Program Book - Master Brewers Association of the Americas

O-37
Fourier-transform infrared (FT-IR) spectroscopy, a costeffective
and high-resolution method to identify, differentiate
and monitor wild and cultured yeasts in a brewing ecosystem
MATHIAS HUTZLER (1), Eberhard Geiger (1), Siegfried Scherer
(1), Mareike Wenning (1)
(1) TU München, Freising, Germany
In brewing microbiology bacteria and yeasts are of major interest
as spoilage and culture organisms. Methods like real-time PCR,
rRNA micro-FISH and RNA-hybridization probes to detect and
identify beer spoiling bacteria are already established in many
breweries to control the presence or absence of beer spoiling
bacteria in the production chain. The monitoring and identification
of wild and brewing yeasts still is an underestimated topic. Several
culture-based, fermentation spectra and DNA-based methods are
proposed in the literature but most of them are time-consuming,
cost-intensive and are mostly developed for one special field of
application (e.g. differentiation of brewing strains). The aim of this
study was to analyze the potential of Fourier-transform infrared (FT-
IR) spectroscopy to identify wild yeasts and to differentiate culture
yeasts including ale and lager strains. The FT-IR spectroscopy
was introduced as a technique to identify microorganisms by the
group of Dieter Naumann, and it has gained growing interest for
identifying microbes on the species and strain levels. The absorption
of infrared light by cell components results in fingerprint-like
spectra which reflect the overall chemical composition of the cells
under investigation. As a standardized physico-chemical technique,
FT-IR spectroscopy benefits from the fact that operating costs are
extremely low, as practically no consumables are required, while
at the same time spectra contain a huge amount of information,
which can be exploited to help solve different kinds of identification
problems. By comparison with large reference data sets, spectra of
microbial cells can be analyzed for identification purposes, or to
reveal certain characteristics or even strain identity. The results
of the present study confirmed that the general benefits of FT-IR
spectroscopy could be transferred to the investigation of a brewing
ecosystem. An already existing yeast FT-IR spectra database
was expanded using spectra of yeast strains isolated in different
breweries and industrial strains from culture collections within a
period of 18 months.
Mathias Hutzler was born in 1978 in Regensburg, Germany. From
1999 to 2004 Mathias pursued a course of study, “Technology and
Biotechnology of Foods,” at the Technische Universität München,
was employed at PIKA Weihenstephan GmbH, and worked on
a diploma thesis on the PCR-based detection of heat-resistant
microorganisms. Since 2004 Mathias has been a scientific assistant
at the Chair for Brewing Technology II (Prof. Geiger), Technische
Universität München, Weihenstephan. The topic of his doctoral thesis
is “Differentiation of Industrial and Spoilage Yeasts Based on Novel
Rapid Methods.” His main fields of research are brewing and food
microbiology and detection of microorganisms in mixed cultures
and on the strain level. A further project is the investigation of the
microbial flora in the processes of indigenous fermented banana
beverages, in cooperation with AIKO, in Costa Rica.
Technical Session XI: Fermentation
Moderator: Katherine Smart, University of Nottingham,
Loughborough, United Kingdom
Katherine Smart completed a B.S. (Hon.) degree in biological sciences
at Nottingham University and was awarded the Rainbow Research
Scholarship to complete a Ph.D. degree in brewing yeast physiology
at Bass Brewers. She held a research fellowship at Cambridge
University and academic posts at Oxford Brookes University before
joining the University of Nottingham in 2005 as the SABMiller
Professor of Brewing Science. Katherine has received several awards
for her research: the IBD Cambridge Prize (1999), the prestigious
Royal Society Industrial Fellowship (2001–2003) and the Save
British Science Award (2003). She has published more than 80
papers, book chapters, and proceedings.
O-38
Production of hydrogen sulfide during secondary
fermentation related to pH value
ATSUSHI TANIGAWA (1), Masahide Sato (1), Kiyoshi Takoi (1),
Katsuaki Maeda (1), Junji Watari (1)
(1) Sapporo Breweries Ltd., Yaizu, Japan
Lager yeast is known to produce higher levels of sulfur compounds,
such as hydrogen sulfide (H S) and sulfite, than those of ale yeast.
2
In particular, “happoshu” and the so-called “third category
beer”, which are Japanese beer-flavor beverages brewed with
little or no malt content, show higher levels of H S and also lower
2
pH values than those of regular beer. At the 71st ASBC Annual
Meeting in Victoria, BC, Canada, based on the results of the gene
expression analysis of the two types (Saccharomyces cerevisiae and
Saccharomyces bayanus types) of genes, we showed that the gene
expression balance of MET3 and MET10 leads to higher levels of
sulfite being produced in the lager yeast. However, it has not been
fully clarified whether the lager yeast produces a higher level of
H S. During our further study of Japanese beer-flavor beverages, we
2
found that H S was detected, in the secondary as well as the main
2
fermentation. Furthermore, the content of H S produced during
2
the secondary fermentation tended to increase further at low pH
values. In this report, we investigated the correlation between the
amounts of H S and pH values during the secondary fermentation
2
related to sulfite. Sulfite is one of the intermediates of sulfur amino
acid biosynthesis. Although sulfate is generally used as a source of
sulfur in wort, sulfite can be utilized only at low concentrations.
– 2– Sulfite exists as a mixture of three forms (SO , HSO and SO3 )
2 3
depending on the pH value, and the yeast can uptake sulfite only
in its molecular state (SO ) by simple diffusion (1) since the yeast
2
membrane causes the anion to become electrically charged, and
the ratio of SO would be higher in fermenting wort as its pH values
2
subside. The results of adjustment trials of the pH value within the
range 3.0 to 5.0 during secondary fermentation indicated a negative
correlation between the contents of H S and the pH values, and the
2
sensory test also showed the same pattern. This suggests that the
increase of H S level during the secondary fermentation would be
2
due to the form of sulfite depending on the pH value and that the
increased H S content of Japanese beer-flavor beverages may be
2
responsible for the low pH values. Based on this result, we propose a
new model for the production of H S during secondary fermentation
2
by the simple diffusion of sulfite. Reference: 1) Malcolm Stratford
and Anthony H. Rose. J. Gen. Microbiol. 132: 1-6, 1986.
Atsushi Tanigawa received a M.S. degree from the Department of
Agricultural and Environmental Biology, Tokyo University. He
found employment with Sapporo Breweries, Ltd. in April 2005 as a
microbiologist in the Frontier Laboratories of Value Creation.
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