U.S. patent application number 12/505723 was filed with the patent office on 2009-11-19 for barley lipoxygenase 1 gene, method of selecting barley variety, material of malt alcoholic drinks and process for producing malt alcoholic drink.
This patent application is currently assigned to SAPPORO BREWERIES LIMITED. Invention is credited to Naohiko HIROTA, Hirotaka Kaneda, Takafumi Kaneko, Hisao Kuroda, Kazuyoshi Takeda, Kiyoshi Takoi.
Application Number | 20090285932 12/505723 |
Document ID | / |
Family ID | 33094979 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090285932 |
Kind Code |
A1 |
HIROTA; Naohiko ; et
al. |
November 19, 2009 |
BARLEY LIPOXYGENASE 1 GENE, METHOD OF SELECTING BARLEY VARIETY,
MATERIAL OF MALT ALCOHOLIC DRINKS AND PROCESS FOR PRODUCING MALT
ALCOHOLIC DRINK
Abstract
A selection method for barley lipoxygenase-1 deficient barley,
comprising a step of distinguishing the barley lipoxygenase-1
deficient barley by whether or not the guanine at the splicing
donor site of the 5th intron of the barley lipoxygenase-1 gene is
mutated to a different base; and a method for production of malt
alcoholic beverages using a material for malt alcoholic beverages
derived from barley obtained by the selection method.
Inventors: |
HIROTA; Naohiko; (Gunma,
JP) ; Kaneko; Takafumi; (Gunma, JP) ; Kuroda;
Hisao; (Yaizu-shi, JP) ; Kaneda; Hirotaka;
(Yaizu-shi, JP) ; Takoi; Kiyoshi; (Yaizu-shi,
JP) ; Takeda; Kazuyoshi; (Kurashiki-shi, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
SAPPORO BREWERIES LIMITED
Tokyo
JP
|
Family ID: |
33094979 |
Appl. No.: |
12/505723 |
Filed: |
July 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10550528 |
Nov 27, 2006 |
|
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PCT/JP04/04217 |
Mar 25, 2004 |
|
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12505723 |
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Current U.S.
Class: |
426/16 ; 426/590;
426/592; 426/598; 800/320 |
Current CPC
Class: |
A01H 5/10 20130101; C12Q
2600/156 20130101; A23L 2/38 20130101; C12C 1/00 20130101; A23V
2002/00 20130101; C12Q 2600/13 20130101; C12Q 1/6895 20130101; C12N
9/0069 20130101 |
Class at
Publication: |
426/16 ; 426/592;
426/590; 426/598; 800/320 |
International
Class: |
C12C 12/00 20060101
C12C012/00; C12C 7/00 20060101 C12C007/00; C12C 1/00 20060101
C12C001/00; A01H 5/00 20060101 A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 25, 2003 |
JP |
2003-083924 |
Claims
1-13. (canceled)
14. A product produced from a barley plant, or a part thereof,
wherein the barley plant has a mutation in the LOX-1 gene causing a
total loss of LOX-1 activity.
15. The product of claim 14, which is a malt composition.
16. The product of claim 15, wherein said part of said barley plant
is a seed.
17. The product of claim 14, which is a wort composition prepared
from: a) the barley plant or part thereof, or b) a malt composition
prepared from said barley plant or part thereof, or c) a mixture of
a) and b).
18. The product of claim 17, wherein said part of said plant is a
seed.
19. The product of claim 17, which is prepared further with an
enzyme.
20. The product of claim 14, which is prepared from a mixture of
(i) a composition comprising a barley plant or a part thereof,
wherein the barley plant has a mutation in the LOX-1 gene causing a
total loss of LOX-1 activity, and (ii) a malt composition
comprising a processed barley plant or a part thereof, wherein the
barley plant has a mutation in the LOX-1 gene causing a total loss
of LOX-1 activity.
21. The product of claim 17, which is a wort composition.
22. The product of claim 14, which is a beverage having stable
organoleptic qualities.
23. The product of claim 22, wherein said beverage is beer.
24. The product of claim 22, wherein said beverage is prepared from
malt prepared from seeds of said barley plant.
25. The product of claim 22, wherein said beverage is prepared from
a wort composition prepared from a barley plant or part thereof, or
from a malt composition prepared from said barley plant or part
thereof.
26. The product of claim 22, wherein said beverage is a
non-fermented beverage.
27. The product of claim 22, wherein said barley plant, or parts
thereof, comprise a LOX-1 gene, said gene comprising a splice site
mutation.
28. A beverage having stable organoleptic qualities, wherein said
beverage is manufactured from a barley plant, and said beverage
having a concentration of trihydroxyoctadecenoic acid of not more
than 14.7 ppm.
29. The beverage of claim 28, wherein said beverage is beer.
30. The beverage of claim 28, wherein said beverage comprises at
most 0.36 ppb trans-2-nonenal (T2N).
31. A method of producing a malt composition; a wort composition; a
beverage; or a combination thereof, the method comprising
processing a barley plant or part thereof, wherein the barley plant
has a mutation in the LOX-1 gene causing a total loss of LOX-1
activity.
32. The method of claim 31, wherein said method is a method for
producing a beverage having stable organoleptic qualities, said
method comprising: (i) preparing a composition comprising the
barley plant or parts thereof, (ii) processing the composition of
(i) into a beverage; thereby obtaining a beverage with stable
organoleptic qualities.
33. The method of claim 32, wherein (i) comprises preparing a malt
composition from seeds of said barley plant or part thereof.
34. The method according to claim 32, wherein processing the
composition into a beverages comprises a mashing step.
35. The method of claim 31, wherein said method is a method for
producing a malt composition with no LOX-1 activity, said method
comprising (i) providing seeds from the barley plant; (ii) adding
water to the seeds; (iii) germinating the seeds from (ii); and (iv)
treating germinated seeds with heat; thereby producing a malt
composition with no or low LOX-1 activity.
Description
TECHNICAL FIELD
[0001] The present invention relates to a barley lipoxygenase-1
gene, a barley selection method, materials for malt alcoholic
beverages and a method for production of malt alcoholic
beverages.
BACKGROUND ART
[0002] Barley lipoxygenase-1 (hereinafter, "LOX-1") is an enzyme
present in malt, which oxidizes malt-derived linoleic acid to
9-hydroperoxyoctadecadienoic acid during mashing for production of
malt alcoholic beverages (Kobayashi, N. et al., J. Ferment.
Bioeng., 76, 371-375, 1993). 9-Hydroperoxyoctadecadienoic acid is
further converted to trihydroxyoctadecenoic acid (THOD) by
peroxygenase-like activity (Kuroda, H., et al., J. Biosci. Bioeng.,
93, 73-77, 2002). It is known that THOD reduces beer foam
stability, imparts an astringent flavor and impairs smoothness of
beer flavor (Kobayashi, N., J. Am. Soc. Brew. Chem. 60: 37-41.
2002; and Kaneda, H. et al., J. Biosci. Bioeng., 92, 221-226.
2001), resulting in lower quality of malt alcoholic beverages. In
addition, 9-hydroperoxyoctadecadienoic acid is converted to
trans-2-nonenal which is the substance responsible for an
unpleasant cardboard flavor in aged malt alcoholic beverages
(Yasui, Journal of the Brewing Society of Japan, 96:94-99
(2001)).
[0003] As a strategy for inhibiting production of trans-2-nonenal
in order to improve flavor stability of malt alcoholic beverages,
there has been proposed a method of producing malt alcoholic
beverages using malt with low LOX-1 activity (Drost, J. Am. Soc.
Brew. Chem. 48:124-131 (1990)).
[0004] Douma et al. have induced mutation in barley by mutagenic
(chemical) treatment to create an induced mutated line exhibiting
9% of lower LOX-1 activity compared to controls, and have attempted
to produce malt alcoholic beverages using such barley
(WO02/053721).
[0005] Even when such barley is used, however, the reduced
trans-2-nonenal concentration of the obtained malt alcoholic
beverages is insufficient and flavor stability is not adequately
improved. Furthermore, absolutely no definite results have been
achieved in terms of reducing THOD or improving foam stability.
DISCLOSURE OF INVENTION
[0006] The present invention has been accomplished in light of the
aforementioned problems of the prior art, and its object is to
provide a barley LOX-1 gene which is useful for production of malt
alcoholic beverages exhibiting improved flavor stability and foam
stability without gene manipulation, a selection method for LOX-1
deficient barley, materials for malt alcoholic beverages derived
from barley obtained by the selection method, and a method for
production of malt alcoholic beverages using the materials for malt
alcoholic beverages.
[0007] As a result of much diligent research conducted with the aim
of achieving the object described above, the present inventors have
completed this invention upon discovering a native barley variety
which is completely deficient in LOX-1 activity, and identifying a
novel LOX-1 mutant gene from the barley variety.
[0008] Specifically, the LOX-1 mutant gene of the present invention
is characterized in that the guanine at the splicing donor site
(5'-GT-3') of the 5th intron of the known barley LOX-1 gene is
mutated to a different base. The different base is preferably
adenine.
[0009] The selection method for barley LOX-1 deficient barley
according to the invention is characterized by distinguishing the
barley LOX-1 deficient barley by whether or not the guanine at the
splicing donor site of the 5th intron of the LOX-1 gene is mutated
to a different base. The different base is preferably adenine.
[0010] Also, the selection method for LOX-1 deficient barley is
characterized by comprising a genomic DNA extraction step wherein
genomic DNA is extracted from a barley sample; a DNA fragment
amplification step wherein a DNA fragment containing the splicing
donor site of the 5th intron of the LOX-1 gene is amplified from
the extracted genomic DNA; and a DNA fragment detection step
wherein the DNA fragment containing the splicing donor site of the
5th intron of the LOX-1 gene amplified in the DNA fragment
amplification step is cleaved with a restriction enzyme, a DNA
fragment having the prescribed number of bases is detected, and the
barley LOX-1 deficient barley is distinguished by whether or not
the guanine at the splicing donor site is mutated to a different
base.
[0011] The restriction enzyme used in the DNA fragment detection
step is preferably AfaI and/or RsaI which recognize the nucleotide
sequence 5'-GTAC-3'.
[0012] According to the invention, the barley variety having the
LOX-1 activity-deficient trait is distinguished based on the
presence or absence of a mutation of guanine at the splicing donor
site of the 5th intron of the LOX-1 gene.
[0013] As a result, it is possible to easily distinguish the LOX-1
activity-deficient barley variety by analysis on the genetic level,
without directly measuring LOX-1 activity. Enzyme activity is
influenced by individual growth stages, environment and other
factors and is therefore difficult to measure precisely, but this
method allows the LOX-1 activity-different barley variety to be
distinguished in a different manner from enzyme measurement, and
therefore independently of environmental and other factors.
Moreover, while enzyme activity cannot be measured until the seeds
have matured, DNA screening can identify the presence or absence of
the activity-deficient trait at an early stage of growth since it
is carried out before flowering, and is thus effective for
continuous back-crossing.
[0014] The material for malt alcoholic beverages of the invention
is characterized by being a seed, a malt, malt extract, barley
decomposition product or processed barley derived from barley
having a LOX-1 mutant gene according to the invention.
[0015] The material for malt alcoholic beverages of the invention
is also characterized by being a seed, a malt, malt extract, barley
decomposition product or processed barley derived from barley
selected by a selection method for LOX-1 deficient barley according
to the invention.
[0016] The method for production of malt alcoholic beverages of the
invention is characterized by using a material for malt alcoholic
beverages according to the invention.
[0017] According to the invention, it is possible to obtain malt
alcoholic beverages with improved flavor stability and foam
stability because LOX-1 is not present in the material, and
therefore 9-hydroperoxyoctadecadienoic acid is not readily produced
from linoleic acid and consequently THOD and trans-2-nonenal are
also not readily produced in the malt alcoholic beverage production
method.
[0018] The invention further provides a nucleic acid comprising the
nucleotide sequence from position 1 to 1554 as set forth in SEQ ID
NO: 10. This nucleotide sequence represents the coding region of
the gene encoding a mutant LOX-1 protein lacking the lipoxygenase
activity of LOX-1 protein. By detecting the presence or absence of
this nucleic acid in a barley sample, it is possible to distinguish
whether or not the barley has the LOX-1 activity-deficient
trait.
[0019] The invention still further provides a nucleic acid
comprising the nucleotide sequence as set forth in SEQ ID NO: 11.
This nucleotide sequence represents the genomic sequence of the
gene encoding a mutant LOX-1 protein lacking the lipoxygenase
activity of LOX-1 protein. By detecting the presence or absence of
this nucleic acid in a barley sample, it is possible to distinguish
whether or not the barley has the LOX-1 activity-deficient
trait.
[0020] The invention still further provides a nucleic acid
comprising the nucleotide sequence of 10 to 60 continuous bases
including the 3178th base in the nucleotide sequence as set forth
in SEQ ID NO: 11. The 3178th base is a single nucleotide
polymorphism which is G in authentic LOX-1 and A in mutant LOX-1.
By detecting the presence or absence of nucleic acid including the
polymorphic site in a barley sample, it is possible to distinguish
whether or not the barley has the LOX-1 activity-deficient
trait.
[0021] The invention still further provides a method for detecting
the presence of LOX-1 activity in barley, comprising a step of
isolating a genomic DNA from a barley sample, and a step of
detecting 3178th base of the nucleotide sequence as set forth in
SEQ ID NO: 11, wherein the presence of the base is an indicator of
the presence of LOX-1 activity in the barley. According to this
method, it is possible to distinguish whether or not tested barley
has the LOX-1 activity-deficient trait.
[0022] A seed, malt, malt extract, barley decomposition product or
processed barley derived from LOX-1 activity-deficient barley
discovered by this method may be used as raw material for
production of malt alcohol beverages, in order to obtain malt
alcoholic beverages with improved flavor stability and foam
stability.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a graph showing the results for LOX-1 activity in
Search Test 1.
[0024] FIG. 2 is a graph showing the results for LOX-1 inhibiting
activity in Verification Test 1.
[0025] FIG. 3 is a pair of electrophoresis images showing the
results for Western analysis of barley seed LOX protein in
Verification Test 2. Image A shows the results of Western analysis
after SDS-PAGE, and B shows the results of Western analysis after
IEF.
[0026] FIG. 4 is an electrophoresis image showing the results for
RT-PCR analysis of barley seed RNA in Verification Test 3.
[0027] FIG. 5 is a diagram showing the structure of the splicing
donor site of the 5th intron of the LOX-1 gene in Verification Test
4.
[0028] FIG. 6 is a pair of electrophoresis images showing the
results of analyzing splicing of the LOX-1 mutant gene in
Verification Test 5. Image A is an electrophoresis image for
amplified fragments containing the 3rd to 5th intron, and B is an
electrophoresis image of the same fragments as A after digestion
with StuI.
[0029] FIG. 7 is an electrophoresis image showing
expression-induced proteins in E. coli in Verification Test 7 and
8.
[0030] FIG. 8 is a graph showing the activity of LOX-1
expression-induced in E. coli in Verification Test 1.
[0031] FIG. 9 is an electrophoresis image showing DNA polymorphism
in the hybrid 2nd filial generation for Kendall.times.SBOU2 in
Verification Test 9.
[0032] FIG. 10 is a table summarizing DNA polymorphism in the
hybrid 2nd filial generation and LOX activity in the hybrid 3rd
filial generation for Kendall.times.SBOU2 in Verification Test
9.
[0033] FIG. 11 is an electrophoresis image showing the results of
analyzing a general barley variety/line by the AfaI method in
Example 1.
[0034] FIG. 12 is an electrophoresis image showing the results of
analyzing LOX-1 deficient barley by the AfaI method in Example
2.
[0035] FIG. 13 is a pair of graphs showing the results for LOX
activity of LOX-1 deficient barley seeds in Example 2. Graph A
shows the results at an enzyme reaction time of 5 minutes, and
graph B shows the results at an enzyme reaction time of 90
minutes.
[0036] FIG. 14 is a table showing the results of malt analysis of
seeds from the LOX+F4 population and LOX-F4 population in Example
5.
[0037] FIG. 15 is a dot graph showing trans-2-nonenal
concentrations and nonenal potentials for wort in Example 5.
BEST MODE FOR CARRYING OUT THE INVENTION
[0038] The present invention will now be explained in greater
detail.
[0039] To begin with, the LOX-1 mutant gene of the invention will
be explained.
[0040] The LOX-1 mutant gene of the invention is a novel gene
discovered by the present inventors, and it is characterized in
that the 60th base G of the known barley LOX-1 gene (SEQ ID NO: 1)
is replaced by A (SEQ ID NO: 2). Since bases 60-61 of SEQ ID NO: 1
constitute the splicing donor site (5'-GT-3'), this base
substitution produces an aberration in LOX-1 splicing so that
active LOX-1 cannot be expressed.
[0041] The nucleotide sequence of the 5th intron region of the
known LOX-1 gene is listed as SEQ ID NO: 1 in the Sequence Listing,
and the nucleotide sequence of the portion of the LOX-1 mutant gene
of the invention corresponding to the 5th intron region of the
LOX-1 gene is listed as SEQ ID NO: 2.
[0042] The selection method for LOX-1 deficient barley according to
the invention will now be explained.
[0043] The selection method for LOX-1 deficient barley according to
the invention is characterized by distinguishing barley LOX-1
deficient barley based on whether or not the guanine at the
splicing donor site of the 5th intron of the LOX-1 gene is mutated
to a different base.
[0044] The method for selecting LOX-1 deficient barley utilizing
the aforementioned base mutation may be, for example, a method of
using a primer containing the aforementioned mutation site either
at the 3' end of the primer sequence or within the primer sequence
for amplification of DNA, detecting the base mutation based on the
presence of amplification or on the amplification efficiency and
selecting the LOX-1 deficient barley, or a method of amplifying a
DNA fragment containing the aforementioned mutation site,
determining the nucleotide sequence to detect the base mutation and
selecting the LOX-1 deficient barley.
[0045] There are no particular restrictions on the method of
detecting the nucleotide sequence mutation so long as the method
allows detection of DNA fragments, but suitable methods to be
employed include agarose gel electrophoresis and polyacrylamide gel
electrophoresis. When the DNA mutation is to be detected based on
the presence of amplification or on amplification efficiency,
quantitative PCR such as the TAQMAN method may be used instead of
electrophoresis.
[0046] The selection method for LOX-1 deficient barley of the
present invention is characterized by comprising, preferably, a
genomic DNA extraction step wherein genomic DNA is extracted from a
barley sample, a DNA fragment amplification step wherein a DNA
fragment containing the splicing donor site of the 5th intron of
the LOX-1 gene is amplified from the extracted genomic DNA, and a
DNA fragment detection step wherein the DNA fragment containing the
splicing donor site of the 5th intron of the LOX-1 gene amplified
in the DNA fragment amplification step is cleaved with a
restriction enzyme, a DNA fragment having the prescribed number of
bases is detected, and the barley LOX-1 deficient barley is
distinguished by whether or not the guanine at the splicing donor
site is mutated to a different base.
[0047] The genomic DNA extraction step of the invention will be
explained first.
[0048] There are no particular restrictions on the method of
extracting the genomic DNA from the barley test sample, and any
publicly known method may be employed. Specifically, the extraction
may be carried out by, for example, the CTAB method (Murray et al.,
1980, Nucleic Acids Res. 8:4321-4325) or the ethidium bromide
method (Varadarajan and Prakash 1991, Plant Mol. Biol. Rep.
9:6-12). The tissue used for extraction of the genomic DNA is not
limited to barley seeds, but may also be leaves, stems, roots or
the like. For example, leaves may be utilized for selection of a
lot of individuals in back-crossing generations.
[0049] The DNA fragment amplification step of the invention will
now be explained.
[0050] There are no particular restrictions on the method of
amplifying the DNA fragment, and for example, the PCR (Polymerase
Chain Reaction) method may be employed. The primers used for the
PCR method are not particularly restricted in their nucleotide
sequences so long as they are of a region allowing amplification of
a DNA fragment containing the splicing donor site of the 5th intron
of the LOX-1 gene, and specifically, they are preferably 10-60
continuous bases and more preferably 15-30 continuous bases of the
LOX-1 gene. Generally, the nucleotide sequence of the primer will
preferably have a GC content of 40-60%. Also, the difference in the
Tm values of the two primers used for the PCR method is preferably
zero, or very small. The primers preferably do not form a secondary
structure with each other.
[0051] The DNA fragment detection step of the invention will now be
explained.
[0052] The LOX-1 mutant gene according to the invention has a
different nucleotide sequence from the known LOX-1 as explained
above, and therefore if a restriction enzyme which recognizes or
cleaves the differing portion is used to cleave the amplification
product, a difference in the sizes of the obtained DNA fragments
will be apparent. There are no particular limitations on the
restriction enzyme used for the invention so long as it recognizes
or cleaves the differing portion, but restriction enzymes AfaI
and/or RsaI, which have already been demonstrated to exhibit such
activity, are preferred.
[0053] In other words, since the LOX-1 mutant gene of the invention
has the guanine at the splicing donor site mutated to a different
base, it lacks the cleavage site for restriction enzymes AfaI and
RsaI (5'-GTAC-3': nucleotides 60-63 of the 5th intron) which are
present in the known LOX-1 gene. As a result, its cleavage pattern
when the gene amplification product containing the cleavage site is
cleaved with AfaI and/or RsaI will differ from that of the known
LOX-1 gene, thereby allowing identification of the LOX-1 mutant
gene.
[0054] The DNA fragment having the prescribed number of bases is
not limited in its number of bases so long as it is a DNA fragment
wherein the presence of the differing portion results in a
difference in the size of the DNA fragment obtained by cleaving the
amplification product with the restriction enzyme.
[0055] The detection in this step is not particularly restricted so
long as it is a method allowing detection of the DNA fragment
cleaved by the restriction enzyme, and specifically, the detection
may be accomplished by agarose gel electrophoresis or
polyacrylamide gel electrophoresis, for example.
[0056] The material for malt alcoholic beverages of the invention
will now be explained.
[0057] The material for malt alcoholic beverages of the invention
is characterized by being a seed, a malt, malt extract, barley
decomposition product or processed barley derived from barley
having the LOX-1 mutant gene according to the invention, and by
being a seed, a malt, malt extract, barley decomposition product or
processed barley derived from barley selected by a selection method
for LOX-1 deficient barley according to the invention.
[0058] Malt extract is the extract from malt, and as examples there
may be mentioned the extract of sugar components or protein
components from malt. Barley decomposition product is the product
of decomposition of barley with enzymes or the like, and it
includes barley mash and the like. Processed barley refers to the
milled barley used as the adjunct for malt alcoholic beverages.
[0059] Since the material for malt alcoholic beverages according to
the invention contains no LOX-1, production of
9-hydroperoxyoctadecadienoic acid from linoleic acid does not
readily occur, and consequently production of THOD and
trans-2-nonenal also does not readily occur, during the malt
alcoholic beverage production method; it is therefore possible to
obtain malt alcoholic beverages with improved flavor stability and
foam stability.
[0060] The method for production of malt alcoholic beverages will
now be explained.
[0061] The method for production of malt alcoholic beverages of the
invention is characterized by using a material for malt alcoholic
beverages according to the invention.
[0062] The malting step according to the invention will be
explained first.
[0063] The malting step according to the invention is a malt
obtaining step characterized by using LOX-1 deficient barley, and
the method is not particularly restricted and may be a publicly
known method. More specifically, for example, steeping to a
steeping degree of 40-45% is followed by germination at
10-20.degree. C. for 3-6 days and kiln-drying to obtain malt.
[0064] The mashing step according to the invention will now be
explained.
[0065] The mashing step according to the invention is a step of
obtaining wort by mashing of the aforementioned malt. More
specifically, it consists of the following four steps.
[0066] The first step is a mashing step in which the
malt-containing material is mixed with water, the obtained mixture
is heated for mashing of the malt, and the wort is obtained from
the saccharified malt.
[0067] The malt used for this step is preferably obtained by
addition of water and air to barley for germination followed by
drying to remove the radicals. The malt is the source of the
necessary enzymes for production of wort, as well as the major
starch source as the material for mashing. Also, kiln-dried
germinated malt is used for production of the wort in order to
impart the characteristic flavor and color of a malt alcoholic
beverage. In addition to such malt, there may be added adjuncts
such as LOX-1 deficient barley according to the invention and/or
ordinary barley, corn starch, corn grits, rice, saccharides or the
like.
[0068] In the wort production step described above, malt extract,
barley decomposition product or processed barley prepared from the
LOX-1 deficient barley of the invention and/or ordinary barley is
mixed with the mashing water, and the aforementioned adjuncts are
added as necessary, to obtain the wort.
[0069] The malt is mixed after addition to the mashing water. When
adjuncts are also added, they may be also mixed in at this point.
Saccharides may be added prior to the boiling described hereunder.
There are no particular restrictions on the mashing water, and
water may be used which is suitable for the malt alcoholic beverage
to be produced. The mashing may be carried out basically under
conventional conditions. After lautering of the malt mash obtained
in this manner, materials which impart flavoring or bitterness such
as hops or herbs are added and the mixture is boiled and then
chilled to obtain a chilled wort.
[0070] The second step is a step of adding yeast to the chilled
wort for fermentation to obtain intermediate products of a malt
alcoholic beverage.
[0071] The yeast used in this step may be any brewer's yeast for
alcohol fermentation, which metabolizes sugars in wort obtained by
malt mashing to produce alcohol and carbon dioxide gas, and
specifically there may be mentioned, for example, Saccharomyces
cerevisiae and Saccharomyces uvarum.
[0072] The fermentation is accomplished by cooling the wort
obtained in the mashing step and adding the yeast thereto. The
fermentation conditions may be basically the same as for
conventional fermentation, and for example, the fermentation
temperature will ordinarily be no higher than 15.degree. C. and
preferably 8-10.degree. C., while the fermentation period is
preferably 8-10 days.
[0073] The third step is a storage step in which the intermediate
products of the malt alcoholic beverage obtained in the
fermentation step is stored.
[0074] In this step, the fermentation solution which has completed
alcoholic fermentation is transferred to a sealed tank and stored.
The secondary fermentation is basically the same as conventional
conditions, and for example, the storing temperature is preferably
0-2.degree. C. and the storing time is preferably 20-90 days.
Storage of the fermented solution allows re-fermentation and
maturation of the residual extract to occur.
[0075] The fourth step is a filtering step in which the
intermediate products of the malt alcoholic beverage obtained in
the storage step is filtered to obtain a malt alcoholic
beverage.
[0076] The filtering conditions are basically the same as
conventional conditions, and for example, the filtering material
used may be diatomaceous earth, PVPP (polyvinyl pyrrolidone),
silica gel, cellulose powder or the like, and the temperature may
be 0.+-.1.degree. C.
[0077] This procedure yields a malt alcoholic beverage. The
filtered malt alcoholic beverage is then tanked, barreled, bottled
or canned for shipping to the market, either directly or after
sterile filtration or heat treatment.
[0078] The proportion of malt used for production of the barley
alcoholic beverage is not particular restricted, and the alcoholic
beverage may be any one produced using malt as the material.
Specifically there may be mentioned, for example, beer and
sparkling malt liquor. Non-alcoholic beer and non-alcoholic
sparkling malt liquor are also considered malt alcoholic beverages
since the production method employed is similar to malt alcoholic
beverages such as beer.
[0079] Since the material according to the invention contains no
LOX-1, production of 9-hydroperoxyoctadecadienoic acid from
linoleic acid does not readily occur, and consequently production
of THOD and trans-2-nonenal is inhibited, during the malt alcoholic
beverage production method, and it is therefore possible to obtain
malt alcoholic beverages with improved flavor stability and foam
stability.
[0080] The nucleic acid and the method for detecting the presence
of LOX-1 activity in barley according to the invention will now be
explained.
[0081] The nucleic acid of the invention is characterized by
comprising the nucleotide sequence as set forth in SEQ ID NO: 11.
SEQ ID NO: 11 represents the genomic sequence for mutant LOX-1 in
the LOX-1 activity-deficient barley variety SBOU2. That is, the
LOX-1 mutant gene of the invention is characterized by being
represented by SEQ ID NO: 11. The base corresponding to position
3178 is G in the authentic LOX-1 gene, whereas the 3178th base is
mutated to A in the mutant LOX-1 gene. This base is also the first
base of the 5th intron of the authentic LOX-1 gene, and the
sequence GT as the sequence of bases 3178-3179 corresponds to the
splicing donor site (FIG. 5). In the mutant LOX-1 gene, however,
the sequence of bases 3178-3179 corresponding to the splicing donor
site is AT, and therefore a splicing aberration occurs which
prevents splicing. Furthermore, the sequence of bases 3176-3178 is
TGA which is a stop codon, and therefore translation ends at this
point.
[0082] Since the mutant LOX-1 protein expressed from the mutant
LOX-1 gene only possesses the portion corresponding up to the 5th
exon, it lacks the amino acid residues at the C-terminal end from
the 5th exon of authentic LOX-1 protein. The molecular weight of
authentic LOX-1 protein is 95 kD, while that of the mutant LOX-1
protein is 57 kD. The mutant LOX-1 protein is deficient in
lipoxygenase activity, and this correlates with the known fact that
the domain corresponding to the exon region near the 5th intron in
authentic LOX-1 protein is the active center of plant LOX (Shibata
and Axelrod (1995) J. Lipid Mediators and Cell Signaling
12:213-218).
[0083] Consequently, if barley possessing the mutant LOX-1 gene is
used as raw material for production of malt alcohol beverages, no
LOX-1 protein will be present in the raw material, and hence
production of 9-hydroperoxyoctadecadienoic acid from linoleic acid
during the production method for malt alcoholic beverages will be
reduced so that, as a result, inhibition of THOD and
trans-2-nonenal production may be achieved in order to obtain malt
alcoholic beverages with improved flavor stability and foam
stability. Thus, nucleic acid comprising the nucleotide sequence as
set forth in SEQ ID NO: 11 according to the invention is highly
useful for obtaining malt alcoholic beverages with improved flavor
stability and foam stability.
[0084] The nucleic acid of the invention provides nucleic acid
comprising the nucleotide sequence of 10 to 60 continuous bases
including the 3178th base in the nucleotide sequence as set forth
in SEQ ID NO: 11. The nucleic acid may be used as a probe to
distinguish between authentic and mutant forms of the barley LOX-1
gene. That is, since the 3178th base of the authentic LOX-1 gene is
G, the resulting mismatch may be utilized to distinguish between
the authentic and mutant forms based on the difference in
hybridization. For example, by forming hybrids between these
nucleic acids and nucleic acid of the LOX-1 gene, gradually
increasing the temperature and measuring the melting temperature of
the hybrids, it is possible to easily distinguish between the
authentic LOX-1 gene and mutant LOX-1 gene since their melting
temperatures will differ. The nucleic acid may also be utilized to
distinguish between LOX-1 gene forms (authentic/mutant forms) by
methods known to those skilled in the art. From the standpoint of
specificity, the nucleic acid preferably comprises a nucleotide
sequence of 20-50 continuous bases including the 3178th base, and
it preferably includes bases 3178-3181. The nucleic acid may also
be labeled with a fluorescent substance, radioisotope or the
like.
[0085] The method for detecting the presence of LOX-1 activity in
barley according to the invention comprises a step of isolating a
genomic DNA from a barley sample, and a step of detecting 3178th
base of the nucleotide sequence as set forth in SEQ ID NO: 11,
wherein the presence of the base is an indicator of the presence of
LOX-1 activity in the barley. According to this method, it is
possible to distinguish whether or not tested barley has the LOX-1
activity-deficient trait.
[0086] The barley sample is not restricted to barley seeds, and it
may be barley leaves, stems, roots or the like. The nucleic acid
may be isolated by a publicly known method, and for example, the
CTAB method or the ethidium bromide method may be used.
[0087] Detection of the 3178th base of the nucleotide sequence as
set forth in SEQ ID NO: 11 may be accomplished by a method known to
those skilled in the art. If necessary, for example, a nucleic acid
containing the 3178th base of the nucleotide sequence represented
by SEQ ID NO: 11 may be amplified by a nucleic acid amplification
method such as PCR. The identity of the base at position 3178 of
isolated nucleic acid or an amplified nucleic acid fragment can be
discriminated, for example, by using nucleic acid comprising the
nucleotide sequence represented by SEQ ID NO: 11 wherein the
nucleic acid comprises a sequence of 10-60 continuous bases
including the 3178th base, as described above.
[0088] However, a method for detecting the 3178th base utilizing
the difference in bases 3178-3181 of the LOX-1 gene is more
convenient and efficient. The site of bases 3178-3181 is the
cleavage site for restriction enzymes AfaI/RsaI in the authentic
LOX-1 gene, but because the 3178th base is A in the mutant LOX-1
gene, it does not form a cleavage site for restriction enzymes
AfaI/RsaI (FIG. 5). In other words, if an isolated nucleic acid or
amplified nucleic acid fragment is treated with restriction enzymes
AfaI/RsaI, the nucleic acid of authentic LOX-1 will be cleaved
whereas nucleic acid of mutant LOX-1 will not be cleaved.
Electrophoretic analysis of the restriction enzyme-treated nucleic
acid sample will allow the form of the LOX-1 gene (authentic or
mutant) to be distinguished based on the difference in
electrophoresis patterns, so that the identity of the base at
position 3178 can be discriminated. In addition, by using nucleic
acid comprising a nucleotide sequence of 10-60 continuous bases
including bases 3178-3181 as a probe for hybridization with the
restriction enzyme-treated nucleic acid, it is possible to
distinguish the form of the LOX-1 gene and thus allow the identity
of the 3178th base to be discriminated.
[0089] If, as a result of discriminating the 3178th base in this
manner, the base is found to be G, then it may be concluded that
the tested barley has LOX-1 activity and is not suitable as a raw
material for malt alcoholic beverages with improved flavor
stability and foam stability. On the other hand, if the base is A,
then it may be concluded that the tested barley does not have LOX-1
activity and is therefore suitable as a raw material for malt
alcoholic beverages with improved flavor stability and foam
stability.
[0090] The nucleic acid of the invention is also characterized by
comprising the nucleotide sequence from position 1 to 1554 as set
forth in of SEQ ID NO: 10. SEQ ID NO: 10 represents the cDNA
sequence for mutant LOX-1 expressed by the LOX-1 activity-deficient
barley variety SBOU2. The nucleotide sequence from position 1 to
1554 represents the coding region. As explained above, the mutant
LOX-1 protein encoded by this cDNA lacks the amino acid residues at
the C-terminal end from the 5th exon of authentic LOX-1 protein,
its molecular weight is 57 kD, and it lacks lipoxygenase
activity.
[0091] Consequently, barley expressing this nucleic acid may be
used as raw material for production of malt alcohol beverages, in
order to obtain malt alcoholic beverages with improved flavor
stability and foam stability, as mentioned above.
EXAMPLES
[0092] The present invention will now be explained in greater
detail through the following examples, with the understanding that
these examples are in no way limitative on the invention.
[0093] Search Test 1 (Search for Lox-1 Deficient Barley by LOX-1
Enzyme Activity Measurement)
[0094] LOX-1 enzyme activity was measured by the method described
below, and a search for LOX-1 deficient barley was conducted from
barley gene resources.
[0095] First, a crude enzyme solution was extracted from barley
seeds by the following method. One mature barley seed was crushed
with a hammer, and 500 .mu.L of extraction buffer (0.1 M sodium
acetate buffer (pH 5.5)) was used for extraction with shaking at
4.degree. C. for 30 minutes. The obtained extract was centrifuged
for 10 minutes at 15,000 rpm, and then the supernatant was taken as
a crude enzyme solution.
[0096] Next, 5 .mu.L of substrate solution (40 mM linoleic acid,
1.0% (W/V) Tween20 aqueous solution) and 85 .mu.L of extraction
buffer were added to 10 .mu.L of the crude enzyme solution and
mixed therewith, and then reaction was conducted at 24.degree. C.
for 5 minutes. The reaction was terminated by adding and mixing 100
.mu.L of stop solution (80 mM 2,6-di-t-butyl-p-cresol, methanol
solution). After allowing the reaction mixture to stand at
-20.degree. C. for 30 minutes, it was centrifuged at 3000 rpm for
20 minutes and the supernatant was used in the following color
developing reaction. A 200 .mu.L portion of a color developing
solution (4 mM 2,6-di-t-butyl-p-cresol, 25 mM sulfuric acid, 0.25
mM ammonium iron(II) sulfate hexahydrate, 100 mM xylenol orange,
90% aqueous methanol) was added to 20 .mu.L of the obtained
supernatant, and after standing for 30 minutes, the absorbance at
550 nm was measured. As a negative control, reaction was conducted
in the same manner using the crude enzyme solution heat treated at
100.degree. C. for 5 minutes for inactivation of LOX-1, while as a
positive control there were used seeds of Kendall, a barley
variety.
[0097] As shown in FIG. 1, the search for gene resources revealed
no significant LOX-1 activity in SBOU2 seeds. Since SBOU2 is a
landrace, it is a spontaneous mutant rather than a artificially
mutagenized line.
[0098] Verification Test 1 (Confirmation of Lack of Lox-1
Inhibiting Activity in SBOU2 Crude Enzyme Solutions)
[0099] SBOU2 crude enzyme solutions were examined for the presence
or absence of LOX-1 inhibiting activity.
[0100] SBOU2 crude enzyme solutions (10 .mu.L, 20 .mu.L, 50 .mu.L)
were added to a crude enzyme solution exhibiting LOX-1 activity
(positive control: PC) and the changes in LOX-1 activity were
examined. The LOX-1 activity was unchanged with addition of the
SBOU2 crude enzyme solution, and therefore no LOX-1 inhibiting
activity was exhibited by the SBOU2 crude enzyme solutions (FIG.
2). This suggested that the cause of LOX-1 activity deficiency in
SBOU2 was not due to a LOX activity-inhibiting substance.
[0101] Verification Test 2 (Confirmation of Lox-1 Protein
Expression Level in SBOU2 Seeds)
[0102] Anti-LOX-1 antibody was used to determine whether or not
LOX-1 protein is expressed in SBOU2 seeds.
[0103] An anti-LOX-1 antibody was prepared first. The LOX-1 protein
used as the antigen was obtained by purifying LOX-1 protein
expressed by E. coli (Kuroda et al. (2002) J. Bioscience and
Bioengineering 93:73-77). The purified protein was used to immunize
rabbits for production of LOX-1 antibody. This antibody recognizes
both LOX-1 and LOX-2.
[0104] Western blotting was then carried out in the following
manner to examine LOX-1 protein expression in SBOU2 seeds. A 3
.mu.g portion of total soluble protein extracted from SBOU2 using
0.1 M sodium acetate buffer (pH 5.5) was fractionated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted
on a PVDF membrane (Millipore). The membrane was rinsed with TTBS
(20 mM Tris-HCl (pH 7.5), 0.15 M sodium chloride, 0.05% (w/v)
Tween20, 0.05% (w/v) sodium azide) and subsequently reacted with a
LOX-1 antibody solution (1000-fold dilution/TTBS) for 30 minutes.
The membrane was rinsed with TTBS three times for 5 minutes each
time, after which reaction was conducted for 30 minutes with alkali
phosphatase-labeled goat anti-rabbit IgG antibody solution (Santa
Cruz Biotechnology, TTBS solution at 1000-fold dilution). After
rinsing the membrane with TTBS for 5 min.times.2 and then with
AP9.5 (10 mM Tris-HCl (pH 9.5), 0.1 M sodium chloride, 5 mM
magnesium chloride) for 5 min.times.1, reaction was conducted with
alkali phosphatase substrate solution (1 mg/ml nitroblue
tetrazolium, 0.5 mg/ml BCIP, AP9.5 solution) for color development.
As a result, a dark band with a molecular weight of about 95 kD was
obtained with the control variety (Kendall), while two very faint
bands were detected at molecular weight regions of about 95 kD and
about 57 kD with the SBOU2 seeds (FIG. 3A).
[0105] The extraction sample was then subjected to isoelectric
focusing (IEF, pI 3-9) using a PhastSystem (Amersham Pharmacia) and
subjected to Western analysis in the same manner as above. As a
result of analysis with SBOU2, a band was detected at the pI
position of LOX-2, but no clear band was present at the pI position
of LOX-1 (FIG. 3B). This suggested that the approximately 95 kD
band appearing with the SBOU2 seed-extracted protein was LOX-2
protein.
[0106] These results confirmed that SBOU2 seeds express virtually
no authentic LOX-1 protein.
[0107] Verification Test 3 (Analysis of SBOU2 Seed Lox-1 RNA)
[0108] RT-PCR was carried out using as the template total RNA
extracted from SBOU2 approximately 4-week ripened seeds and 3-day
germinated seeds. The reaction was conducted using commercially
available kits (Roche Diagnostics, Perkin Elmer), according to the
kit manuals. The primers were 5'-GGAGAGGAGGCCAAGAACAAGATG-3' (SEQ
ID NO: 3) and 5'-GGTTGCCGATGGCTTAGAT-3' (SEQ ID NO: 4), designed
based on the published sequence (DNA Databank: Accession No.
L35931). After incubation at 94.degree. C. for 2 min, PCR was
carried out with reaction at 94.degree. C., 1 min, 60.degree. C., 2
min and 72.degree. C., 4 min, repeated 31 times, followed by
extension reaction at 72.degree. C., 7 min.
[0109] Upon electrophoresis of the amplified DNA, amplification of
an approximately 2.5 Kb band was detected for the RNA from the
maturing and germinating seeds, although at a slightly lower amount
than the control (Kendall variety) (FIG. 4). This indicated that
the LOX-1 gene was properly transcribed.
[0110] Since the aforementioned results for the SBOU2 seeds
indicated that (1) LOX-1 activity was not detected, (2) only a
trace amount of antigen protein reacting with the LOX-1 antibody
was present, (3) the presence of a protein with a molecular weight
of approximately 57 kD was detected and (4) LOX-1 mRNA was
detected, it was concluded that the mechanism for the deficiency of
LOX-1 activity in SBOU2 was an aberration after transcription.
[0111] Verification Test 4 (Structural Analysis of SBOU2 Lox-1 Gene
Intron Regions)
[0112] In order to analyze the structure of the intron and exon
regions of the LOX-1 gene, genomic DNA of a region comprising all
of the exons was isolated. Total DNA from SBOU2 was used as the
template. The primers (5'-CACGTCGCCGTCCGATCCATC-3' (SEQ ID NO: 5),
5'-GGTTGCCGATGGCTTAGAT-3' (SEQ ID NO: 4)) were designed based on
the reported sequence (DNA Databank: Accession Nos. U83904,
L35931). The PCR was carried out with reaction at 94.degree. C., 1
min, 65.degree. C., 2 min and 72.degree. C., 3 min, repeated 31
times, followed by extension reaction at 72.degree. C., 7 min. The
obtained DNA fragment was cloned in pCR2.1 (pGLXABAL1) for use as
the template for structural analysis. The structural analysis was
carried out using an ABI sequencer, and the sequencing reaction was
conducted by the dye terminator method. Whole structure is shown in
SEQ ID NO: 11 in Sequence Listing.
[0113] SEQ ID NO: 1 of the Sequence Listing shows the structure of
the region of the reported nucleotide sequence of the LOX-1 gene
(WO 02053721) containing the 5th intron. The splicing donor site is
the nucleotide sequence 5'-GT-3' at positions 60-61.
[0114] The nucleotide sequence of the corresponding region of SBOU2
determined from the analysis results is listed as SEQ ID NO: 2.
Clearly in SBOU2, the guanine at position 60 in the splicing donor
site is mutated to adenine.
[0115] Replacement of the 60th base of SEQ ID NO: 2 with adenine
forms a new stop codon (the nucleotide sequence 5'-TGA-3' at
positions 58-60 of SEQ ID NO: 2), and therefore presumably
translation of the LOX-1 protein ends at that point if the splicing
site never changes to the upstream to the 5' end (FIG. 5).
[0116] The exon region near the 5th intron is known to be the
active center of plant LOX (Shibata and Axelrod (1995), J. Lipid
Mediators and Cell Signaling 12:213-228), and it is believed that
the aforementioned splicing aberration has a major effect on the
LOX-1 enzyme activity.
[0117] Verification Test 5 (Analysis of Splicing at 5Th Intron)
[0118] RT-PCR analysis was conducted in order to confirm that a
splicing aberration had actually occurred in the 5th intron.
[0119] Total RNA was extracted from germinating SBOU2 and Kendall,
and a commercially available kit (Roche Diagnostics) was used for
synthesis of cDNA to prepare template DNA. PCR was conducted using
two different primers (5'-CCATCACGCAGGGCATCCTG-3' (SEQ ID NO: 6),
5'-GCGTTGATGAGCGTCTGCCG-3' (SEQ ID NO: 7)) designed to give an
amplified fragment containing the 3rd intron (106 bp), 4th intron
(132 bp) and 5th intron (79 bp) in genomic DNA sequence. The PCR
was carried out with reaction at 94.degree. C., 1 min, 65.degree.
C., 2 min and 72.degree. C., 3 min, repeated 31 times, followed by
extension reaction at 72.degree. C., 7 min. Agarose gel
electrophoresis of the amplified DNA fragments indicated that the
SBOU2 amplified fragment was approximately 80 bp larger than the
Kendall amplified fragment (FIG. 6A). An approximately 1.2 Kb
fragment was amplified from the SBOU2 genomic DNA, suggesting that
the results of the RT-PCR were the results for the expressed
RNA.
[0120] Next, in order to investigate in which of the introns the
splicing aberration had occurred (the 3rd intron (106 bp), 4th
intron (132 bp) or 5th intron (79 bp)), the amplified fragment was
digested at the restriction enzyme StuI site present in the exon
region between the 4th intron (132 bp) and 5th intron. As a result,
the DNA fragment containing the 5th intron had mobility roughly
equivalent to that of one of the amplification fragments from
genomic DNA, and therefore an aberration had obviously occurred in
splicing of the 5th intron which had either prevented splicing or
had shifted the splicing site toward the 3' end (FIG. 6B). That is,
clearly, the stop codon newly formed as shown in FIG. 5 had halted
translation of the SBOU2 LOX-1 protein at that codon, resulting in
loss of LOX-1 activity.
[0121] Verification Test 6 (Structural Analysis of Lox-1 cDNA of
SBOU2)
[0122] In order to elucidate the structure of LOX-1 derived from
SBOU2, cDNA was isolated by the same method described above.
Amplification was carried out using a primer comprising the BamHI
site and start codon (5'-GGATCCATGCTGCTGGGAGGGCTG-3' (SEQ ID NO:
8)) and a primer designed to include the HindIII site and the stop
codon (5'-AAGCTTTTAGATGGAGATGCTGTTG-3' (SEQ ID NO: 9). The
amplified fragment was cloned in pT7 Blue T-Vector (Novagen)
(pBDC1) and then provided for structural analysis. The results of
the structural analysis yielded the cDNA nucleotide sequence shown
as SEQ ID NO: 10. This cDNA clone clearly includes the entire
region of the 5th intron (bases 1554 to 1632 of SEQ ID NO: 10).
[0123] Verification Test 7 (Transformation of E. Coli and Induced
Expression)
[0124] For expression of LOX-1 derived from the Steptoe variety
retaining wild type LOX-1, Steptoe-derived cDNA was also isolated
in the same manner as above and cloned in E. coli (pSDC1),
separately from SBOU2. The BamHI-HindIII fragment was cut out from
each of the clones pSDC1 and pBDC1 and each of the obtained
fragments was inserted at the BamHI-HindIII site of E. coli
expression vector pQE80L (Qiagen) to obtain E. coli expression
vectors (pSQE1 (Steptoe cDNA-inserted) and pBQE1 (SBOU2
cDNA-inserted)). These vectors were used to transform E. coli
JM109, and then expression was induced by IPTG according to the
instruction manual by Qiagen. As a result, expression of an
approximately 95 kD band was induced with pSQE1/JM109, while
expression of an approximately 57 kD band was induced with
pBQE1/JM109 (FIG. 7). The E. coli cells were disrupted by
sonication and the LOX activities of the crude enzyme extracts were
assayed. As a result, high LOX activity was found with pSQE1/JM109
while no LOX activity was found with pBQE1/JM109 (FIG. 8). These
results perfectly matched the LOX-1 activity and LOX-1 protein
analysis results for SBOU2 plants, indicating that the SBOU2 LOX-1
deficiency can be reproduced in E. coli.
[0125] Verification Test 8 (Exchange Insertion and Expression
Test)
[0126] Next, a PstI-StuI fragment containing a mutation at the
splicing donor site of the 5th intron of pBQE1 (the StuI site is at
bases 1502-1507 of SEQ ID NO: 10, and the PstI site is at bases
2048-2053 of SEQ ID NO: 10) was mutually exchanged with the
PstI-StuI fragment of the wild type pSQE1 (pSQE1+BPS, pBQE1+SPS),
and expression was induced in E. coli in the same manner described
above. As a result, with pSQE1+BPS/JM109 having the pBQE1-derived
PstI-StuI fragment containing a mutation at the splicing donor site
of the 5th intron inserted into pSQE1, expression of an
approximately 57 kD protein was induced (FIG. 7) and LOX activity
was lost (FIG. 8), similar to pBQE1/JM109. Conversely, with
pBQE1+SPS/JM109 having the pSQE1-derived PstI-StuI fragment
inserted into pBQE1, expression of an approximately 95 kD protein
was induced (FIG. 7) and LOX activity was restored (FIG. 8),
similar to pSQE1/JM109. The nucleotide sequence of the PstI-StuI
fragment of pBQE1 was exactly identical to the wild LOX-1 gene
except for the splicing donor site of the 5th intron (G is
substituted to A at the 1554th base of SEQ ID NO: 10). These
results clearly demonstrated that the presence or absence of the
mutation at the splicing donor site of the 5th intron of SBOU2
determines whether LOX-1 activity is present.
[0127] Verification Test 9 (Mapping and Selection of Barley Hybrid
Variety Using 5th Intron Mutation)
[0128] In the reported LOX-1 nucleotide sequence, a sequence
containing the splicing donor site of the 5th intron (nucleotides
60-63 of SEQ ID NO: 1: 5'-GTAC-3') can be digested by restriction
enzyme AfaI (or RsaI) which recognizes the sequence GTAC. The LOX-1
deficient gene was mapped utilizing the fact that a mutation is
present in the sequence of this region in the SBOU2 line
(nucleotides 60-63 of SEQ ID NO: 2: 5'-ATAC-3') which prevents
digestion by AfaI and/or RsaI (FIG. 5). DNA was extracted from the
leaves of 144 individuals of a hybrid 2nd filial generation (F2) of
a cross between Kendall and SBOU2 line, and PCR was conducted using
two different primers (5'-CCATCACGCAGGGCATCCTG-3' (SEQ ID NO: 6),
5'-GCGTTGATGAGCGTCTGCCG-3' (SEQ ID NO: 7)) designed so that the
amplified fragment would contain the AfaI site. The PCR was carried
out with reaction at 94.degree. C., 1 min, 65.degree. C., 2 min and
72.degree. C., 3 min, repeated 31 times, followed by extension
reaction at 72.degree. C., 7 min. Each of the amplified fragments
was cleaved with AfaI and analyzed by 2.5% agarose gel
electrophoresis (this will hereinafter be referred to as the "AfaI
method"). As a result, it was possible to easily distinguish
between the SBOU2, Kendall and hetero types (FIG. 9). In addition
to the polymorphism examination by the AfaI method, polymorphism in
each variety was also examined using a DNA marker (JBC970) near the
LOXA gene locus of the barley 4H chromosome bearing the LOX-1 gene
(FIG. 10).
[0129] A mature seed of each F2 individual was used to examine the
LOX activity of the seeds. For lines exhibiting no LOX activity,
the activity was measured using several (4-7) seeds (FIG. 10).
[0130] As a result of the AfaI method polymorphism examination of
the F2 generation and LOX activity measurement of F3 seeds,
segregation of the LOX-1 deficient trait of SBOU2 perfectly matched
segregation of the AfaI method polymorphism. Thus, this series of
genetic analyses clearly showed that the LOX-1 deficient gene of
SBOU2 is at the LOXA gene locus.
[0131] The results also demonstrated that using the AfaI method as
an example of a barley selection method utilizing DNA mutation
allows selection of LOX-1 deficient progeny lines at the early
stage of the growth, eliminating the need to wait until the seeds
have matured.
Example 1
AfaI Polymorphism Examination of Other Barley Varieties
[0132] General barley varieties/lines were used for AfaI
polymorphism examination. A total of 32 varieties/lines were used:
Mikamo Golden, Golden Melon, Haruna Nijo, Myogi Nijo, Sakitama
Nijo, Wasedori Nijo, Agurimochi, Harupin Nijo, Ryofu, Hokuiku 33,
Hokuiku 35, Prior, Schooner, Sloop, Lofty Nijo, Franklin, Betzes,
Harrington, Manley, B1251, CDC Kendall, CDC Stratus, CDC Copeland,
Hanna, Merit, AC Metcalfe, TR145, Chariot, Stirling, Proctor, Koral
and Heartland. As a result of AfaI method polymorphism examination,
it was determined that the tested varieties were not the SBOU2
type, but instead were digested at the restriction enzyme AfaI site
containing the splicing donor site of the 5th intron (nucleotides
60-63 of SEQ ID NO: 1: 5'-GTAC-3') (FIG. 11). This indicated that
these viable lines did not possess a DNA mutation at the AfaI site
(nucleotides 60-63 of SEQ ID NO: 1: 5'-GTAC-3'), and therefore that
the AfaI method can be effectively utilized for selection of LOX-1
deficient genes among progeny lines.
Example 2
Gene Resource Search
[0133] Worldwide gene resources of barley (landrace) stocked at
Okayama University were examined by the AfaI method. As a result,
five new lines (SBOU1, SBOU3, SBOU4, SBOU5 and SBOU6 stocked at
Okayama University) were discovered which were not digested at the
restriction enzyme AfaI site containing the splicing donor site of
the 5th intron (nucleotides 60-63 of SEQ ID NO: 1: 5'-GTAC-3')
(FIG. 12).
[0134] The LOX-1 activity of seeds of these lines was measured by
the method described in Search Test 1. The activity measurement for
SBOU5 and SBOU6 was carried out by reaction for 5 minutes (FIG.
13A), while activity measurement for SBOU1, SBOU3 and SBOU4 was
with an extended reaction time of 90 minutes for clear
identification of activity (FIG. 13B). As a result, no significant
activity was found in any of the lines (FIG. 13).
[0135] This clearly demonstrated that SBOU2 as well as SBOU1,
SBOU3, SBOU4, SBOU5 and SBOU6 (SBOU2 type LOX-1 deficient barley)
were LOX-1 deficient barley lines. Since all of the lines are
landraces and not artificially mutagenized lines, they represent
spontaneous mutants for the LOX-1 gene.
[0136] The results described above demonstrated that the AfaI
method, as an example of a barley selection method utilizing DNA
mutation, is a technique allowing not only selection of LOX-1
deficient barley progeny lines, but also efficient selection of
LOX-1 deficient barley from barley gene resources.
Example 3
Growth of Barley for Test Brewing
[0137] A barley variety, Taishomugi, was crossed with SBOU2, and
the obtained first filial generation (F1) was self-pollinated to
obtain an F2 generation. The LOX-1 deficient trait was confirmed by
the LOX-1 enzyme activity measuring method described in Search test
1 and the AfaI method described in Verification test 9 above, and
the group of LOX-1 deficient lines and the group of LOX-1 retaining
lines were provided as populations for the seed propagation
described below.
[0138] Seed propagation was carried out for each line (population)
using a uniform plot or greenhouse until F4 seeds were obtained.
When the LOX-1 enzyme activity of the F4 seeds was assayed, it was
found that the respective LOX-1 activity traits of the F2
individuals were maintained, indicating that the LOX-1 deficient
trait is stably passed on to progeny.
[0139] The F4 seeds were used for the following malt production
test and malt alcoholic beverage production test.
Example 4
Production of Malt for Test Brewing
[0140] A LOX-1 deficient barley F4 population (LOX-F4) comprising
barley seeds having no LOX-1 activity and a LOX activity-retaining
barley F4 population (LOX+F4) from barley seeds with LOX-1, both
derived from the Taishomugi.times.SBOU2 cross, were prepared and
used for malting.
[0141] The malting was carried out using an Automatic Micromalting
System (Phoenix Systems) under conditions with a steeping
temperature of 16.degree. C. for a total of 82 hours (5 hr WET/7 hr
DRY cycle), germination at 15.degree. C. for 139 hours, and
kiln-drying for 29 hours (55.degree. C. for 13.5 hrs, 65.degree. C.
for 8 hrs, 75.degree. C. for 3.5 hrs, 83.degree. C. for 4 hrs).
Example 5
Analysis of Malt and Congress Wort
[0142] The malt was analyzed according to the EBC Standard Method
(European Brewery Convention ed., Analytica EBC (4th Ed), 1987). As
a result, no significant difference in analysis values of malt was
found between the malts using LOX-F4 and LOX+F4, demonstrating that
they can be used for brewing of malt alcoholic beverages for the
purpose of comparing the presence or absence of LOX-1 activity
(FIG. 14).
[0143] Next, 50 g of the malt was used to produce wort by the
Congress Method (European Brewery Convention ed., Analytica EBC
(4th Ed), 1987), and the lipid oxidation in the congress wort was
analyzed.
[0144] First, the amount of trans-2-nonenal in the congress wort
was measured by the following method. An 8 mL wort sample was
placed in a vial, 3 g of NaCl was added, and the vial was capped.
Next, a polydimethylsiloxane SPME fiber (Supelco, Inc.) was
inserted into the head space of the vial and the vial was incubated
at 40.degree. C. for 15 minutes.
[0145] The fiber was then inserted into a injection port of a gas
chromatography/mass spectrometry equipped with a J&W DB-1
column as the capillary column (30 m.times.0.25 mm, film thickness:
1 .mu.m). Helium was used as the carrier gas (1 mL/min) and oven
conditions was from 60.degree. C. to 225.degree. C. (5.degree.
C./min), in select ion mode (m/z: 70). The quantitation was
performed by the standard addition method using trans-2-nonenal
(Sigma) as standard sample.
[0146] As a result, the trans-2-nonenal concentrations of the
congress worts produced using LOX-F4 and LOX+F4 were 0.36 ppb and
3.85 ppb, respectively. Thus, it was demonstrated that production
of wort using malt according to the present invention allows
production of trans-2-nonenal to be inhibited by as much as 1/10 or
less of conventional malt.
[0147] The nonenal potential of the congress wort was measured by
the following method. First, the congress wort was boiled for 2
hours by the method of Drost et al. (Drost, B. W., van den Berg,
R., Freijee, F. J. M., van der Velde, E. G., and Hollemans, M., J.
Am. Soc. Brew. Chem., 48, 124-131, 1990). The amount of
trans-2-nonenal in the sample was then measured by the
trans-2-nonenal measuring method described above, and the nonenal
potential of the congress wort was calculated.
[0148] As a result, the nonenal potentials of the congress worts
produced using LOX-F4 and LOX+F4 were 2.74 ppb and 11.9 ppb,
respectively. Since the nonenal potential is known as an index of
product aging (Drost, B. W., et al., J. Am. Soc. Brew. Chem., 48,
124-131, 1990; Ueda et al. (2001) EBC-proceedings 55:p3 28th
Congress), malt produced from barley according to the invention can
be utilized for brewing of malt alcoholic beverages to
significantly improve flavor stability of malt alcoholic
beverages.
[0149] The trans-2-nonenal concentrations and nonenal potentials of
the congress worts produced using LOX-F4 are shown in FIG. 15 in
comparison with those of congress worts produced using commercially
available malt. As is clear by the results shown in FIG. 15, the
worts of the present invention exhibited values that have not been
achievable with conventional barley.
[0150] These results demonstrated that utilizing barley according
to the present invention allows production of malt with a superior
level of quality not exhibited by conventional products.
[0151] The THOD concentrations of the congress worts were measured
by high performance liquid chromatography-mass spectrometry
(HPLC-MS) analysis. The HPLC conditions were as follows. The flow
rate of the mobile phase was 0.3 mL/min, using a mixed solution of
0.5% acetic acid (Solution A) and acetonitrile (Solution B) as the
mobile phase, with a linear gradient of Solution A: Solution B=35:
65 (0 min) to Solution A: Solution B=5: 95 (30 min). The column
used was a Waters Asymmetry column (No. 106005; C18, 3.5 .mu.m:
2.1.times.150 mm), the column temperature was 50.degree. C., and a
Model 1100 HPLC system (Hewlett-Packard) was used for separation of
5 .mu.L sample of wort or malt alcoholic beverages. Mass analysis
was performed using a Waters ZQ, with monitoring of mass 329 under
ES ionization negative mode. The THOD standard solution used was a
beer extract sample (Kobayashi, N., et al., J. Biosci. Bioeng., 90,
69-73, 2000).
[0152] As a result, the THOD concentrations of the congress worts
produced using LOX-F4 and LOX+F4 were 6.5 ppm and 14.7 ppm,
respectively, thus demonstrating that production of wort using malt
according to the present invention can inhibit the wort THOD
concentration to 1/2 or below.
[0153] As mentioned above, THOD is produced by conversion from
linoleic acid by the action of malt LOX-1 and malt peroxygenase
activity in the mashing step, but since malt peroxygenase activity
is thought to be the rate-limiting step for THOD production
(Kuroda, H., et al., J. Biosci. Bioeng., 93, 73-77, 2002), it has
not been clear to what degree reduced malt LOX-1 activity inhibits
production of THOD. However, the results in the examples provided
in the present specification have demonstrated that using malt
produced from barley seeds with no LOX-1 activity reduces THOD
concentration in wort. Since THOD survives to the final product
without being metabolized by yeast (Kobayashi, N., et al., J. Inst.
Brew., 106, 107-110 (2000)), the use of malt derived from barley
according to the present invention can clearly result in production
of malt alcoholic beverages with good flavor quality and foam
quality.
Example 6
Test Brewing of Malt Alcoholic Beverages
[0154] 1. Production and Analysis of Wort
[0155] The LOX-F4 malt and LOX+F4 malt obtained in Example 4 above
were mashed with a 50 L scale mashing apparatus according to the
standard mashing methods for Happoshu, low malt alcoholic beverage
(malt content: 24%). The mashing conditions were as follows. 1.5 kg
of each malt was mashed alone with 15 L of mashing water according
to a diagram of 50.degree. C. for 20 min, 65.degree. C. for 30 min
and 75.degree. C. for 3 min. After mashing, wort lautering was
carried out with a lauter tun. 35 L of lautered worts were
obtained.
[0156] Before boiling, the lautered wort was mixed with 5 kg of
starch syrup (75% saccharides). 13 g of hop pellets (bitterness
unit: 87.0 BU (EBC)) was added into the wort. After boiling for 70
minutes, the boiled wort was cooled to 10.degree. C. The extract
content of cooled worts were adjusted by water addition to
11.6-11.8%.
[0157] The obtained worts were analyzed according to the EBC
Standard Method (European Brewery Convention ed., Analytica EBC
(4th Ed), 1987). The analysis values are shown in Table 1. As seen
in Table 1, no distinct difference was found between LOX-F4 and
LOX+F4 with regard to parameters.
TABLE-US-00001 TABLE 1 LOX - Variety LOX + F4 F4 Specific gravity
1.0475 1.0467 Extract (%) 11.78 11.60 Real nonfermented extract (%)
3.45 3.38 Real attenuation (%) 70.7 70.9 Apparent nonfermented
extract (%) 1.54 1.52 Apparent attenuation limit (%) 86.9 86.9 pH
5.88 5.93 Color (.degree.EBC) 2.1 2.1 BU 31.2 27.3 Total nitrogen
(mg/100 ml) 24 22 Polyphenol (mg/L) 44 48 FAN (mg/L) 46 51
[0158] 2. Production of Malt Alcoholic Beverage (Happoshu, Low Malt
Alcoholic Beverage)
[0159] The wort obtained in 1. above was transferred to a
steam-sterilized 30 L scale cylindroconical tank, and then yeast
was added to an initial concentration of 30 million cells/ml for
main fermentation at 13.degree. C. When the extract content in the
fermentation liquid fell to 2.5%, it was transferred to a new
similar tank for a storage step. The storage step was carried out
at 13.degree. C. for the first 6 days and then at 0.degree. C. for
2 weeks thereafter.
[0160] After finishing the storage step, fermentation liquid was
supplied to a beer filtration and filling apparatus. The malt
alcoholic beverage was filtered and filled into bottles.
[0161] 3. Analysis of Malt Alcoholic Beverage
[0162] The malt alcoholic beverage obtained in 2. above was
analyzed as follows.
[0163] Analysis according to the EBC Standard Method shows that no
significant difference was found between LOX-F4 and LOX+F4 in terms
of the general analysis parameters other than the lipid oxidation
parameters (Table 2).
TABLE-US-00002 TABLE 2 Variety LOX + F4 LOX - F4 Specific gravity
1.00562 1.00565 Original wort extract (%) 11.82 11.56 Real extract
(%) 3.43 3.38 Real attenuation (%) 71.0 70.7 Apparent extract (%)
1.44 1.45 Apparent attenuation (%) 87.8 87.4 Alcohol (vol %) 5.5
5.35 Alcohol (w/w %) 4.33 4.21 pH 3.51 3.28 Gas pressure
(20.degree. C.) kg/cm 2.35 2.55 Color (.degree.EBC) 1.5 1.7 Total
nitrogen (mg/100 ml) 16 19 BU 11.6 9.5 Polyphenol (mg/L) 45 43 FAN
(mg/L) 10 12
[0164] The foam stability of the malt alcoholic beverage obtained
in 2. above was analyzed by the following method.
[0165] The foam stability analysis was conducted by the NIBEM
method. Upon analysis of the foam stability using a Haffmans Foam
Stability Tester (Table 3), the LOX-F4 barley clearly had higher
foam stability, with a NIBEM value of 21 points higher than the
LOX+F4 barley.
[0166] Also, as a result of measuring the THOD concentration by the
method described in Example 5 above, the THOD content of LOX-F4
exhibited a reduction to less than half of that of LOX+F4 (Table
3).
[0167] These results clearly demonstrated that the malt alcoholic
beverage production method of the invention enable to produce the
malt alcoholic beverage with reduced THOD content and improved foam
retention.
TABLE-US-00003 TABLE 3 Variety LOX + F4 LOX - F4 NIBEM 239 260 THOD
(mg/L) 3.6 1.7
[0168] The malt alcoholic beverage obtained in 2. above was then
subjected to the following sensory test by 13 panelists for
comparison of the flavor stability.
[0169] First, the LOX-F4 and LOX+F4 malt alcoholic beverages were
stored at 37.degree. C. for one week. They were then poured into
cups at ordinary drinking temperature and provided for sensory test
by panelists for evaluation of the off-flavor and total staleness
on a scale of 0-4 (with a higher value representing progressive
aging) (Tables 4A, B).
[0170] As a result, 10 of the 13 panelists assigned lower scores to
LOX-F4 for off-flavor, and therefore LOX-F4 exhibited a lower score
(average) than LOX+F4. The difference according to a paired t test
was determined to be significant at the 5% probability (Table
4A).
[0171] For the total staleness, 11 of the 13 panelists assigned
lower scores to LOX-F4, and therefore LOX-F4 exhibited a lower
score (average) than LOX+F4. The difference according to a paired t
test was determined to be significant at the 5% probability level
(Table 4B).
[0172] The above sensory test and statistical analysis demonstrated
that LOX-F4 had lower off-flavor and total staleness than
LOX+F4.
TABLE-US-00004 TABLE 4A Off-flavor LOX + F4 LOX - F4 Panelist 1 3 2
Panelist 2 1 2 Panelist 3 3 2 Panelist 4 2.5 2.5 Panelist 5 2 2
Panelist 6 2.5 2 Panelist 7 2.5 1.5 Panelist 8 2.5 1 Panelist 9 1
0.5 Panelist 10 2.5 2 Panelist 11 2 1.5 Panelist 12 2.5 2 Panelist
13 2 1.5 Average 2.2 1.7
TABLE-US-00005 TABLE 4B Total staleness LOX + F4 LOX - F4 Panelist
1 2.5 2 Panelist 2 1 2 Panelist 3 3 2 Panelist 4 3 2.5 Panelist 5
2.5 2 Panelist 6 2.5 2 Panelist 7 2.5 1.5 Panelist 8 2.5 1.5
Panelist 9 1 0.5 Panelist 10 2.5 2 Panelist 11 2 1.5 Panelist 12
2.5 2.5 Panelist 13 2 1.5 Average 2.3 1.8
[0173] As a result of measuring the trans-2-nonenal content of the
malt alcoholic beverage obtained in 2 above, LOX-F4 had a lower
trans-2-nonenal content than LOX+F4 before and after storage at
37.degree. C. for one week. The trans-2-nonenal content of LOX-F4
was reduced to approximately 1/3 compared to that of LOX+F4 after
storage (Table 5).
TABLE-US-00006 TABLE 5 trans-2-nonenal conc. LOX + F4 LOX - F4
Before storage 0.02 0.01 After storage 0.35 0.12 (Unit: ppb)
[0174] These results of the sensory test and results of analysis of
the trans-2-nonenal content in the malt alcoholic beverages
demonstrated that the malt alcoholic beverage production method of
the invention enable to produce the malt alcoholic beverages with
improved flavor stability.
[0175] Finally, the body and smoothness of the malt alcoholic
beverage obtained in 2. above were analyzed by sensory test and
with a lipid membrane sensor.
[0176] First, an sensory test was carried out by 13 well-trained
panelists for comparison of the flavor quality. LOX-F4 and LOX+F4
malt alcoholic beverages were provided for sensory test, and the
body and smoothness were evaluated on a scale of 0-4 (with higher
scores for fuller body and better smoothness) (Table 6).
[0177] For body, no significant difference (5% probability level)
was found between LOX-F4 and LOX+F4 (Table 6A).
[0178] For smoothness, 8 of 13 panelists assigned higher scores to
LOX-F4 (Table 6B). LOX-F4 had a higher (average) score than LOX+F4,
and the difference according to a paired t test was determined to
be significant at the 5% probability level.
[0179] These results demonstrated that brewing of malt alcoholic
beverages using LOX-F4 can improve smoothness without affecting
body.
TABLE-US-00007 TABLE 6A Body LOX + F4 LOX - F4 Panelist 1 2 1
Panelist 2 3 2 Panelist 3 3 2.5 Panelist 4 3.5 3.5 Panelist 5 3 3
Panelist 6 2 2 Panelist 7 2 2 Panelist 8 3 2 Panelist 9 3 2
Panelist 10 2.5 2.5 Panelist 11 3 2 Panelist 12 2 3 Panelist 13 2 3
Average 2.6 2.3
TABLE-US-00008 TABLE 6B Smoothness LOX + F4 LOX - F4 Panelist 1 1 2
Panelist 2 1 3 Panelist 3 1.5 3 Panelist 4 3 3 Panelist 5 1 1
Panelist 6 2 2 Panelist 7 2 3 Panelist 8 1.5 3 Panelist 9 1 2
Panelist 10 1.5 2 Panelist 11 2 3 Panelist 12 3 2 Panelist 13 3 2
Average 1.8 2.4
[0180] The body and smoothness were also evaluated using a lipid
membrane sensor, according to the method of Kaneda et al. (Kaneda,
H. et al., J. Biosci. Bioeng., 92, 221-226, 2001) (Table 7).
[0181] The body was evaluated based on adsorption onto the lipid
membrane, and the results showed no statistically significant
difference (5% probability level) between adsorption with LOX-F4
and LOX+F4 (Table 7A).
[0182] The smoothness was evaluated based on the duration of
adsorption onto the lipid membrane (with a higher duration of
adsorption indicating poorer smoothness), and LOX-F4 exhibited a
residue of approximately 1/4 compared to LOX+F4, with a significant
difference at a probability level of 1% (Table 7B).
TABLE-US-00009 TABLE 7A Body Adsorption S.D. LOX + F4 189 4 LOX -
F4 187 3 (Unit: Hz)
(No Significant Difference at 5% Probability Level)
TABLE-US-00010 [0183] TABLE 7B Smoothness Residue S.D. LOX + F4 12
3 LOX - F4 3 3 (Unit: Hz)
(Significant Difference at 1% Probability Level)
[0184] No correlation has been hitherto found between barley LOX-1
activity and THOD production levels in mashing steps (Kobayashi, N.
et al., (2000) J. Biosci. Bioeng. 90:69-73), and it has been
unclear to what extent THOD production is reduced by inhibition of
barley LOX-1. Moreover, it has not been possible in the prior art
to predict whether smoothness can be enhanced as a result of such
inhibition of barley LOX-1. However, the results of the sensory
test and analysis of product body and smoothness using a lipid
membrane sensor in the examples described in the present
specification has demonstrated for the first time that barley
containing the gene claimed herein can be utilized to enhance
product smoothness without affecting product body.
Example 7
Test Brewing of Malt Alcoholic Beverages Using Processed Barley
[0185] 1. Production and Analysis of Wort
[0186] Using processed barley of LOX-F5 and LOX+F5, the next
generations of the lines obtained in Example 3 above, as the
adjunct mashing was carried out with a 50 L scale mashing apparatus
according to the standard mashing methods for Happoshu, low malt
alcoholic beverage (malt content: 24%, processed barley content:
76%). The mashing conditions were as follows.
[0187] 1.2 kg of a commercially available malt for the brew and 3.8
kg of each processed barley was mashed with 20 L of mashing water
according to a diagram of 50.degree. C. for 30 min, 65.degree. C.
for 60 min and 75.degree. C. for 3 min (enzymes such as
.alpha.-amylase and .beta.-glucanase were added because of the high
content of the processed barley). After mashing, wort lautering was
carried out with a lauter tun. 40 L of lautered worts were
obtained.
[0188] 53 g of hop pellets (bitterness unit: 25.6 BU (EBC)) was
added into the obtained lautered worts. After boiling for 80
minutes, the boiled wort was cooled to 10.degree. C. The extract
content of cooled worts were adjusted by water addition to
7.5-7.6%.
[0189] The obtained worts were analyzed according to the EBC
Standard Method. The analysis values are shown in Table 8. As seen
in Table 8, no distinct difference was found between LOX-F5 and
LOX+F5 with regard to parameters.
TABLE-US-00011 TABLE 8 LOX - Variety LOX + F5 F5 Specific gravity
1.0303 1.0296 Extract (%) 7.63 7.46 Real nonfermented extract (%)
2.00 2.06 Real attenuation (%) 73.8 72.4 Apparent nonfermented
extract (%) 0.67 0.71 Apparent attenuation limit (%) 91.2 90.5 pH
5.69 5.71 Color (.degree.EBC) 5.7 6.5 BU 31.7 30.9 Total nitrogen
(mg/100 ml) 45 47 Polyphenol (mg/L) 159 137 FAN (mg/L) 72 72
[0190] 2. Production of Malt Alcoholic Beverage (Happoshu, Low Malt
Alcoholic Beverage)
[0191] The wort obtained in 1. above was transferred to a
steam-sterilized 30 L scale cylindroconical tank, and then yeast
was added to an initial concentration of 30 million cells/ml for
main fermentation at 15.degree. C. When the extract content in the
fermentation liquid fell to 1.3%, it was transferred to a new
similar tank for a storage step. The storage step was carried out
at 13.degree. C. for the first 5 days and then at 0.degree. C. for
2 weeks thereafter.
[0192] After finishing the storage step, fermentation liquid was
supplied to a beer filtration and filling apparatus. The malt
alcoholic beverage was filtered and filled into bottles.
[0193] 3. Analysis of Malt Alcoholic Beverage
[0194] The malt alcoholic beverage obtained in 2 above was analyzed
as follows.
[0195] Analysis according to the EBC Standard Method shows that no
significant difference was found between LOX-F5 and LOX+F5 in terms
of the general analysis parameters other than the lipid oxidation
parameters (Table 9).
TABLE-US-00012 TABLE 9 Variety LOX + F5 LOX - F5 Specific gravity
1.00307 1.00338 Original wort extract (%) 7.77 7.60 Real extract
(%) 2.11 2.14 Real attenuation (%) 73.7 72.7 Apparent extract (%)
0.79 0.87 Apparent attenuation (%) 89.9 88.6 Alcohol (vol %) 3.62
3.49 Alcohol (w/w %) 2.86 2.75 pH 4.58 4.59 Gas pressure
(20.degree. C.) kg/cm 2.29 2.38 Color (.degree.EBC) 4.0 4.2 Total
nitrogen (mg/100 ml) 28 27 BU 16.8 15.3 Polyphenol (mg/L) 111 91
FAN (mg/L) 16 16
[0196] The foam stability of the malt alcoholic beverage obtained
in 2 above was analyzed by the following method.
[0197] The foam stability analysis was conducted by the NIBEM
method. Upon analysis of the foam stability using a Haffmans Foam
Stability Tester (Table 10), the LOX-F5 barley clearly had higher
foam stability, with a NIBEM value of 17 points higher than the
LOX+F5 barley.
[0198] Also, as a result of measuring the THOD concentration by the
method described in Example 5 above, the THOD content in the malt
alcoholic beverage of LOX-F5 exhibited a reduction to less than
half of that of LOX+F5.
[0199] These results clearly demonstrated that the malt alcoholic
beverage production method of the invention enable to produce the
malt alcoholic beverage with reduced THOD content and improved foam
retention.
TABLE-US-00013 TABLE 10 Variety LOX + F5 LOX - F5 NIBEM 279 296
THOD (peak area ratio) 728 237
[0200] The values of THOD indicate the relative values, where the
peak areas of the internal standard are 100.
[0201] The malt alcoholic beverage obtained in 2 above was then
subjected to the following sensory test by 13 panelists for
comparison of the flavor stability. The specific method of the
sensory test is the same as that described in Example 6.
[0202] As a result, 11 of the 13 panelists assigned lower scores to
LOX-F5 for off-flavor, and therefore LOX-F5 exhibited a lower score
(average) than LOX+F5. The difference according to a paired t test
was determined to be significant at the 5% probability (Table
11A).
[0203] For the total staleness, 12 of the 13 panelists assigned
lower scores to LOX-F5, and therefore LOX-F5 exhibited a lower
score (average) than LOX+F5. The difference according to a paired t
test was determined to be significant at the 5% probability level
(Table 11B).
[0204] The above sensory test and statistical analysis demonstrated
that LOX-F5 had lower off-flavor and total staleness than
LOX+F5.
TABLE-US-00014 TABLE 11A Off-flavor LOX + F5 LOX - F5 Panelist 1 2
1.5 Panelist 2 3 2 Panelist 3 3 2 Panelist 4 2 1.5 Panelist 5 3 2
Panelist 6 2 1 Panelist 7 2.5 3 Panelist 8 2 1 Panelist 9 2.5 2
Panelist 10 2 1 Panelist 11 3 2 Panelist 12 2.5 1.5 Panelist 13 2 3
Average 2.4 1.8
TABLE-US-00015 TABLE 11B Total staleness LOX + F5 LOX - F5 Panelist
1 2 1.5 Panelist 2 3 1 Panelist 3 3.5 1.5 Panelist 4 2 1.5 Panelist
5 3 2 Panelist 6 2 1 Panelist 7 2.5 3 Panelist 8 2 1 Panelist 9 3 2
Panelist 10 2 1 Panelist 11 3 2 Panelist 12 2.5 1.5 Panelist 13 3
1.5 Average 2.6 1.6
[0205] As a result of measuring the trans-2-nonenal content of the
malt alcoholic beverage obtained in 2 above before and after
storage at 37.degree. C. for one week, LOX-F5 had a similar
trans-2-nonenal content as LOX+F5 before the storage. The
trans-2-nonenal content of LOX-F5 was reduced to approximately 1/2
compared to that of LOX+F5 after the storage (Table 12).
TABLE-US-00016 TABLE 12 trans-2-nonenal conc. LOX + F5 LOX - F5
Before storage 0.06 0.06 After storage 0.16 0.09 (Unit: ppb)
Example 8
Test Brewing of Malt Alcoholic Beverages
[0206] 1. Production and Analysis of Wort
[0207] The LOX-F4 malt and LOX+F4 malt obtained by the same method
as that described in Example 4 above were mashed with a 50 L scale
mashing apparatus according to the standard mashing methods for
beer (malt content: 71%). The mashing conditions were as
follows.
[0208] 5.0 kg of the test malt above and 2.0 kg of adjunct (corn
starch, corn grits and broken rice) were mashed with 23 L of
mashing water according to a diagram of 50.degree. C. for 20 min,
65.degree. C. for 40 min and 75.degree. C. for 3 min. After
mashing, wort lautering was carried out with a lauter tun. 40 L of
lautered worts were obtained.
[0209] 40 g of hop pellets (bitterness unit: 44.9 BU (EBC)) was
added into the obtained lautered worts. After boiling for 90
minutes, the boiled wort was cooled to 10.degree. C. The extract
content of cooled worts were adjusted by water addition to
10.8-11.1%.
[0210] The obtained worts were analyzed according to the EBC
Standard Method. The analysis values are shown in Table 13. As seen
in Table 13, no distinct difference was found between LOX-F4 and
LOX+F4 with regard to parameters.
TABLE-US-00017 TABLE 13 Variety LOX + F4 LOX - F4 Specific gravity
1.0444 1.0433 Extract (%) 11.05 10.79 Real nonfermented extract (%)
3.05 3.05 Real attenuation (%) 72.4 71.7 Apparent nonfermented
extract (%) 1.25 1.31 Apparent attenuation limit (%) 88.7 87.9 pH
5.71 5.68 Color (.degree.EBC) 6.3 6.5 BU 38.0 38.2 Total nitrogen
(mg/100 ml) 77 78 Polyphenol (mg/L) 150 147 FAN (mg/L) 153 148
[0211] 2. Production of Malt Alcoholic Beverage (Beer)
[0212] The wort obtained in 1 above was transferred to a
steam-sterilized 30 L scale cylindroconical tank, and then yeast
was added to an initial concentration of 15 million cells/ml for
main fermentation at 10.5.degree. C. When the extract content in
the fermentation liquid fell to 2.5%, it was transferred to a new
similar tank for a storage step. The storage step was carried out
at 8.degree. C. for the first 8 days and then at 0.degree. C. for 2
weeks thereafter.
[0213] After finishing the storage step, fermentation liquid was
supplied to a beer filtration and filling apparatus. The malt
alcoholic beverage was filtered and filled into bottles.
[0214] 3. Analysis of Malt Alcoholic Beverage
[0215] The foam stability of the malt alcoholic beverage obtained
in 2 above was analyzed by the following method. The foam stability
analysis was conducted by the NIBEM method. Upon analysis of the
foam stability using a Haffmans Foam Stability Tester (Table 14),
the LOX-F4 barley clearly had higher foam stability, with a NIBEM
value of 30 points higher than the LOX+F4 barley.
[0216] Also, as a result of measuring the THOD concentration by the
method described in Example 5 above, the THOD content in the malt
alcoholic beverage of LOX-F4 exhibited a reduction to less than
half of that of LOX+F4.
[0217] These results clearly demonstrated that the malt alcoholic
beverage production method of the invention enable to produce the
malt alcoholic beverage with reduced THOD content and improved foam
retention.
TABLE-US-00018 TABLE 14 Variety LOX + F4 LOX - F4 NIBEM 273 303
THOD (peak area ratio) 499 221
[0218] The values of THOD indicate the relative values, where the
peak areas of the internal standard are 100.
[0219] The malt alcoholic beverage obtained in 2 above was then
subjected to the following sensory test by 13 panelists for
comparison of the flavor stability. The specific method of the
sensory test is the same as that described in Example 6.
[0220] As a result, 11 of the 13 panelists assigned lower scores to
LOX-F4 for off-flavor, and therefore LOX-F4 exhibited a lower score
(average) than LOX+F4. The difference according to a paired t test
was determined to be significant at the 5% probability (Table
15A).
[0221] For the total staleness, 12 of the 13 panelists assigned
lower scores to LOX-F4, and therefore LOX-F4 exhibited a lower
score (average) than LOX+F4. The difference according to a paired t
test was determined to be significant at the 5% probability level
(Table 15B).
[0222] The above sensory test and statistical analysis demonstrated
that LOX-F4 had lower off-flavor and total staleness than
LOX+F4.
TABLE-US-00019 TABLE 15A Off-flavor LOX + F4 LOX - F4 Panelist 1
2.5 2 Panelist 2 3 3.5 Panelist 3 3.5 2 Panelist 4 2.5 2 Panelist 5
1.5 1 Panelist 6 3 1 Panelist 7 2.5 3 Panelist 8 3 2 Panelist 9 2
1.5 Panelist 10 2 1 Panelist 11 3 1.5 Panelist 12 1.5 1 Panelist 13
2 1 Average 2.5 1.7
TABLE-US-00020 TABLE 15B Total staleness LOX + F4 LOX - F4 Panelist
1 3 2 Panelist 2 3 3.5 Panelist 3 3 1.5 Panelist 4 2.5 2 Panelist 5
1.5 1 Panelist 6 3 1 Panelist 7 3 2.5 Panelist 8 3 2 Panelist 9 2 1
Panelist 10 2 1 Panelist 11 3 1 Panelist 12 1.5 1 Panelist 13 2 1
Average 2.5 1.6
[0223] These results of the sensory test and results of analysis of
the trans-2-nonenal content in the malt alcoholic beverages
demonstrated that the malt alcoholic beverage production method of
the invention enable to produce the malt alcoholic beverages with
improved flavor stability.
INDUSTRIAL APPLICABILITY
[0224] It is possible to provide a LOX-1 mutant gene which is
useful for production of malt alcoholic beverages exhibiting
improved flavor stability and foam stability without gene
manipulation, a selection method for LOX-1 deficient barley,
materials for malt alcoholic beverages derived from barley obtained
by the selection method, and a method for production of malt
alcoholic beverages using the materials for malt alcoholic
beverages.
Sequence CWU 1
1
141240DNAHordeum vulgare 1ctcgccaagg cctacgtcgc cgtcaatgac
tccgggtggc accagctcgt cagccactgg 60tacgttctcc acggtcgatg tgattcagtc
agtcgatgca caacaactga tcgaaatatg 120attgattgaa acgcgcaggc
tgaacactca cgcggtgatg gagccgttcg tgatctcgac 180gaaccggcac
cttagcgtga cgcacccggt gcacaagctg ctgagcccgc actaccgcga
2402240DNAHordeum vulgare 2ctcgccaagg cctacgtcgc cgtcaatgac
tccgggtggc accagctcgt cagccactga 60tacgttctcc acggtcgatg tgattcagtc
agtcgatgca caacaactga tcgaaatatg 120attgattgaa acgcgcaggc
tgaacactca cgcggtgatg gagccgttcg tgatctcgac 180gaaccggcac
cttagcgtga cgcacccggt gcacaagctg ctgagcccgc actaccgcga
240324DNAArtificialprimer 3ggagaggagg ccaagaacaa gatg
24419DNAArtificialprimer 4ggttgccgat ggcttagat
19521DNAArtificialprimer 5cacgtcgccg tccgatccat c
21620DNAArtificialprimer 6ccatcacgca gggcatcctg
20720DNAArtificialprimer 7gcgttgatga gcgtctgccg
20824DNAArtificialprimer for LOX-1 with BamHI site 8ggatccatgc
tgctgggagg gctg 24925DNAArtificialprimer for LOX-1 with HindIII
site 9aagcttttag atggagatgc tgttg 25102668DNAHordeum vulgare
10atgctgctgg gagggctgat cgacaccctc acgggggcga acaagagcgc ccggctcaag
60ggcacggtgg tgctcatgcg caagaacgtg ctggacctca acgacttcgg cgccaccatc
120atcgacggca tcggcgagtt cctcggcaag ggcgtcacct gccagcttat
cagctccacc 180gccgtcgacc aagacaacgg cggtcgcggg aaggtgggcg
cggaggcgga gctggagcag 240tgggtgacga gcctgccgtc gctgacgacg
ggggagtcca agttcggcct caccttcgac 300tgggaggtgg agaagctcgg
ggtgccgggc gccatcgtcg tcaacaacta ccacagctcc 360gagttcctgc
ttaaaaccat caccctccac gacgtccccg gccgcagcgg caacctcacc
420ttcgtcgcca actcatggat ctaccccgcc gccaactacc gatacagccg
cgtcttcttc 480gccaacgaca cgtacctgcc gagccagatg ccggcggcgc
tgaagccgta ccgcgacgac 540gagctccgga acctgcgtgg cgacgaccag
cagggcccgt accaggagca cgaccgcatc 600taccgctacg acgtctacaa
cgacctcggc gagggccgcc ccatcctcgg cggcaactcc 660gaccaccctt
acccgcgccg cggccgcacg gagcgcaagc ccaacgccag cgacccgagc
720ctggagagcc ggctgtcgct gctggagcag atctacgtgc cgcgggacga
gaagttcggc 780cacctcaaga cgtccgactt cctgggctac tccatcaagg
ccatcacgca gggcatcctg 840ccggccgtgc gcacctacgt ggacaccacc
cccggcgagt tcgactcctt ccaggacatc 900atcaacctct atgagggcgg
catcaagctg cccaaggtgg ccgccctgga ggagctccgt 960aagcagttcc
cgctccagct catcaaggac ctcctccccg tcggcggcga ctccctgctt
1020aagctccccg tgccccacat catccaggag aacaagcagg cgtggaggac
cgacgaggag 1080ttcgcacggg aggtgctcgc cggcgtcaac ccggtcatga
tcacgcgtct cacggagttc 1140ccgccaaaaa gtagtctgga ccctagcaag
tttggtgacc acaccagcac catcacggcg 1200gagcacatag agaagaacct
cgagggcctc acggtgcagc aggcgctgga aagcaacagg 1260ctgtacatcc
ttgatcacca tgaccggttc atgccgttcc tgatcgacgt caacaacctg
1320cccggcaact tcatctacgc cacgaggacc ctcttcttcc tgcgcggcga
cggcaggctc 1380acgccgctcg ccatcgagct gagcgagccc atcatccagg
gcggccttac cacggccaag 1440agcaaggttt acacgccggt gcccagcggc
tccgtcgaag gctgggtgtg ggagctcgcc 1500aaggcctacg tcgccgtcaa
tgactccggg tggcaccagc tcgtcagcca ctgatacgtt 1560ctccacggtc
gatgtgattc agtcagtcga tgcacaacaa ctgatcgaaa tatgattgat
1620tgaaacgcgc aggctgaaca ctcacgcggt gatggagccg ttcgtgatct
cgacgaaccg 1680gcaccttagc gtgacgcacc cggtgcacaa gctgctgagc
ccgcactacc gcgacaccat 1740gaccatcaac gcgctggcgc ggcagacgct
catcaacgcc ggcggcatct tcgagatgac 1800ggtgttcccg ggcaagttcg
cgttggggat gtcggccgtg gtgtacaagg actggaagtt 1860caccgagcag
ggactgccgg acgatctcat caagaggggc atggcggtgg aggacccgtc
1920gagcccgtac aaggtgcggt tgctggtgtc ggactacccg tacgcggcgg
acgggctggc 1980gatctggcac gccattgagc agtacgtgag cgagtacctg
gccatctact acccgaacga 2040cggcgtgctg cagggcgata cggaggtgca
ggcgtggtgg aaggagacgc gcgaggtcgg 2100gcacggcgac ctcaaggacg
ccccatggtg gcccaagatg caaagtgtgc cggagctggc 2160caaggcgtgc
accaccatca tctggatcgg gtcggcgctg catgcggcag tcaacttcgg
2220gcagtacccc tacgcggggt tcctcccgaa ccggccgacg gtgagccggc
gccgcatgcc 2280ggagcccggc acggaggagt acgcggagct ggagcgcgac
ccggagcggg ccttcatcca 2340caccatcacg agccagatcc agaccatcat
cggcgtgtcg ctgctggagg tgctgtcgaa 2400gcactcctcc gacgagctgt
acctcgggca gcgggacacg ccggagtgga cctcggaccc 2460aaaggccctg
gaggtgttca agcggttcag cgaccggctg gtggagatcg agagcaaggt
2520ggtgggcatg aaccatgacc cggagctcaa gaaccgcaac ggcccggcta
agtttcccta 2580catgctgctc taccccaaca cctccgacca caagggcgcc
gctgccgggc ttaccgccaa 2640gggcatcccc aacagcatct ccatctaa
2668114393DNAHordeum vulgare 11cacgtcgccg tccgatccat ctctccaaag
ccgagcgcca caccaccggg accggacccg 60gaccggccta taaattgccc ggaccgagct
gcaagcagct cctcacacac actcacgcaa 120cacacatcca tcttcactga
aaagtgaaaa acagtgtgct ggtgccattg gttggagcag 180tgaaagcgag
gagaggaggc caagaacaag atgctgctgg gagggctgat cgacaccctc
240acgggggcga acaagagcgc ccggctcaag ggcacggtgg tgctcatgcg
caagaacgtg 300ctggacctca acgacttcgg cgccaccatc atcgacggca
tcggcgagtt cctcggcaag 360ggcgtcacct gccagcttat cagctccacc
gccgtcgacc aaggtaatca ctaccctcct 420ccggccttct cctctgttta
caagatatag tatttctttc gtgtgggccg gcggccatgg 480atggatggat
gtgtctggat cggctaaaga agataggata gctagccctg gccggtcgtc
540tttacctgag catgggcata tgccatcgaa aaaagagaca acagcatgca
tgcatggtgc 600gcgcaccaga ccacgcagag caccggatgc tcgagacaaa
gcaacacaac aagcaaggac 660gacacgtcaa aagcaacaca acaagcaagg
acggcacgtc aaaagcaaca caaacctaaa 720ctaaagcaca aagacgtaag
agcaagcaca caatcagcag gctataaaca gttgtcatca 780aaaacaacgc
tggaagagag agagaaggaa ggaagtagta gccatgaaaa attaaatcac
840cgggcgttgc tctttgccca acaattaatc aagcagggta cgtggcatgt
atagttcttg 900taagtaaact aagcatgtga tatgagaagg tacgtggtgg
tgcagacaac ggcggtcgcg 960ggaaggtggg cgcggaggcg gagctggagc
agtgggtgac gagcctgccg tcgctgacga 1020cgggggagtc caagttcggc
ctcaccttcg actgggaggt ggagaagctc ggggtgccgg 1080gcgccatcgt
cgtcaacaac taccacagct ccgagttcct gcttaaaacc atcaccctcc
1140acgacgtccc cggccgcagc ggcaacctca ccttcgtcgc caactcatgg
atctaccccg 1200ccgccaacta ccgatacagc cgcgtcttct tcgccaacga
cgtgcgtgga ttttcctcta 1260ctttcctctc ctttcatttt caccgccttc
gtcattcatg gtcgatcatt aagtcttgcc 1320aggacaatag atgatgagct
aggagtggtt accacttagc agtacgtaca ttatttattc 1380cgtgttggta
gaaaaggata tggtttggtg cagatcgaca caagattgaa tgaaagttgc
1440accgtggcac cgtggcagcg tggtaggtga aaataactgt tgcacggatc
cacccacatg 1500attgttttca tgaataaact ttttaaggat gtgtctagcc
acatctagat gcatgtcaca 1560taattattgc ataccaaaac gattaaatta
agcataaaaa gaaaaggaaa aaaatactca 1620catatctcga cgtaagatca
atgatatagt atttagatat gcaatattta tcttacatct 1680aaacctttct
tcattcctaa atataagaca tttgtaagat ttcactatgg acaacatacg
1740aaacaaaatc agtggatctc tctatgcatt cattatgtag tctataataa
aatctttaaa 1800agatcgtata ttttgcaacg gagggagtaa aacataactt
tttaatagta atgttgcacg 1860gctccacact cgcagacgta cctgccgagc
cagatgccgg cggcgctgaa gccgtaccgc 1920gacgacgagc tccggaacct
gcgtggcgac gaccagcagg gcccgtacca ggagcacgac 1980cgcatctacc
gctacgacgt ctacaacgac ctcggcgagg gccgccccat cctcggcggc
2040aactccgacc acccttaccc gcgccgcggc cgcacggagc gcaagcccaa
cgccagcgac 2100ccgagcctgg agagccggct gtcgctgctg gagcagatct
acgtgccgcg ggacgagaag 2160ttcggccacc tcaagacgtc cgacttcctg
ggctactcca tcaaggccat cacgcagggc 2220atcctgccgg ccgtgcgcac
ctacgtggac accacccccg gcgagttcga ctccttccag 2280gacatcatca
acctctatga gggcggcatc aagctgccca aggtggccgc cctggaggag
2340ctccgtaagc agttcccgct ccagctcatc aaggacctcc tccccgtcgg
cggcgactcc 2400ctgcttaagc tccccgtgcc ccacatcatc caggagaaca
agcaggcgtg gaggaccgac 2460gaggagttcg cacgggaggt gctcgccggc
gtcaacccgg tcatgatcac gcgtctcacg 2520gtgagtcagc gattatttgt
tcattgtgtg tgtatggtgt ccatggtgag aaagtgcaga 2580tcttgatttg
cgttgggtcg catgcacgca tgctgcatgc atgcaggagt tcccgccaaa
2640aagtagtctg gaccctagca agtttggtga ccacaccagc accatcacgg
cggagcacat 2700agagaagaac ctcgagggcc tcacggtgca gcaggtaatt
ggtccaagcc atcgacatca 2760actatgattt acctaggagt aattggtagc
tgtagataat ttggcttcgt tgcaattaat 2820ttgatgctgg ccgatcaagt
gatcgtattg ggtttgaaat ttgcaggcgc tggaaagcaa 2880caggctgtac
atccttgatc accatgaccg gttcatgccg ttcctgatcg acgtcaacaa
2940cctgcccggc aacttcatct acgccacgag gaccctcttc ttcctgcgcg
gcgacggcag 3000gctcacgccg ctcgccatcg agctgagcga gcccatcatc
cagggcggcc ttaccacggc 3060caagagcaag gtttacacgc cggtgcccag
cggctccgtc gaaggctggg tgtgggagct 3120cgccaaggcc tacgtcgccg
tcaatgactc cgggtggcac cagctcgtca gccactgata 3180cgttctccac
ggtcgatgtg attcagtcag tcgatgcaca acaactgatc gaaatatgat
3240tgattgaaac gcgcaggctg aacactcacg cggtgatgga gccgttcgtg
atctcgacga 3300accggcacct tagcgtgacg cacccggtgc acaagctgct
gagcccgcac taccgcgaca 3360ccatgaccat caacgcgctg gcgcggcaga
cgctcatcaa cgccggcggc atcttcgaga 3420tgacggtgtt cccgggcaag
ttcgcgttgg ggatgtcggc cgtggtgtac aaggactgga 3480agttcaccga
gcagggactg ccggacgatc tcatcaagag gtacgtacct ggtaaatgtt
3540atgaatgtgt aaaacaaatt gggcgtctcg ctcactgaca ggaacgtggt
aaaaaaaatg 3600caggggcatg gcggtggagg acccgtcgag cccgtacaag
gtgcggttgc tggtgtcgga 3660ctacccgtac gcggcggacg ggctggcgat
ctggcacgcc attgagcagt acgtgagcga 3720gtacctggcc atctactacc
cgaacgacgg cgtgctgcag ggcgatacgg aggtgcaggc 3780gtggtggaag
gagacgcgcg aggtcgggca cggcgacctc aaggacgccc catggtggcc
3840caagatgcaa agtgtgccgg agctggccaa ggcgtgcacc accatcatct
ggatcgggtc 3900ggcgctgcat gcggcagtca acttcgggca gtacccctac
gcggggttcc tcccgaaccg 3960gccgacggtg agccggcgcc gcatgccgga
gcccggcacg gaggagtacg cggagctgga 4020gcgcgacccg gagcgggcct
tcatccacac catcacgagc cagatccaga ccatcatcgg 4080cgtgtcgctg
ctggaggtgc tgtcgaagca ctcctccgac gagctgtacc tcgggcagcg
4140ggacacgccg gagtggacct cggacccaaa ggccctggag gtgttcaagc
ggttcagcga 4200ccggctggtg gagatcgaga gcaaggtggt gggcatgaac
catgacccgg agctcaagaa 4260ccgcaacggc ccggctaagt ttccctacat
gctgctctac cccaacacct ccgaccacaa 4320gggcgccgct gccgggctta
ccgccaaggg catccccaac agcatctcca tctaatttaa 4380gccatcggca acc
43931260DNAHordeum vulgaremisc_feature(30)..(30)G in the authentic
LOX-1 gene 12tccgggtggc accagctcgt cagccactgg tacgttctcc acggtcgatg
tgattcagtc 601360DNAHordeum vulgaremisc_feature(30)..(30)A in the
mutant LOX-1 gene 13tccgggtggc accagctcgt cagccactga tacgttctcc
acggtcgatg tgattcagtc 60149PRTHordeum vulgare 14Ser Gly Trp His Gln
Leu Val Ser His1 5
* * * * *