Program Book - Master Brewers Association of the Americas

P-70
Identification of novel foam-related proteins through twodimensional
gel electrophoresis analysis of the beer proteins
TAKASHI IIMURE (1), Kiyoshi Takoi (2), Yoshihiro Okada (3),
Takafumi Kaneko (2), Makoto Kihara (1), Katsuhiro Hayashi (1),
Kazutoshi Ito (1), Kazuhiro Sato (4), Kazuyoshi Takeda (4)
(1) Bioresources Research and Development Department, Sapporo
Breweries Ltd., Ota, Japan; (2) Frontier Laboratories of Value
Creation, Sapporo Breweries Ltd., Yaizu, Japan; (3) National
Agricultural Research Center for Kyushu Okinawa Region, Kikuchi,
Japan; (4) Barley Germplasm Center, Research Institute for
Bioresources Okayama University, Kurashiki, Japan
Foam stability is one of the important characteristics in beer
brewing. The purpose of this study was to identify foam-related
proteins using two-dimensional gel electrophoresis (2DE) analysis
of the beer proteins. We brewed a total of 25 beer samples, each
brewed from a malt with different modification in one of the three
cultivars (cultivars A, B and C). In cultivar A, the foam stability
did not change even when malt modification increased. However,
the foam stability decreased in cultivars B and C with increased
modification. To investigate foam-related proteins, we collected
beer proteins in three fractions, namely beer whole proteins, saltprecipitated
proteins and the proteins concentrated from beer foam.
2DE analysis of these protein fractions revealed that protein spot
b2 in cultivar A did not change in any of the three protein fractions
even when the malt modification increased, although spot b2 in both
cultivars B and C decreased. Pre-spot intensity, each spot intensity
against all spots, was calculated by vol. % as a unit. The spot intensity
was calculated by multiplying the beer protein concentration and
the pre-spot intensity by dimensionless as a unit. The foam stability
of 25 beer samples significantly correlated with the intensity of spot
b2 at the 5% level (r = 0.503). Furthermore, we focused on other
2 major protein spots (b0 and b5) observed in 2DE gels in all-malt
beer samples with different foam stability. Subsequently, multiple
regression was analyzed on 25 beer samples by the spot intensities
of spots b0, b2 and b5. As a result, 72.1% of the variation in beer
foam stability was explained by the intensities of spots b0 and b2 as
positive, and spot b5 as negative explanatory variables. Similarly,
85.6% of the variation in beer foam stability of 10 commercially
available beer samples was explained by the intensities of spots
b0 and b2 as positive and the spot b5 as negative explanatory
variables. MALDI TOF-MS and LC-MS/MS analyses followed by
database search revealed that the protein spots b0, b2 and b5 were
identified as protein Z originating from barley, barley dimeric
α-amylase inhibitor I (BDAI-I) and thioredoxin originating from
yeast, respectively. These results suggest that BDAI-I and protein Z
are foam-positive proteins (or indicators), and yeast thioredoxin is a
foam-negative protein (or indicator). We identified BDAI-I and yeast
thioredoxin as novel foam-related proteins.
Takashi Iimure received a M.S. degree in molecular chemistry from
Hokkaido University, Sapporo, Japan, in 2001. He has worked in
the Bioresources Research and Development Department (formerly
Bioresources Research and Development Laboratories) of Sapporo
Breweries Ltd. since 2004. He has researched storage substances
in barley seeds by means of protein and DNA techniques, such as
proteomics, transformation, RFLP, and so on.
P-71
The determination of intact acetolactate concentrations in
fermented products without prior conversion to diacetyl or
acetoin
TAKASHI INOUE (1), Yoshihiro Ohgaki (2), Tetsuya Hoshino (2),
Koichi Miyake (2), Hiroshi Maeda (3)
(1) Tokyo, Japan; (2) Chiba Industrial Technology Research
Institute, Chiba, Japan; (3) Tohoku University, Sendai, Japan
Diacetyl is a key flavor compound in fermented foods and beverages.
It is not formed directly by bacterial or yeast metabolism, but is
formed by the spontaneous conversion of acetolactate produced by
microorganisms during fermentation. Therefore, in order to control
the concentration of diacetyl in the finished product, it is necessary
not only to measure the concentration of diacetyl itself, but also
that of its precursor acetolactate. As acetolactate is a highly labile
compound, it is usually measured after being converted to diacetyl.
However, this method of analysis has two major shortcomings;
1) the percentage conversion is not always consistent, and 2) it
has a relatively long conversion time (a 90 min reaction time is
recommended in the official BCOJ analytical method). In order to
eliminate these obstacles, the authors have developed a method
of measuring intact acetolactate without the need to convert
the compound into diacetyl or acetoin. The method utilizes the
measurement of NADPH oxidation brought about by the conversion
of acetolactate into dihydroxyisovalerate by the enzyme acetolactate
reductoisomerase. We produced a recombinant Aspergillus oryzae
harboring the acetolactate reductoisomerase of A. oryzae (AoIlvC)
on an over-expression plasmid, and purified the recombinant AoIlvC
from the soluble cytoplasmic fraction. This assay method was not
influenced by the presence of high concentrations of diacetyl or
acetoin. This method of analysis makes it possible to measure the
concentrations of acetolactate in fermenting mash during sake (rice
wine) brewing and in milk products produced by fermentation with
lactic acid bacteria.
Takashi Inoue is a consultant for the Chiba Industrial Technology
Research Institute. He worked for 37 years for the Kirin Brewery
Co., Ltd. until his retirement in 1997. At Kirin, he mostly carried out
his job in the research laboratories. His major achievement was the
identification of acetolactate as a precursor of diacetyl in beer and
development of its control technologies and their applications. He
was presented the MBAA Award of Distinction in 1997 and the ASBC
Award of Merit in 2001. He is now a member of the Directors of the
Brewing Society of Japan.
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