good beer flavor.

It is important to understand that, “Yeast is
alive. It is not a spice. It is not an additive. It is a live cell and that is
why you should look at yeast as a living being with all its good and bad
points, needs, wishes,” says Vesko Perovic, the supply chain manager at
Heineken. As yeast is alive it is genetically complex.

            Many types of
beer exist, but the majority can be put into one of the two categories ale and
lager, reflected on the type of yeast used. The ale type is made fermentation
happens on on the temperature from twenty to twenty-five degrees Celsius by
using “top-fermenting” and has a low aging cycle. The lager, on the other hand,
happens on the temperature from eight to fifteen degrees Celsius by using
“bottom-fermenting” and has a long aging cycle.

            From the very
beginning of brewing history, it was known that lager yeast was different from
other types. The first noticeable thing was that brewing yeast strains did not
produce any meiotic offspring. Today, we have learned that a hybrid was formed
from S. cerevisiae and is now closely
related Saccharomyces species. Of all
the genes researched, two of them appeared almost exclusively of which one
invariably showed a restriction and hybridization pattern identical to that
found in the corresponding S. cerevisiae gene, while the other showed diversity.
The finding of these two genes suggests that lager yeast contains two types of
chromosomes Sc- and Non-Sc-. The chromosomes derived from lager brewing yeast
fell in one of the three categories homologous chromosomes, which recombined
normally with S. cerevisiae chromosomes, homoeologous chromosomes, which rarely
recombined with S. cerevisiae chromosomes, and mosaic chromosomes that were
composed of homologous and homoeologous segments.

The hybrid nature of lager brewing yeast has
also been confirmed by hybridization of radioactive probes to chromosome-sized
DNA separated by pulsed-field electrophoresis (Casey 1986b; Tamai et al. 1998;
Yamagishi and Ogata 1999). Experiments indicated at an early point that the
lager brewing yeast Sc-type of any given gene is identical to the corresponding
S. cerevisiae gene (Holmberg 1982; Nilsson-Tillgren et al. 1986; Petersen et
al. 1987). The complicated genetic nature of lager brewing yeast makes it
difficult to the breeding process. The deficiency in production of offspring would
seem to destroy classical breeding efforts. However, in the early 1980s, a method
to select for the few viable spores formed by lager brewing yeast and to
reconstruct functional brewing yeast from such offspring was devised (Gjermansen
and Sigsgaard 1981). Such spores could be used to form a heterogeneous
population for potential brewing strains, but these strains (Johannesen and
Hansen 2002; Hansen et al. 2002; Hansen and Kielland-Brandt 1996, 1996b;
Nilsson-Tillgren et al. 1986; Petersen et al. 1987) could be used for the
selection of recessive mutants (Gjermansen 1983). Out of many, none of the
techniques of analyzing genomes has been found, however times are changing.
With a rapid increase in DNA sequencing technology, such projects are now
achievable. The whole genome sequence of one strain of lager brewing yeast has
been obtained. A combination of different kinds of sequencing were used to
perform a total of 348,001 sequence reads of the genome of lager brewing yeast.
This sequence also consists of 160 million base pair of DNA. The sequences were
assembled into contigs. It was found that lager brewing yeast’s genome was 23.2
million base pairs, which is twice the size of S. cerevisiae genome
(Saccharomyces Genome Database; SGD). Contigs are classified into two groups
those with DNA and those with identities around 85%. It is now evident that two
yeast species came together to make a lager brewing yeast hybrid. One of these
ORFs (Open Reading Frame) has reported a gene consisting of a specific fructose
(Gonçalves et al. 2000). Fructose transport is one of the markers tha
distinguish S. pastorianus and S. bayanus from other Sacchromyces sensu stricto
species (Rodrigues de Sousa et al.

1995). S. bayanus is generally isolated
from oenological environments, rich in fructose. Although this sugar does not
play a major role in brewing, the gene for has stayed around in the lager
brewing yeast. In contrast to the protein-encoding regions, the non-Sc-type
intergenic regionsin the lager brewing yeast are diverged from the Sc-type. In
fact, such differential expression of homoeologues in lager brewing yeast has
been reported for the BAP2 gene (encoding a branchedchain amino acid permease)
homoeologues (Kodama et al. 2001), and for MET2 (encoding homoserine O-acetyl transferase)
and MET14 (encoding adenosylphosphosulphate kinase) (Johannesen and Hansen
2002). “These findings tell us that the lager brewing yeast is not a polyploid
with two divergent but similarly functioning genome parts, but is in fact a unique
organism with a biological complexity larger than any of the species that took
part in its formation.” (Panoutsopoulou et al. 2001; Olesen et al. 2002; James
et al. 2003). In addition an expression analysis during beer fermentation using
signature sequencing has been performed. More than 1400 genomes have been
tested and almost half of them showed a different gene expression. In order to
study this gene difference, DNA arrays containing lager brewing yeast have been
tested (Nakao Y, Kodama Y, Fujimura T, Nakamura N, and Ashikari T). The size of
DNA molecule was a little smaller than that of S. cerevisiae, but the same as S.
pastorianus .Also the gene order of lager DNA is different from S. cerevisiae but
the same as S. bayanus. Moreover, with the introduction of RNA analysis, genes
of lager brewing yeast are different from those of S. cerevisiae (Foury et al.
1998). These results tell us that lager brewing yeast got its mitochondrial
genome from a non-S. cerevisiae ancestor, thus containing two different nuclear

mentioned before the mosaic structure of lager brewing yeast chromosomes has
been discovered a long time ago. The number of recombination events, however,
has not been discovered, not to mention combinational points. Such information
is required to establish the optimal strategies for targeted molecular
breeding. These answers have been answered through analysis of the sequence
contigs obtained by the lager brewing yeast whole genome sequence analysis, as
described before, and partly by hybridisation experiments with S. cerevisiae

gene arrays (Kodama Y, Nakao Y, Nakamura
N, Fujimura T, Shirahige K and Ashikari T). The results confirm that there are
three types of chromosomes in lager brewing yeast: Sc-type, non-Sc-type, and
various chemical types.

precise structures of the chimera-type chromosomes were determined by the links

of forward-reverse shotgun read pairs.
The recombination break points between Sc-type and non-Sc-type chromosomes were
also confirmed by PCR using Sc-type and non-Sc-type sequences as primers and
subsequent sequencing of PCR fragments. “Polymerase chain reaction (PCR)

is a testing method
that can be used to assess the health and purity of yeast cultures”(” Polymerase
Chain Reaction”. These analyses showed that the lager brewing yeast contains at
least eight chimerical chromosomes. The chromosomes VIII, X,
and XI, have a complicated situation, as they appear even more complicated.
They come in three types: pure Sc-type, pure non-Sc-type, and chimerical ones.
The report of competitive genome hybridization using S. cerevisiae DNA with
Cy3-, Cy5-labelled DNA (Bond et al. 2004). Some of the chromosomal breakpoints
were found to be inside ORFs, which means that some hybrid ORFs exist. Most of
these were classified as “non-Sc” ORFs according to the relatively low nucleotide
identities to S. cerevisiae ORFs. It is to be anticipated that the further
investigation of such hybrid ORFs will be highly rewarding in terms of new knowledge
on protein function as well as hybrid speciation.