U.S. patent application number 14/694757 was filed with the patent office on 2015-08-13 for yeast mutant and yeast extract.
The applicant listed for this patent is KOHJIN CO., LTD.. Invention is credited to Shogo FURUE, Setsuko HIRAKURA, Sakiko IKEDA, Ryo IWAKIRI, Hiroko KODERA, Hirokazu MAEKAWA, Naohisa MASUO, Masahiro NISHIDA, Masanori UCHIDA.
Application Number | 20150225690 14/694757 |
Document ID | / |
Family ID | 41135398 |
Filed Date | 2015-08-13 |
United States Patent
Application |
20150225690 |
Kind Code |
A1 |
IWAKIRI; Ryo ; et
al. |
August 13, 2015 |
YEAST MUTANT AND YEAST EXTRACT
Abstract
A natural yeast extract which is rich in glutamic acid and
therefore has an impact at first taste. Further, provided is a
yeast extract which is also rich in 5'-guanylic acid or 5'-inosinic
acid and therefore has strong umami. Further, provided is a yeast
mutant capable of accumulating a large amount of glutamic acid,
glutamine and ribonucleic acid for obtaining such a yeast extract.
A yeast mutant to which resistance to organic acids and analogues
thereof has been imparted by inducing spontaneous mutation,
accumulates a significant amount, i.e., 10% by weight or more of
the total of free glutamic acid and glutamine in the cell, and
further accumulates 5% by weight or more of a ribonucleic acid. The
yeast extract produced by using this strain contains 20% by weight
or more of L-glutamic acid, and further contains 3% by weight or
more of 5'-IG.
Inventors: |
IWAKIRI; Ryo; (Oita-shi,
JP) ; MAEKAWA; Hirokazu; (Saika-shi, JP) ;
MASUO; Naohisa; (Saika-shi, JP) ; FURUE; Shogo;
(Saika-shi, JP) ; KODERA; Hiroko; (Saika-shi,
JP) ; HIRAKURA; Setsuko; (Saika-shi, JP) ;
NISHIDA; Masahiro; (Saika-shi, JP) ; UCHIDA;
Masanori; (Saika-shi, JP) ; IKEDA; Sakiko;
(Saitama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOHJIN CO., LTD. |
Tokyo |
|
JP |
|
|
Family ID: |
41135398 |
Appl. No.: |
14/694757 |
Filed: |
April 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12810624 |
Aug 4, 2010 |
|
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|
PCT/JP2009/056168 |
Mar 26, 2009 |
|
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14694757 |
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Current U.S.
Class: |
435/255.4 ;
435/255.1 |
Current CPC
Class: |
A23L 33/14 20160801;
C12R 1/72 20130101; C12N 15/01 20130101; C12N 1/16 20130101; A23L
27/10 20160801; A23V 2002/00 20130101; A23L 33/145 20160801; C12P
19/32 20130101; A23L 31/15 20160801; C12P 13/14 20130101 |
International
Class: |
C12N 1/16 20060101
C12N001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2008 |
JP |
2008-091709 |
Apr 22, 2008 |
JP |
2008-111232 |
Claims
1. A yeast mutant, wherein free glutamic acid and glutamine are
accumulated in a total amount of 10% or more, based on a weight of
a dry cell.
2. The yeast mutant according to claim 1, wherein ribonucleic acid
(RNA) is further accumulated in an amount of 5% or more, based on a
weight of a dry cell.
3. The yeast mutant according to claim 1, wherein the yeast mutant
is tolerant to ethyl bromopyruvate.
4. The yeast mutant according to claim 1, wherein the yeast mutant
is tolerant to ethyl bromopyruvate and 2-oxoglutaric acid.
5. The yeast mutant according to claim 1, wherein the yeast mutant
is Candida utilis 36D61 (accession number: FERM BP-11103).
6. A yeast extract produced from the yeast mutant as claimed in
claim 1, wherein the yeast extract contains L-glutamic acid in an
amount of 20% by weight or more converted into an amount of a
sodium salt thereof.
7. The yeast extract according to claim 6, wherein the yeast
extract further contains 5'-guanylic acid and 5'-inosinic acid or
5'-adenylic acid in a total amount of 3% by weight or more.
Description
[0001] This is a continuation of application Ser. No. 12/810,624
filed Aug. 4, 2010, which is a National Stage Application of
PCT/JP2009/056168 filed Mar. 26, 2009, and claims the benefit of
Japanese Patent Application Nos. 2008-091709 and 2008-111232 filed
on Mar. 31, 2008 and Apr. 22, 2008. The entire disclosures of the
prior applications are hereby incorporated by reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to: a yeast mutant
accumulating free L-glutamic acid and L-glutamine, and ribonucleic
acid in significant amounts in a cell; and a yeast extract obtained
using the yeast containing natural L-glutamic acid in a high
concentration and exhibiting good taste (umami) having a strong
first taste.
BACKGROUND ART
[0003] Following in addition to the trend towards healthy products,
natural products, and additive-free products, problems on the
safety of foods, such as bovine spongiform encephalopathy (BSE),
expectations for a yeast extract that is a natural seasoning have
been rapidly increased. As a yeast extract, a nucleic acid-based
extract in which a rich taste or an aftertaste is strengthened, and
a peptide-based extract imparting a body or thickness of the taste
to foods, which are recently attracting attentions, are actively
developed.
[0004] On the other hand, as a method of adding a yeast extract in
which umami having first taste is strengthened and which is
represented by glutamic acid, a method of adding inexpensive
purified sodium glutamate (MSG) from the outside is well known and
examples for developing the yeast extract itself are not so
many.
[0005] For example, as shown in Patent Document 1, there is a
method of producing a yeast extract in which a glutamic acid
content is enhanced by enlarging an amount of free glutamic acid
accumulated in a yeast through mutation breeding, or as shown in
Patent Document 2, there is also an attempt to enhance a glutamic
acid content by keeping an extract extraction rate low and further
by improving the production method such as treating with an
enzyme.
[0006] However, the glutamic acid content in thus produced yeast
extract is 14.5% or less converted into the content of a sodium
salt thereof. Since the value is lower than that of a wheat gluten
hydrolysate that is typically used as a glutamic acid-containing
seasoning, it is not at a thoroughly satisfying level as for
tastes.
[0007] Further, there are such problems as the lowering of the cell
yield per sugar by a mutation breeding in Patent Document 1, and
the limitation of the production method due to a low amount of
glutamic acid accumulated in a yeast in Patent Document 2.
[0008] For developing a yeast extract having a high glutamic acid
content by various production methods corresponding to a production
design with productivity capable of meeting the industrial
application, it is necessary to spectacularly enlarge the amount of
glutamic acid accumulated in a yeast without lowering the
productivity of the cell.
[0009] While sodium glutamate produced by using bacteria is
accumulated outside of a cell, in the production of a yeast
extract, glutamic acid is necessary to be accumulated within a
yeast cell. However, it is conjectured that in the cell, a
metabolism control system such as a feedback inhibition acts, so
that it is not easy to accumulate glutamic acid in a high
concentration within the cell.
[0010] There is such a report that by an analysis of the pool of
free amino acids in a yeast cell, it is found that glutamic acid is
present in large quantity in a cytoplasm and glutamine is present
in large quantity in a vacuole (Eur. J. Biochem. Vol. 108, p. 439
(1980)). Thus, if a yeast accumulating in a high concentration, not
only glutamic acid, but also glutamine that is
differentially-localized can be developed, by an enzymatic
conversion of glutamine, which is a usual method, the production of
a yeast extract in which the glutamic acid content is extremely
enhanced, is possible.
[0011] In Patent Document 3, by a gene recombination of
Saccharomyces cerevisiae which is a monoploid laboratory yeast, a
recombinant strain accumulating free glutamic acid and glutamine in
a total amount of 5.4% is built and from this yeast, a yeast
extract having a glutamic acid content of 16.2% is produced.
However, even by using a gene recombination, yet the total amount
of glutamic acid and glutamine is low and it is not more than that
the glutamic acid content of the obtained yeast extract is a little
over 20% converted into the content of a sodium salt thereof. In
addition, the similar gene recombination by industrial yeasts with
high ploidy and with a lowered sporulation ability or by Candida
utilis yeast having no sporulation ability is difficult. Further,
it is not a desirable method at present to apply the produced gene
recombinant to foods, because there are barriers such as the
control of a law and a resistance of consumers.
[0012] Thus, there is such a necessity that for producing a yeast
extract containing glutamic acid in a high concentration, the gene
recombination is not used, and a yeast accumulating free glutamic
acid and glutamine in a high concentration in a cell by a natural
mutation is bred.
[0013] Further, a synergistic effect of glutamic acid and
5'-guanylic acid or 5'-inosinic acid for the taste is well known
and for producing a yeast extract having strong umami, it is
preferred to use a strain containing besides the above amino acids,
ribonucleic acid (RNA), which is a raw material for nucleotide,
also in a high concentration.
Patent Document 1: Japanese Patent Application Publication No.
JP-A-9-294581 Patent Document 2: Japanese Patent Application
Publication No. JP-A-2006-129835 Patent Document 3: Japanese Patent
Application Publication No. JP-A-2002-171961
DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
[0014] In view of the above, the present invention provides a
natural yeast extract having a high-impact first taste by
containing glutamic acid in a large amount. In addition, the
present invention provides a yeast extract having strong umami by
containing 5'-guanylic acid or 5'-inosinic acid in a large amount.
Further, the present invention provides a yeast mutant accumulating
glutamic acid, glutamine and ribonucleic acid in a large amount for
obtaining such a yeast extract.
[0015] The present inventors have made extensive and intensive
studies toward eliminating the above drawbacks. As a result, the
inventors find that a yeast to which organic acid-tolerance or
organic acid analogue-tolerance is imparted by inducing mutation
surprisingly accumulates free glutamic acid and glutamine in such a
significant total amount as 10% by weight or more in the cell and
further accumulates ribonucleic acid in an amount of 5% by weight
or more, and completed the present invention.
[0016] In other words, the present invention provides the following
aspects:
(1) a yeast mutant, in which free glutamic acid and glutamine are
accumulated in a total amount of 10% or more, based on a weight of
a dry cell; (2) the yeast mutant according to (1), in which
ribonucleic acid (RNA) is further accumulated in an amount of 5% or
more, based on a weight of a dry cell; (3) the yeast mutant
according to (1) or (2), in which the yeast mutant is tolerant to
ethyl bromopyruvate; (4) the yeast mutant according to (1) or (2),
in which the yeast mutant is tolerant to ethyl bromopyruvate and
2-oxoglutaric acid; (5) the yeast mutant according to any one of
(1) to (4), in which the yeast mutant is Candida utilis 36D61
(accession number: FERM BP-11103); (6) a yeast extract produced
from the yeast mutant as described in any one of (1) to (5), in
which the yeast extract contains L-glutamic acid in an amount of
20% by weight or more converted into an amount of a sodium salt
thereof; and (7) the yeast extract according to (6), in which the
yeast extract further contains 5'-guanylic acid and 5'-inosinic
acid or 5'-adenylic acid in a total amount of 3% by weight or
more.
Effects of the Invention
[0017] Although the yeast mutant according to an embodiment of the
present invention is a strain obtained without performing a gene
recombination, it accumulates glutamine and glutamic acid in
significant amounts, further accumulates also ribonucleic acid in a
large amount. A yeast extract obtained using this strain exhibits
umami having a strong first taste due to a high glutamic acid
content and further exhibits a strong umami due to a synergistic
effect of inosinic acid and guanylic acid.
[0018] By adding such a yeast extract to foods, satisfying umami
can be imparted to foods with a small amount of the yeast extract
and without adding glutamic acid from the outside, and particularly
umami property having a strong first taste can be imparted.
[0019] Here, "first taste" according to an embodiment of the
present invention means a taste spreading swiftly in the mouth at
the moment a food is contained in the mouth, a taste corresponding
mainly to amino acids, not to a sugar or a common salt, which is
based on the sensitive time of monosodium glutamate (MSG). In
addition, "continuity" means the length of time in which a taste
felt after the first taste is retained.
BEST MODES FOR CARRYING OUT THE INVENTION
[0020] As the yeast used according to an embodiment of the present
invention, an edible yeast is preferred and examples thereof
include a yeast belonging to Saccharomyces, a yeast belonging to
Kluyveromyces, a yeast belonging to Candida and a yeast belonging
to Pichias. Preferably recommended is a yeast belonging to Candida,
that is, Candida utilis known to have high ribonucleic
acid-accumulating function. It is also possible to apply another
yeast that has improved according to a procedure shown in Japanese
Examined Patent Application Publication No. JP-B-07-93871 to
enlarge the amount of accumulated ribonucleic acid.
[0021] Although the present invention has characteristic in terms
of using a yeast accumulating free glutamic acid, glutamine and
ribonucleic acid in a high concentration for producing a yeast
extract containing glutamic acid exhibiting a strong first taste in
a high concentration, such a yeast can be obtained by using a
mutagenizing agent such as ultraviolet rays, X-rays, nitrous acid,
nitrosoguanidine and ethylmethane sulfonate and by selecting a
strain capable of growing on a synthetic medium containing organic
acids or organic acid analogues in a concentration with which the
parent strain thereof cannot grow. As the organic acid analogue
used, those related to the biosynthesis of glutamic acid are
preferred. Specific examples of the organic acid analogue include
ethyl bromopyruvate (hereinafter, abbreviated to BPE) and
2-oxoglutaric acid (hereinafter, abbreviated to 2OG). With respect
to the former, although bromopyruvic acid known as a pyruvic acid
analogue is used for causing a yeast to accumulate glutamic acid in
a high concentration in Japanese Patent Application Publication No.
JP-A-9-313169, for breeding a Candida yeast, an ethyl compound
thereof, that is, ethyl bromopyruvate is effective. The latter is
known as an inhibitor of citric acid synthase and as shown in
Japanese Patent Application Publication No. JP-A-2001-103958, 2OG
is used in improving a yeast accumulating malic acid or succinic
acid in the outside of a cell for improving the flavor of sake.
However, a yeast Candida utilis has tolerance to such a high
concentration as more than 2,000 ppm of this agent, so that it is
difficult to obtain a tolerance strain by using an individual
agent. By using both agents in combination, it is possible to breed
a yeast accumulating free glutamic acid and glutamine in a high
concentration more effectively than using the agent individually.
Further, by repeating a mutation treatment to impart tolerance to
both agents in a high concentration, a yeast accumulating free
glutamic acid and glutamine in a total amount of 10% by weight or
more can be obtained.
[0022] Thus isolated Candida utilis 36D61 strain is extremely
preferred for the production of a yeast extract accumulating free
glutamic acid and glutamine in a total amount of 14% by weight and
ribonucleic acid in an amount of 9% by weight, containing glutamic
acid in a high concentration and having a strong first taste, or
for the production of a yeast extract containing glutamine.
[0023] For example, as shown in Japanese Patent Application
Publication No. JP-A-9-294581, there is such a fear that the
productivity of the cell may be lowered by a mutation breeding,
however, the yeast mutant used in the embodiment of the present
invention maintains a high cell yield per sugar and there is no
problem with respect to the industrial production thereof.
[0024] A culture form according to an embodiment of the present
invention may be any of a batch-wise culture and a continuous
culture, however, the latter is adopted in terms of industrial
productivity.
[0025] For a medium for culturing the mutant according to an
embodiment of the present invention, as a carbon source, glucose,
acetic acid, ethanol, glycerol, molasses, sulfite waste liquid,
etc. are used and as a nitrogen source, urea, ammonia, ammonium
sulfate, ammonium chloride, nitrate salts, etc. are used. As a
source for phosphoric acid, potassium and magnesium, a usual
industrial raw material such as calcium superphosphate, ammonium
phosphate, potassium chloride, potassium hydroxide, magnesium
sulfate and magnesium chloride may be used. Besides them, to the
medium, inorganic salts of zinc, copper, manganese, iron, or the
like are added. As others, vitamins, amino acids, nucleic
acid-related substances, etc. are not specially used, however,
needless to say, these substances or organic substances such as
corn steep liquor, casein, yeast extract, meat extract and peptone
may be added.
[0026] A culture temperature is preferably 21 to 37.degree. C.,
more preferably 25 to 34.degree. C. and pH for the culture is
preferably 3.0 to 8.0, more preferably 3.5 to 7.0. Since the
productivity of amino acids or nucleic acids varies depending on
the culture condition, it is necessary to adopt conditions suitable
for the production specification of an objective yeast extract.
[0027] By using the above obtained yeast accumulating free glutamic
acid and glutamine in a high concentration, the production of a
yeast extract containing glutamic acid in an amount of 20 to 50%
converted into the amount of a sodium salt thereof can be easily
performed according to a production method known for the production
of the yeast extract.
[0028] The extraction can be performed by any of methods typically
used in the production of a yeast extract, for example, a
heating-extraction method, an enzymolysis method and an autolysis
method. Each of the taste of the obtained yeast extracts has
characteristics and an extraction method corresponding to a
production design can be selected. Hereinafter, a hot water
extraction and an enzyme extraction having extraction rates largely
different from each other are exemplified as follows, however the
extraction method is not limited thereto.
[0029] In a production method by the hot water extraction, a cell
suspension prepared in a concentration of 10% is heated at
50.degree. C. or more, preferably 60.degree. C. or more. The
extraction time varies depending on the extraction condition and is
around several seconds to several hours. Glutamine has low thermal
stability and thus, is preferably subjected to small heat history.
In the hot water extraction, a yeast extract having a low peptide
content and thus, having a clear taste can be produced.
[0030] On the other hand, for the enzyme extraction, an enzyme
typically used in the digestion of a yeast, a cell wall digesting
enzyme and protease are applicable. A yeast extract produced by an
enzyme extraction contains more extracted components than that
produced by a hot water extraction. Therefore, since the former
yeast extract has a higher peptide ratio, a body is imparted to the
taste.
[0031] After the extraction, solid contents are removed by a method
such as a centrifugation to obtain an extracted component.
[0032] For further enhancing a glutamic acid concentration in the
yeast extract, as frequently used for foods, a commercially
available glutaminase is made to act on the yeast extract during
extracting the extract or after the isolation of the extracted
component to convert glutamine to glutamic acid.
[0033] This yeast can accumulate, besides the above compounds, RNA
in an amount of 9% by weight or more. For example, by a method
described in Japanese Examined Patent Application Publication No.
JP-B-7-93871, RNA is extracted from the yeast and ribonuclease is
made to act, and depending on the case deaminase is further made to
act. Thus, a yeast extract containing 5'-adenylic acid and
5'-guanylic acid (hereinafter, expressed as 5'-AG) or 5'-inosinic
acid and 5'-guanylic acid (hereinafter, expressed as 5'-IG) can
also be produced at a time. For the extraction of RNA, the heat
inactivation of an enzyme by heating is effective, however, since
glutamine has poor thermal stability, it is advantageous, for
example, to set an optimal temperature or time for the extraction
or to heat and reextract the cell after a hot water extraction.
[0034] When 5'-phosphodiesterase is made to act for decomposing RNA
in the extracted liquid to 5'-nucleotide or when deaminase is made
to act, if desired for converting 5'-adenylic acid in a liquid
containing thus obtained 5'-nucleotide to 5'-inosinic acid, it may
react under recommended conditions using a commercially available
enzyme. Specific examples of the reaction conditions include
conditions disclosed in Japanese Examined Patent Application
Publication No. JP-B-7-93871.
[0035] 5'-nucleotide exhibits a synergistic action of glutamic acid
and the taste and is effective for strengthening umami of first
taste.
[0036] After the completion of the reaction, for inactivating the
enzyme utilized, the reaction solution is heated at 90 to
100.degree. C. for around 30 to 60 minutes and thereafter, the
solid content is removed by a method such as a centrifugation.
Next, a supernatant is concentrated to paste or is processed to
powder by further drying. A concentration method and a drying
method are not particularly limited, however, a drying method, by
which the reaction solution is not subjected to an excessively high
temperature, such as a vacuum concentration method, a freeze-drying
method and a spray drying method, is used.
EXAMPLES
[0037] Hereinafter, the present invention is described in detail
referring to examples.
[0038] Here, an analysis method and an evaluation method are as
follows.
(Quantification Method of Glutamic Acid and Glutamine in Yeast
Cell)
[0039] The quantification of free glutamic acid and glutamine in a
yeast cell was performed as follows.
[0040] A washed cell was heated in boiling water for 4 minutes, was
cooled in running water, and then was centrifuged. The resultant
supernatant liquid was appropriately diluted to quantify glutamic
acid and glutamine by using a bio-sensor (trade name: BF-5;
manufactured by Oji Scientific Instruments) equipped with each
enzyme electrodes for glutamic acid and for glutamine.
(Quantification Method of RNA in Yeast Cell)
[0041] The quantification of RNA in the yeast cell was performed
according to Schmidt-Tanhaeuser-Schneider method (J. Biol. Chem.
Vol. 164, p. 747 (1946)).
[0042] The dry weight of the cell was measured by a method
including: taking a sample of 10 mL of a washed yeast suspension
into a weighing bottle; volatilizing a water content from the
suspension by heating at 105.degree. C. for 20 hours; leaving the
resultant residue to be cooled to room temperature in a desiccator;
and measuring the difference in the weight of the residue between
before and after the heating using a precision electronic balance.
Based on this dry weight of the yeast cell, the content (in % by
weight) of each component in the cell was calculated.
(Quantification Method of Free Glutamic Acid and Other Free Amino
Acids in Powder Yeast Extract)
[0043] The concentrations of free glutamic acid and free other
amino acids, and the total amino acid concentration in the dry
powder yeast extract were measured according to a common procedure
using an amino acid analyzer (trade name: L8800; manufactured by
Hitachi, Ltd.).
(Quantification Method of 5'-Nucleotide in Powder Yeast
Extract)
[0044] 5'-nucleotide in the dry powder yeast extract was quantified
under conditions disclosed in Japanese Examined Patent Application
Publication No. JP-B-7-93871 using a high performance liquid
chromatography.
(Sensory Evaluation)
[0045] The sensory evaluation of the extract was performed as
follows.
[0046] A sample was prepared by dissolving in warm water, 1% of a
powder extract and a common salt so that the concentration of the
common salt during drinking the sample becomes 0.3%. By panelists
capable of recognizing umami of MSG in a concentration of 0.05
g/dL, the difference between "strength of first taste",
"continuity" and "strength of umami" and those of the control plot
were evaluated in seven criteria and the result of the evaluation
was expressed in the average value. Here, the criteria of the
evaluation of first taste, continuity and umami in seven criteria
evaluation are as follows. "+3 points: extremely strong, +2 points:
considerably strong, +1 point: somewhat strong, 0 point: there was
no difference with "control plot", -1 point: somewhat weak, -2
points: considerably weak, -3 points: extremely weak".
Example 1
Obtaining Mutant
[0047] The strain of Candida utilis ATCC 9950 was cultured in a
test tube containing a YPD medium (yeast extract 1%, polypeptone
2%, glucose 2%) till the logarithmic growth phase. This cell was
recovered and washed and thereafter, the cell was subjected to a
mutagenizing treatment according to a method of Adelberg et. al.
using nitrosoguanidine (Biochem. Biophys. Res. Comm. Vol. 18, p.
788 (1965)). The cell subjected to a mutagenizing treatment was
washed twice and then was cultured on a YPD medium at 30.degree. C.
over one night to prepare a mutagenizing-treated cell.
[0048] The cell was cultured on a selected medium prepared by
adding to a synthetic medium (containing 2% by weight of glucose,
2% by weight of monopotassium phosphate, 0.1% by weight of ammonium
sulfate, 0.05% by weight of magnesium sulfate, 0.2% by weight of
urea, 8.6 ppm of ferric sulfate, 14.6 ppm of zinc sulfate, 0.7 ppm
of copper sulfate, 3.3 ppm of manganese sulfate and 2% by weight of
agar), 40 to 45 ppm of ethyl bromopyruvate (BPE) or further 100 to
200 ppm of 2-oxoglutaric acid (2OG) at 30.degree. C. for 3 to 7
days. As a result, colonies growing on a selected medium on which
the parent strain thereof cannot grow were isolated. These colonies
were cultured on the above synthetic liquid medium and strains
exhibiting advantageous cell-productivity and accumulating free
glutamic acid and glutamine in a large amount were selected.
Specifically, first, BPE0128 strains having BPE 40 ppm-tolerance
were obtained and next, by repeating the above operations relative
to these strains, 2OG3D4 strains having double tolerance of BPE 40
ppm and 2OG 100 ppm were obtained, and by further repeating
mutagenizing treatment in the same manner, 36D61 strains which are
the yeast mutant according to an embodiment of the present
invention and which have double tolerance of BPE 45 ppm and 2OG 200
ppm were obtained.
[0049] Next, the parent strain and the obtained mutant were
cultured in a 30 L fermenter-scale to confirm the productivity. A
test strain was yeast cultured beforehand in a conical flask
containing a YPD medium and was 0.5 to 1.5%-inoculated in the 30 L
fermenter. At this time, the medium composition was "glucose: 6% by
weight, monopotassium phosphate: 2% by weight, ammonium sulfate:
1.4% by weight, magnesium sulfate: 0.06% by weight, ferric sulfate:
10 ppm, zinc sulfate: 18 ppm, copper sulfate: 1 ppm, manganese
sulfate: 8 ppm". The culture was performed under conditions "liquid
amount in the fermenter: 15 L, pH (automatically controlled with
ammonia): 4.3, temperature for culture: 30.degree. C., aeration: 1
vvm, stirring: 400 rpm". The obtained cells were analyzed and the
result is shown in Table 1.
TABLE-US-00001 TABLE 1 Productivity of culturing mutant Cell yield
Marker Glu Gln Glu + Gln per sugar Strain (ppm) (%) (%) (%) (%)
ATCC9950 -- (parent strain) 2.13 0.95 3.08 51.19 BPE0128 BPE40 2.63
1.46 4.09 50.37 2OG3D4 BPE40 + 2OG-100 3.32 1.44 4.76 50.70 36D61
BPE45 + 2OG-200 4.30 10.08 14.38 52.66
[0050] When the total amount of free glutamic acid and glutamine
accumulated of the mutant is compared with the amount of the parent
strain, while in an ethyl bromopyruvate-tolerant strain (BPE0128),
the total amount was enlarged by 30%, in high concentration ethyl
bromopyruvate and 2-oxoglutaric acid-tolerant strain (36D61), the
total amount was enlarged surprisingly to about 4 times. At this
time, the RNA content of 36D61 strain was 9.7%.
[0051] The yeast Candida utilis 36D61 strain has the identical
mycological characteristics, except drug tolerance, to those of the
parent strain ATCC 9950. In addition, this strain exhibited vital
growth also on a medium containing glycerol and ethanol as carbon
sources.
[0052] Candida utilis 36D61 strain was deposited in National
Institute of Advanced Industrial Science and Technology;
International Patent Organism Depositary as accession number: FERM
BP-11103 on Mar. 18, 2008.
Example 2
Obtaining Yeast Extract
[0053] For the experimental production of the yeast extract, a
continuous culture liquid of Candida utilis mutant 36D61 strain was
used. A test strain was yeast cultured beforehand in a conical
flask containing a YPD medium and was 0.5 to 1.5%-inoculated in a 5
L-volume fermenter. The medium composition was the same as that in
the above 30 L batch-wise culture. The culture was performed under
conditions "liquid amount in the fermenter: 2 L, pH: 4.3
(automatically controlled with ammonia), temperature for culture:
30.degree. C., aeration: 1 vvm, stirring: 600 rpm". At this time,
the specific growth rate was 0.24 to 0.25 hr.sup.-1. The obtained
cell was analyzed and the results were as follows. Total amount of
free glutamic acid and glutamine: 13.9% by weight (glutamic acid:
4.7% by weight, glutamine: 9.2% by weight), cell yield per sugar:
56.8%.
[0054] The continuous culture liquid was recovered while cooling it
with ice and yeast cells were collected by centrifugation to obtain
wet yeast cell. The obtained cell was re-suspended in water and was
centrifuged to obtain about 160 g as a dry weight of cell. The
obtained yeast cell was suspended in water and the total volume
thereof was messed up to 1.6 L. Next, the suspension of the yeast
cell was heated in a hot water bath and after the temperature
reached 70.degree. C., an extract was extracted while maintaining
the temperature at 70.degree. C. and stirring the suspension for 10
minutes. Immediately thereafter, the suspension was cooled in
running water and insoluble solid contents were removed by
centrifugation to obtain an extract. The temperature of the
obtained extract was lowered to 50.degree. C. and then, a solution
in which 4.4 g of glutaminase (trade name: Daiwa C100S;
manufactured by Daiwa Fine Chemicals Co., Ltd.) were dissolved in a
small amount of water was added to the extract, followed by
reacting the resultant mixture at 40 to 60.degree. C. for 5 hours
while stirring the mixture. The resultant extract was heated at 90
to 95.degree. C. for 30 minutes and was cooled, and thereafter,
insoluble solid contents in the extract were removed again by
centrifugation. Next, the resultant extract was concentrated using
a rotary evaporator and the resultant concentrate was freeze-dried
to obtain about 49 g of a powder yeast extract. The extract
extraction rate at this time was about 25% and the free glutamic
acid content in the extract was 54.5% by weight converted into the
content of a sodium salt thereof. In addition, at this time, the
free amino acid content was 64.2% by weight and the total amino
acid content was 72.3% by weight. From these contents and the
following equation, the peptide content was calculated, and was
found to be 8.1% by weight.
Peptide content (%)=total amino acid content (%)-free amino acid
content (%)
[0055] The obtained yeast extract aqueous solution has considerably
strong MSG-like umami and first taste, which are high-impact tastes
never present in a conventional yeast extract. In addition, the
yeast extract has a little yeast odor, a moderate thickness of
taste and a clear aftertaste, so that the yeast extract has
preferred tastes.
[0056] In the experimental production, a sensory evaluation of the
extracts before and after the glutaminase reaction was performed. A
part of the extracts before and after the glutaminase reaction was
collected as a sample and was dissolved in warm water so that the
concentration of the dissolved solid content becomes 1%. In the
resultant solution, a common salt was also dissolved so that the
concentration of the common salt during drinking the sample becomes
0.3%. "Strength of first taste", "continuity" and "strength of
umami" of the extract after the glutaminase reaction were evaluated
by 5 panelists relative to those of the extract before the
glutaminase reaction as the control plot. Here, the glutamic acid
concentration measured with BF-5 was 20% before the glutaminase
reaction and 51% after the glutaminase reaction as converted into
the content of a sodium salt thereof.
[0057] As a result, the sample after the glutaminase reaction had
first taste, continuity and umami of respectively +2.6, +1.8 and
+2.2 and it could be confirmed that the umami and the first taste
were markedly enhanced. In addition, the sample after the reaction
had a totally strong taste and a thickness of taste was also
felt.
Example 3
[0058] In substantially the same manner as in Example 2, a cell of
Candida utilis mutant 36D61 strain having a dry weight of about 21
g was obtained. The obtained cell was analyzed and as a result, the
total amount of glutamic acid and glutamine was found to be 14.3%
by weight (glutamic acid: 5.0% by weight, glutamine: 9.3% by
weight) and the cell yield per sugar was found to be 56.1%.
[0059] Water was added to the above obtained yeast and the total
volume was messed up to 200 mL. Next, the resultant suspension was
heated in a warm water bath and after the temperature reached
90.degree. C., the suspension was heated at 90.degree. C. for 2
minutes. Immediately thereafter, the suspension was cooled in
running water to lower the temperature of the suspension to
50.degree. C. and thereafter, a solution in which 0.2 g of a cell
wall digesting enzyme (trade name: Tunicase; manufactured by Amano
Enzyme Inc.) were dissolved in a small amount of water was added
thereto, followed by reacting the resultant mixture at 50.degree.
C. for 1 hour while stirring the mixture. Insoluble solid contents
were removed from the reaction mixture after the completion of the
reaction by centrifugation to obtain an extract. To the obtained
extract, 0.56 g of glutaminase C100S were added and the reaction,
concentration and drying were performed in substantially the same
manner as in Example 2 to obtain about 11 g of a powder yeast
extract. The extract extraction rate was about 51% and the free
glutamic acid content in the extract was 31.9% by weight converted
into the content of a sodium salt thereof. In addition, at this
time, the free amino acid content was 39.0% by weight; the total
amino acid content was 53.1% by weight; and the peptide content was
14.1% by weight.
[0060] Although this solution had a little poor strength, it had
the same tastes as those in Example 2.
Example 4
[0061] In substantially the same manner as in Example 2, a cell of
Candida utilis mutant 36D61 strain having a dry weight of about 20
g was obtained. The obtained cell was analyzed and as a result, the
total amount of glutamic acid and glutamine, and the amount of RNA
were found to be respectively 12.5% by weight (glutamic acid: 4.7%
by weight, glutamine: 7.8% by weight), and 9.0% by weight, and the
cell yield per sugar was found to be 56.2%.
[0062] Water was added to the above obtained yeast and the total
volume was messed up to 200 mL. Next, the resultant suspension was
heated in a warm water bath and the suspension was heated at
90.degree. C. for 5 minutes. Immediately thereafter, the suspension
was cooled in running water to lower the temperature of the
suspension to 50.degree. C. and thereafter, a solution in which 0.2
g of a cell wall digesting enzyme (trade name: Tunicase;
manufactured by Amano Enzyme Inc.) were dissolved in a small amount
of water was added thereto, followed by reacting the resultant
mixture at 50.degree. C. for 6 hour while stirring the mixture.
Insoluble solid contents were removed from the reaction mixture
after the completion of the reaction by centrifugation to obtain an
extract. To the obtained extract, 0.47 g of glutaminase C100S was
added and in a similar manner as in Example 2, after the completion
of the reaction, the reaction mixture was warmed to 65.degree. C.
To the reaction mixture, a solution in which 30 mg of
5'-phosphodiesterase (trade name: ribonuclease P; manufactured by
Amano Enzyme Inc.) was dissolved in a small amount of water was
added and the reaction was effected at the same temperature for 3
hours while stirring the reaction mixture. Next, the temperature of
the reaction mixture was lowered to 45.degree. C. and to the
reaction mixture, a solution in which 20 mg of deaminase (trade
name: deamizyme; manufactured by Amano Enzyme Inc.) were dissolved
in a small amount of water was added. The reaction mixture was
maintained at this temperature for 2 hours while stirring the
reaction mixture. Thereafter, the reaction mixture was heated at 90
to 95.degree. C. for 30 minutes and was left to be cooled.
Subsequently, insoluble solid contents were removed by
centrifugation and the reaction mixture was concentrated and dried
to obtain about 12 g of a powder yeast extract. The extract
extraction rate was about 60% and the free glutamic acid content in
the extract was 23.2% by weight converted into the content of a
sodium salt thereof. The 5'-IG content was 3.3% by weight. In
addition, at this time, the free amino acid content was 32.1% by
weight, the total amino acid content was 44.8% by weight, and the
peptide content was 12.7% by weight.
[0063] The obtained yeast extract aqueous solution had a little
yeast odor, a high-impact taste, strong umami, and further
continuity and a thickness of the taste, so that the yeast extract
has extremely well balanced tastes.
Example 5
[0064] In substantially the same manner as in Example 2, a cell of
Candida utilis mutant 36D61 strain having a dry weight of about 20
g was obtained. The obtained cell was analyzed and as a result, the
total amount of glutamic acid and glutamine, and the amount of RNA
were found to be respectively 12.1% by weight (glutamic acid: 4.8%
by weight, glutamine: 7.3% by weight), and 8.8% by weight, and the
cell yield per sugar was found to be 56.6%.
[0065] Water was added to the above obtained yeast and the total
volume was messed up to 200 mL, followed by heating the resultant
suspension in a warm water bath. Next, the suspension was subjected
to a heating treatment in substantially the same manner as in
Example 4 and was cooled in running water to 50.degree. C. of the
suspension temperature. Subsequently, a 6N sodium hydroxide was
added to make pH of the suspension 10 and the suspension was
stirred for 3 hours. Insoluble solid contents were removed from the
reaction mixture after the completion of the reaction by
centrifugation to obtain an extract. The obtained extract was
subjected to glutaminase, 5'-phosphodiesterase and deaminase
reactions in substantially the same manner as in Example 4.
Thereafter, the reaction mixture was heated at 90 to 95.degree. C.
for 30 minutes and was left to be cooled. Subsequently, insoluble
solid contents were removed by centrifugation and the reaction
mixture was concentrated and dried to obtain about 10 g of a powder
yeast extract. The extract extraction rate was about 43% and the
free glutamic acid content in the extract was 28.1% by weight
converted into the content of a sodium salt thereof. The 5'-IG
content was 5.4% by weight. In addition, in this extract, the free
amino acid content was 36.6% by weight, the total amino acid
content was 42.6% by weight, and the peptide content was 6.0% by
weight.
[0066] The obtained yeast extract aqueous solution had the same
glutamic acid content as that in Example 3, however, by an effect
of 5'-IG, a considerably strong first taste and umami were felt and
further, the continuity of taste was also felt. The solution had
smaller rough tastes than that in Example 4 and had clear
flavor.
[0067] The yeast extract according to an embodiment of the present
invention was subjected to a sensory evaluation.
[0068] A yeast extract was experimentally produced from 2OG3D4
strain in substantially the same manner as in Example 2, except
that the glutaminase reaction was omitted. "Strength of umami",
"strength of first taste" and "continuity" of the powder extract
obtained by each production method were evaluated by 7 panelists
relative to a powder yeast extract containing glutamic acid in an
amount of 12.0% by weight converted into the amount of a sodium
salt thereof as the control plot.
TABLE-US-00002 TABLE 2 Content in powder extract Evaluation (% by
weight) Umami First taste Yeast extract Glu 5'-IG Peptide strength
strength Continuity Control plot 12.0 <1% 8.00 0 0 0 (2OG3D4)
Example 2 54.5 <1% 8.1 2.6 2.2 1.5 Example 3 31.9 <1% 14.1
1.0 1.1 0.4 Example 4 23.2 3.3 12.7 2.0 2.0 1.0 Example 5 29.2 5.2
6.0 2.1 2.3 1.4 * The Glu and 5'-IG contents are expressed in a
content calculated by converting into that as a sodium salt
thereof.
[0069] As is apparent from Table 2, the yeast extract obtained from
the yeast mutant obtained according to an embodiment of the present
invention has a free glutamic acid content apparently higher than
that of 2OG3D4 strain as the control plot irrespective of the
extraction method used and has a higher 5'-IG content. Then, it is
also apparent that the yeast extract according to an embodiment of
the present invention having such characteristics exhibits umami
having a strong first taste.
INDUSTRIAL APPLICABILITY
[0070] According to some aspects of the present invention, a yeast
extract having an extremely high glutamic acid content can be
produced and a natural umami seasoning having a strong first taste
can be provided. The seasoning is preferably used to foods
requiring umami having a strong first taste.
[0071] In addition, the yeast mutant according to some aspects of
the present invention accumulates glutamine in a significant
amount. Glutamine is considered to be effective for the
proliferation and the functional expression of an immunocyte and
can be utilized as a yeast cell and a yeast extract as a supplement
utilizing the function.
Accession Number
[0072] Accession number: FERM BP-11103
* * * * *