Program Book - Master Brewers Association of the Americas

P-214
Functional analysis of mitochondria in fermentation: Role of
mitochondrial DNA (mtDNA) copy number in resistance of
brewing yeast to fermentation stresses
STEPHEN LAWRENCE (1), Wendy Box (1), Katherine Smart (1)
(1) University of Nottingham, Sutton Bonington Campus,
Loughborough, United Kingdom
Brewery fermentations and handling of yeast populations between
successive fermentations exposes brewing yeast cells to a number of
biological, chemical and physical stresses. It is generally accepted
that repitching of yeast in subsequent fermentations leads to an
increase in incidence of petite mutations, which result from the loss
of mitochondrial DNA (mtDNA) integrity. Eventually this can lead
to aberrant fermentation profiles and impaired product quality. Ale
and lager yeasts exhibit different susceptibilities to elicit stress and
repair responses to the conditions which favor petite formation.
Since all mtDNA must be damaged for a petite mutation to be
formed, susceptibility of a given strain to forming petite mutations
may also be a function of the mtDNA copy number (typically 20–50
in Saccharomyces species). We have explored the effect of serial
repitching of warm and cold cropped yeast from production scale
cylindroconical vessels on mtDNA copy number using real time
PCR and % petite mutations. Samples were collected from different
portions of the cone, and mtDNA copy number was shown to vary.
Restriction fragment length polymorphism (RFLP) assessment of
petite mutations isolated from different crop generations showed
variability, demonstrating the instability of the yeast mtDNA when
exposed to stress in the cone. The role of oxidative, ethanol and
acetaldehyde stresses in mtDNA copy number and mtDNA integrity
has been assessed and hence the propensity of yeast to form petite
mutations.
Stephen Lawrence was awarded an upper second-class honors degree
in biochemistry and microbiology at the University of Sheffield
in 2002. He then joined Professor Katherine Smart’s research
group at Oxford Brookes University to start a Ph.D. program in
brewing yeast systems biology, completing it at the University of
Nottingham. Stephen is currently a post-doctoral research fellow at
the University of Nottingham. Stephen’s research interests include
yeast heterogeneity in the cone of fermentation vessels, formation of
brewing yeast mitochondrial DNA (mtDNA) mutants during yeast
handling processes, and the influence of mitochondrial copy number
on susceptibility of brewing yeast mtDNA to damage. He has recently
begun a new research project in association with SABMiller.
170
P-215
The development of a simultaneous measurement of yeast
viability and vitality by flow cytometry
MAYURA MOCHIZUKI (1), Mao Sugihara (1), Hiroyuki Yoshimoto
(1), Takeo Imai (1), Yutaka Ogawa (1)
(1) Research Laboratories for Brewing, Kirin Brewery Company,
Limited, Yokohama-shi, Japan
Yeasts with high vitality are very important for brewing high-quality
beer. Accordingly, techniques for yeast vitality measuring are
considered to be basic to the understanding of the yeast condition,
which can lead to the production of high-quality beer. In the many
methods that have been developed until now, the intracellular pH
(ICP) method, which achieves the highest sensitivity by using the
principle of H + extrusion activity, has been used for handling of high
activity yeasts in our breweries. However, the ICP method targets
only “vitality” and not “viability”. Consequently, low-viability yeasts
with 99% dead cells and 1% high-vitality yeasts might be determined
to be active yeasts. In this study, we combined two different concepts
of vitality and viability and developed a new technique that measures
them simultaneously, which could solve these problems. This
technique uses a flow cytometer with the ICP method for measuring
vitality and a method using TO-PRO 3 (TP3), which enables the
determination of viability by measuring the permeability of the
plasma membrane of the yeast. In the ICP method, a pH-sensitive
fluorescence reagent “5(6)-carboxyfluorescenceindiacetate
(CFDA)” with an excitation wavelength of 488 nm is used, and
for the viability measurement TP3 with an excitation wavelength
of 633 nm is used. TP3 can stain the nucleic DNA by using its
permeability through the plasma membrane of the yeast, which
enables very sensitive viability measurement without interference
with fluorescence of CFDA. We could accurately analyze both
vitality and viability simultaneously by flow cytometric measurement
after the treatment of yeast suspensions with CFDA and TP3 in
citrate-phosphate buffer (pH 3). In contrast to the existing methods
that cannot provide vitality and viability measurements of the
same yeasts group, this method offers an accurate simultaneous
measurement of “vitality” and “viability” which combines the two
different concepts of yeast’s physiological state.
Mayura Mochizuki is a researcher at the Research Laboratories
for Brewing, Kirin Brewery Co., Ltd. She graduated from Japan
Women’s University in 2003 with B.A. degree in home economics
(food and nutrition) and joined Kirin Brewery. Her main research
activity is yeast physiology and beer filtration.