Program Book - Master Brewers Association of the Americas

Technical Session XVI: Yeast
Moderator: David Ryder, Miller Brewing Company, Milwaukee, WI
David Ryder is vice president of brewing, research, and quality
assurance for the Miller Brewing Company. David began his brewing
career in England at Associated British Maltsters. He then joined the
South African Breweries beer division and was later named director
of research and development for brewing and malting concerns
at Delta Corporation, Ltd. He was subsequently an international
technical consultant with Artois Breweries S.A. in Belgium. Prior to
joining Miller Brewing Company, he was vice president, technical
services at J.E. Siebel Sons’ Co. Inc. in Chicago and director of
education of the Siebel Institute. He joined Miller Brewing Company
in 1992. David is the current president of the Institute of Brewing
& Distilling and past president of the American Society of Brewing
Chemists. He is also a member of the Master Brewers Association
of the Americas and the Brewing Science Group of the European
Brewery Convention, where he was past chair of the Sub-group for
Studying Emerging Fermentation Systems. David has published
widely in the brewing literature.
O-56
Isolation of lager yeast mutants with low proteinase A for foam
stability of Chinese draft beer
DELIANG WANG (1), Huiping Li (2), Wujiu Zhang (1)
(1) China National Research Institute of Food and Fermentation
Industries, Beijing, China; (2) Guangzhou Zhujiang Brewery Group
Co., Guangzhou, China
The foam stability of draft beer is a critical character that reflects
beer quality. Although there are many factors that positively and
negatively influence foam stability, the most important negative
factor is proteinase A. Brewing yeast excretes proteinase A into the
fermenting wort during fermentation. Proteinase A diminishes the
hydrophobicity of foam-positive polypeptides and reduces beer foam
stability. The study mainly focuses on breeding brewing yeast with a
low ability to excrete proteinase A during wort fermentation by way
of a mutagenic agent, including both nitrosoguanidine (NTG) and
ethyl methanesulfonate (EMS). Compared with that of the parent
yeast, the results showed a more than 30% decline in protenase A
with the mutant yeast at the end of 100 L of pilot fermentation. It
opens up the possibility of producing draft beer with foam stability
by utilizing mutant strains in future.
Deliang Wang received a Ph.D. degree in food science from China
Agriculture University in Beijing. He began employment with
the China National Research Institute of Food and Fermentation
Industries (CNRIFFI) in July 1999 as a technical assistant. Since
February 2003, he has functioned as the technical manager in charge
of technical consulting for most Chinese breweries. More than onethird
of total Chinese beer production depends on technical support
from CNRIFFI at the present time. He is a member of ASBC.
O-57
Elimination of diacetyl production in brewer’s yeast by
relocation of the ILV2 gene
CHRISTINA CHRISTENSEN (1), Carolina Kristell (1), Kjeld Olesen
(1), Troels Felding (1), Claes Gjermansen (1)
(1) Carlsberg Research Laboratory, Copenhagen, Denmark
Diacetyl has a strong butter-like taste and its presence in lager
beer is undesirable. It is produced during fermentation by a nonenzymatic
decarboxylation of α-acetolactate in the media. During
the maturation period the yeast takes up diacetyl and reduces it to
the less flavor-active acetoin. α-Acetolactate (an intermediate of
valine biosynthesis) is derived from pyruvate by the action of the
ILV2 gene product. The enzymes involved in valine biosynthesis are
expressed in the nucleus with a targeting sequence that directs the
gene product to the mitochondria, where valine biosynthesis takes
place. Thus far, it has been believed that α-acetolactate formed in
the mitochondria is responsible for the diacetyl production observed
during fermentation. However, we hypothesized that the precursor
of diacetyl might be produced in the cytoplasm. This hypothesis is
based on the assumption that Ilv2p in its pro-form is active in the
cytoplasm during transport to the mitochondria. In the cytoplasm,
the pro-Ilv2p is believed to convert pyruvate to α-acetolactate,
which diffuses out of the cell and subsequently is converted to
diacetyl. It was speculated that this effect could be circumvented by
deleting the nuclear ILV2 gene and simultaneously, expressing the
ILV2 gene in the mitochondria, thus preventing protein transport
through the cytoplasm. A modified ILV2 m gene was constructed
by changing the DNA codons using in vitro genetic modification
techniques allowing for expression in the mitochondria. The
resulting ILV2 m gene was inserted into the mitochondrial genome
of a laboratory ilv2 yeast strain using biolistic bombardment
techniques and selection for valine prototrophy. Cultivation of
the transformants in YPD media showed that diacetyl production
was eliminated. Hence, having demonstrated the ability to abolish
diacetyl production in a laboratory tester strain, the modified
ILV2 m gene was inserted into the mitochondrial genome of a
Saccharomyces pastorianus (Dilv2/Dilv2) spore clone. The
resulting transformants regained their ability to grow without supply
of valine. Upon mating, hybrids expressing the modified ILV2 m gene
in the mitochondria were selected, all having a valine prototrophic
phenotype. Two hybrids were subjected to fermentation trials, and
diacetyl production was monitored daily. Both hybrids produced
very little diacetyl compared to wild type S. pastorianus lager yeast.
The results of this study are consistent with the precursor of diacetyl
(α-acetolactate) being produced in the cytoplasm in contrast to
previous theory. This result may lead to future savings in beer
production.
Christina Lund Christensen received her Ph.D. degree in molecular
microbiology from the Technical University of Denmark in May
1997. After graduation, she worked as post-doctoral researcher in
the Department of Bacterial Gene Technology at Novozymes A/S.
In February 1999, she joined the Carlsberg Research Laboratory as
a research fellow in the Barley Technology group. Since November
2000, she has worked as a scientist in the Yeast Breeding group.
During this period, she has worked with different aspects of yeast
physiology and yeast handling in the brewing process.
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