JP4638591B2 - New yeast and yeast extract - Google Patents

Description

【００５２】
以上のデータより酵母エキスの味の厚みは、原料酵母細胞内の遊離グルタミンの濃度が少なくとも１５ｍｇ／ｇ乾燥菌体以上の場合に発現していることが確認された。この細胞内遊離グルタミンの値は酵母エキスを製造した場合エキス固形分当たり３％のグルタミン酸に相当する。
【００５３】
【発明の効果】
酵母におけるグルタミン酸およびグルタミンの代謝に関与する遺伝子を変異することにより、細胞内に多量に遊離グルタミンと遊離グルタミン酸を蓄積する酵は母が育種できた。また該酵母をグルタミナーゼを併用して消化することでグルタミン酸含量が高く呈味力価に優れ、さらに従来の酵母エキスにはない味の厚みが付与された呈味バランスの良い酵母エキスが製造できるようになった。
【００５４】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel yeast extract and a method for producing the same. Yeast extract is used as a natural seasoning.
[0002]
[Prior art]
Industrial production of yeast extract is performed mainly by digesting baker's yeast, beer yeast, and yeast of the genus Candida, and has been used as a natural seasoning consisting of amino acid, peptide, and nucleotide taste components produced. . These yeast extracts are used as beef substitutes or hidden tastes to give complex tastes. These umami tastes largely depend on the taste of nucleotide components such as peptide components, inosinic acid and guanylic acid, and the umami titer is not so high, and due to the presence of unpleasant yeast odor possessed by yeast extract, Since the amount of addition to can not be increased so much, its use has been limited. Furthermore, the taste quality of yeast extract is mainly peptides and nucleotides, and is characterized by a wide and relatively rich aftertaste.
[0003]
In order to increase the umami titer of the yeast extract, and to aim for a complex seasoning characteristic consisting of a so-called precocious amino acid and a post-taste nucleotide component, sodium glutamate or the like is added to the yeast extract as an additive (hereinafter “ The seasoning is also manufactured. However, with the recent trend toward natural and health consciousness, an increasing number of consumers dislike seasonings containing sodium glutamate, and the seasoning industry is also trending towards natural seasonings. From such a background, a yeast extract having a high umami titer without adding sodium glutamate, that is, a yeast extract having a high glutamic acid content has been desired.
[0004]
Industrial production methods of glutamic acid using prokaryotes, that is, coryneform bacteria, Escherichia coli, and the like have been widely known. For example, an example in which a large amount of glutamic acid is released and accumulated in the production medium by reducing the activity of 2-ketoglutarate dehydrogenase, which is one enzyme of the bacterial tricarboxylic acid cycle, has been reported. However, it was not known whether the glutamic acid concentration in these bacterial cells was increased. Furthermore, even if the KGD activity of yeast cells is reduced, eukaryotic yeast is controlled by a complex metabolic control system, and it is not considered that glutamic acid is accumulated only by a decrease in KGD activity. It was.
[0005]
On the other hand, it has also been attempted to accumulate glutamic acid directly in yeast cells by culturing a mutant strain of yeast having resistance to glutamic acid analogs. Although it is disclosed in EP592785 that a yeast extract having a high glutamic acid content can be produced by digesting a yeast having a high intracellular free glutamic acid concentration, there is no description regarding the balance of taste and yeast odor of the yeast extract. Although the price increase can be imagined, other effects cannot be confirmed.
[0006]
[Problems to be solved by the invention]
The present inventors established a technique for producing a yeast extract having a high glutamic acid content without externally adding glutamic acid, and started studies for the purpose of confirming the effect on the taste quality.
[0007]
That is, in the present invention, the content of glutamic acid that is not externally added is preferably increased to about 3% or more based on the solid content of the extract. The yeast extract with the thickness of
[0008]
The present invention further provides a yeast containing 15 mg or more of free glutamine per gram of dry cells in the cells, which is used for the production method of the yeast extract and the yeast extract.
[0009]
The present invention further provides a method for obtaining the yeast.
[0010]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have (1) yeast cells with reduced activity of 2-ketoglutarate dehydrogenase (KGD) and / or glutamate decarboxylase (GAD). (2) that a yeast extract having a high glutamic acid content can be produced by digesting such yeast in the presence of glutaminase, (3) The taste of the yeast extract thus produced was surprisingly found to have a stronger “thickness” than the conventional yeast extract with glutamic acid added externally. Furthermore, (4) yeast with reduced glutamine synthetase (GS) activity as well as KGD activity selectively accumulates glutamic acid in the cell, but the yeast extract obtained therefrom is the same as that with external addition of glutamic acid. It was confirmed that the thickness was small, and the present invention was reached.
[0011]
That is, the essence of the present invention is that when a yeast extract is produced from a yeast that accumulates free glutamine in cells, the “thickness” of the taste is specifically increased. Furthermore, it is another element that constitutes the present invention that the umami titer can be increased simultaneously by converting glutamine accumulated in cells into glutamic acid with glutaminase. The present invention will be described in detail below.
Yeast that accumulates free glutamine
The yeast to be used may be any yeast that is acceptable as a food, and yeasts belonging to the genus Saccharomyces, Candida, Hansenula, Pichia, etc. are exemplified, and these can be used as original strains. It is desirable for carrying out the breeding described below that the yeast is capable of forming a haploid spore and capable of doubling by sexing.
[0012]
The yeast having a high intracellular free glutamine concentration of the present invention can be bred by introducing mutations into genes of enzyme systems involved in glutamate and glutamine metabolism. Examples of the gene into which the mutation is introduced include 2-ketoglutarate dehydrogenase gene kgd1 and glutamate decarboxylase gene gad1. However, even if mutations are introduced into these genes, the amount of intracellular free glutamine does not increase in all yeasts. It is important to select a yeast strain that increases the amount of intracellular free glutamine by introducing mutations. It is.
[0013]
With recent advances in gene manipulation technology, it is easy to introduce a specific mutation into a specific gene in yeast. By using a gene disruption technique or a site-specific mutation technique, kgd1 or Mutations can be introduced into gad1 to produce strains in which the activity of these gene products has been reduced or have been deleted. In particular, in Saccharomyces cerevisiae, the entire base sequence of the genome is known, and the kgd1 and gad1 genes can be easily cloned using PCR technology. A DNA fragment containing at least a part of the target gene is amplified by PCR and cloned using an E. coli host-vector system. After confirming the base sequence of the cloned fragment and confirming that it is a fragment of a gene to be introduced with mutation, it is used for introducing mutation. Even when yeast other than Saccharomyces cerevisiae is used, it may be possible to amplify the target gene by performing PCR using primers designed based on the Saccharomyces cerevisiae genome sequence. If cloning by PCR is difficult, the target gene product (enzyme) can be purified, and its amino acid sequence can be examined to design primers and probes for cloning so that cloning can be performed with high accuracy. It is.
[0014]
As a method of causing mutations in these genes using cloned gene fragments containing kgd1, gad1, etc., gene disruption is convenient. That is, a DNA fragment that is a part of the coding region of the target gene and does not contain a portion corresponding to the N-terminus and C-terminus of the protein is cut out using a restriction enzyme or the like, and this is cut into a yIp type for yeast. Connect to a vector. Using this, yeast is transformed to select a gene-disrupted strain. A yeast transformation vector that uses a drug resistance gene such as an antibiotic as a marker is advantageous in selecting a transformant.
[0015]
As a method other than gene disruption, site-specific mutation (Nucleic Acids Res. 10, 6487-6500 (1982)) can also be used. Using a known standard method, mutations such as deletion, insertion and substitution are introduced into the coding region of the cloned DNA fragment. In this case, in order to efficiently detect the strain into which the mutation has been introduced, by performing simultaneous transformation with a yEp type or yRp type plasmid having a drug resistance marker, and selecting from colonies showing drug resistance, It becomes possible to select a strain into which the target mutation has been introduced at a high frequency.
[0016]
The transformation method can also be used for the step of introducing the DNA thus introduced with mutation into the yeast genome. That is, yeast is transformed using a DNA fragment into which a mutation has been introduced, and a yeast in which the mutant DNA has been replaced with genomic DNA is selected. For transformation, a known standard method, that is, a protoplast method, an alkali cation method, or an electroporation method can be used. Whether the target mutation has been introduced into the genome can be confirmed by PCR, and the activity of the enzyme that is the gene product can be determined by a standard method (α-ketoglutarate dehydrogenase: J. Biol. Chem 249, 3660-3670 (1969)). , Advance in Biophysics (Kotani, M., ed) Vol 9, pp. 187-227, Univ. Of Tokyo press, Glutamate decarboxylase: Meth. Biochem. Anal. 4,285-306 (1957), Biochem. Biophys. Res. Commun 28,525-530 (1967), Biochemistry 9,226-233 (1970), Glutamine synthetase: Anal. Biochem., 95, 275-285 (1979)).
[0017]
As mentioned earlier, there are some yeasts that do not accumulate glutamine and glutamic acid at high concentrations in cells by reducing the activity of KGD and GAD. By using technology, that is, gene disruption or site-specific mutation, these differences can be easily discriminated, and by using the selected excellent strain as a source strain for breeding, the present invention can be used with high accuracy. It means that yeast can be bred. Specifically, after introducing these mutations (gene disruption) using gene disruption or site-specific mutation, the yeast is cultured and the intracellular free glutamine and free glutamate concentrations are measured using known methods By doing so, the yeast which can be utilized for this invention can be selected. The criterion for selecting the mutant strain (gene disruption strain) is that a large amount of glutamic acid or glutamine is accumulated in the cell. By digesting such yeast in the presence of glutaminase, a yeast extract having a high glutamic acid content and a thick taste can be produced.
[0018]
The intracellular glutamine and glutamic acid concentrations are measured as follows. The yeast cells are collected from the yeast culture solution by centrifugation, washed with water as appropriate, and then lyophilized. The freeze-dried yeast cells (known weight) are suspended in water, crushed with glass beads, and the centrifugal supernatant is extracted with hot water. By measuring the glutamic acid concentration by using a known method such as amino acid analysis for both this and the glutaminase reaction, and comparing the glutamic acid concentration before and after the glutaminase reaction, the concentrations of intracellular free glutamic acid and free glutamine are Calculated. It is also possible to simultaneously quantify glutamine and glutamic acid in the extract before glutaminase reaction by changing the conditions of the amino acid analyzer.
[0019]
When the kgd1 or gad1 mutation is introduced alone into yeast, 16-22 mg of free glutamine and 21-30 mg of free glutamic acid are accumulated per 1 g of dry weight of the yeast, and 12 to 12 per solid matter by digesting this yeast. Yeast extract containing 15% glutamic acid can be produced. The taste characteristics of the yeast extract obtained in this way are characterized by a longer umami sensitivity time than those produced by simply adding the required amount of glutamic acid, accompanied by a kokumi taste and a salt-free effect. In addition, thickness is added to the taste, and it is harmonized as a whole. The characteristic of this taste is presumed to have a critical significance that at least 3% or more of glutamic acid in the yeast extract is derived from intracellular free glutamine. And when this 3% or more free glutamine is converted into the amount in 1 g of dry yeast, it becomes at least 15 mg.
[0020]
Although it is not clear why digesting yeast that accumulates glutamine in the cell produces a thick yeast extract, the accumulation of glutamine changes the balance of intracellular nitrogen metabolism, that is, the balance between amino acids and organic acids. As a result, it is assumed that a thick yeast extract can be produced. Moreover, free glutamine can be efficiently converted to glutamic acid by adding a step of glutaminase reaction when digesting yeast, and a yeast extract excellent in umami titer can be produced. In order to produce a yeast extract with a high umami titer, it is desirable that the total concentration of intracellular free glutamine and free glutamic acid be 30 mg / g dry cells or more.
[0021]
In the present invention, Saccharomyces cerevisiae IFO10150 is a specific example of yeast in which intracellular glutamine is increased by reducing the activity of KGD and GAD. This strain can be purchased as laboratory yeast by anyone from the Fermentation Research Institute. By carrying out kgd1 gene disruption on Saccharomyces cerevisiae IFO10150, this strain accumulates 16 mg / g dry cells or more in the cells, and about 2.5 times as much free glutamine as the original strain. In addition, when the gad1 gene is disrupted for this strain, the intracellular free glutamine content becomes 21 mg / g dry cells. Furthermore, when both kgd1 and gad1 are destroyed, free glutamine of 24 mg / g dry cells can be accumulated.
[0022]
On the other hand, even when the same Saccharomyces cerevisiae strain YPH499, YPH500, YPH501 (all available from Stratagene) was subjected to kgd1 disruption, the amount of intracellular free glutamine was 4.0 to 4.8 mg / g. There is almost no increase compared to the original strain, about the dry cell. When gad1 gene disruption is performed on these strains, the intracellular free glutamine slightly increases to 6.0 to 6.4 mg / g dry cells, but even if both kgd1 and gad1 are disrupted, gad1 disruption occurs. The amount of glutamine does not increase compared to the strain.
[0023]
Similarly, in practical yeasts, it is possible to select a strain in which the amount of glutamine accumulated by gene disruption changes and a strain that does not change. Usually, practical yeasts exist in the form of diploids. From this, spores are formed and evaluated by gene disruption in the form of haploids. Among the haploid yeast obtained by sporulation, a strain in which the amount of intracellular free glutamine is increased by gene disruption can be selected. The selected haploid yeast is subjected to site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500 (1982)) or mutagenesis using ordinary mutagens, and KGD or GAD The yeast of the present invention can be bred by selecting a strain having reduced activity. Since haploid yeast generally has a slower growth rate than diploid yeast, it is also possible to produce diploid yeast based on bred haploid yeast using crossing and cell fusion techniques. Is possible.
[0024]
In addition, although it is possible to use yeast introduced with gene disruption or site-directed mutation as it is for the production of yeast extract, guidelines for using yeast produced using recombinant DNA technology as food ingredients Not yet enacted. Therefore, it is practically important to breed the yeast as described above without using recombinant DNA technology. The original strain selected by an operation such as gene disruption, i.e., the original strain selected by site-specific mutation or gene disruption, i.e., a strain capable of increasing the amount of intracellular glutamine, is used as an original strain. By mutating with a mutagen and selecting a strain with reduced KGD or GAD activity, it is possible to breed a yeast that is not problematic in terms of food hygiene. Fortunately, kgd1-disrupted strains often have a phenotype that cannot grow using ethanol, glycerin, pyruvic acid, or the like as a single carbon source, and it is possible to select a target mutant strain using this as an index. The target mutant can also be selected by directly measuring the intracellular glutamine concentration. The selected mutants often have poor growth rates and yields as they are, so it is possible to breed strains with high growth rates and yields and high intracellular glutamine concentrations by repeated hybridization and cell fusion. it can.
Preparation of yeast extract
The yeast culture method is no different from known methods. The yeast is cultured in a medium containing sucrose, glucose, liquid sugar, molasses, etc. as a carbon source and ammonia or urea as a nitrogen source. In order to suppress alcohol fermentation by yeast, it is necessary to supply oxygen sufficiently. However, if oxygen is supplied excessively, the accumulation of glutamic acid and glutamine may decrease. It is good to ask for the amount. After culturing, the yeast cells are collected by centrifugation or filtration.
[0025]
A known method can also be used to obtain a yeast extract from the collected yeast cells. As a typical example, water is added to yeast, and the components contained therein are extracted by dissolving the cell walls of the yeast. Thereafter, the enzyme is degraded by an enzyme of yeast and / or an enzyme added from the outside.
[0026]
In order to self-digest with the enzyme which yeast has, what is necessary is just to react for 6 to 72 hours at 40-55 degreeC.
When digesting with the addition of enzymes, proteases are required for protein degradation, cell wall lytic enzymes are required for cell wall lysis, glutaminase is required for conversion of glutamine to glutamic acid, and deaminase and nuclease are required for the production of taste nucleic acids. Add as appropriate. When adding an enzyme, the enzyme derived from raw material yeast may exist or may be deactivated.
[0027]
The order of adding the enzyme is such that cell wall lysing enzyme is added first, and then protease is added as necessary to react. Thereafter, glutaminase may be further added and reacted. The reaction with glutaminase is a preferred embodiment of the present invention because the amount of glutamic acid is increased to increase the taste titer of the yeast extract. Furthermore, in order to improve the umami taste due to the nucleic acid component of the obtained yeast extract, it is also preferable to extract the yeast nucleic acid into the extract. In this case, the nucleic acid in the yeast can be extracted into the extract sufficiently efficiently by heating the reaction product at about 65 ° C. for about 2 hours at a pH of about 8.5. It is preferable that the yeast extract thus obtained further inactivates the enzymes in the cells and various added enzymes by heating at, for example, about 90 ° C. for about 30 minutes.
[0028]
As the various enzymes described above, commercially available enzymes can be used as they are. For example, protease A “Amano” (Amano Pharmaceutical), protease N “Amano” (Amano Pharmaceutical) and the like, and cell wall lytic enzymes such as YL-15 (Amano Pharmaceutical), Kitalase M (Kumiai Kasei) and the like, Examples of glutaminase include glutaminase Daiwa C100 (Daiwa Kasei), glutaminase F “Amano” (Amano Pharmaceutical), deaminase includes deamizyme (Amano Pharmaceutical), and nuclease includes nuclease “Amano” (Amano Pharmaceutical). These enzymes are usually added in an amount of 0.01% to 5% (preferably 0.1 to 2%) with respect to the raw material. Protease reaction is performed at pH 4-7 (preferably 5-6), temperature 40-60 ° C (preferably 40-55 ° C), and reaction time is suitably 1-72 hours (preferably 16-24 hours). . There is no problem if the temperature of each other enzyme reaction is the optimum temperature and pH range of each enzyme. Each reaction time is suitably 0.5 to 8 hours (preferably 1 to 4 hours).
[0029]
Accordingly, the amount of glutamic acid in the dry solid content of the extract obtained by digesting the yeast of the present invention is 12% or more. After the enzyme reaction, the enzyme is heat-inactivated, and the cell residue is removed by centrifugation or a filter press to obtain a supernatant. Depending on the purpose, the supernatant may be clarified by membrane filtration with UF, MF or the like. The obtained liquid is concentrated to the target solid content. Powdering may be performed in addition to concentration. For powdering, spray drying or freeze drying may be selected as appropriate. You may use an auxiliary agent in the case of powdering. The yeast extract thus produced has a strong taste of glutamic acid and a good taste coupled with the complex taste of the yeast extract, and has a thickness that cannot be seen when glutamic acid is added externally, It shows a very favorable taste as a seasoning.
[0030]
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
[0031]
【Example】
The method shown below was used for preparation and confirmation of the gene disruption strain.
(1) Cloning and gene disruption of 2-ketoglutarate dehydrogenase gene (kgd1)
Genomic DNA was extracted from Saccharomyces cerevisiae IFO10150 using a genome extraction kit (ISOPLANT) manufactured by Nippon Gene. About 1 μg of the primer DNA shown in SEQ ID NO: 1 in the sequence listing and about 1 μg of the primer DNA shown in SEQ ID NO: 2 in the sequence listing were added to about 100 ng of genomic DNA. PCR was performed using LA-Taq manufactured by Takara Shuzo Co., Ltd. in a liquid volume of 50 μl. PCR conditions were 25 cycles of 95 ° C. for 0.5 minutes, 55 ° C. for 1 minute, and 72 ° C. for 1.5 minutes. The amplification of a fragment of about 1.3 kb was confirmed and purified with a DNA purification kit manufactured by QIAGEN. The PCR product was digested with the restriction enzyme SacI, incorporated at the SacI breakpoint of pKF18, and introduced into Escherichia coli JM109 strain (purchased from Takara Shuzo) by electroporation. After preparing the recombinant plasmid, the DNA sequence of the inserted fragment was confirmed. As a result, it was confirmed that the inserted fragment had the DNA sequence shown in SEQ ID NO: 3 in the sequence listing. The inserted fragment was the portion corresponding to the 80th to 497th amino acid residues in the coding region of Saccharomyces cerevisiae kgd1 gene (encoding 1015 amino acid residues).
[0032]
This insert fragment was again excised with SacI to prepare a recombinant plasmid pAUR101-KGD1 which was integrated into the SacI breakpoint of pAUR101 (Takara Shuzo), a yIp type shuttle vector. Since this plasmid has a restriction enzyme NruI cleavage point in the inserted fragment, yeast was transformed by electroporation using the plasmid digested with this restriction enzyme. A transformant resistant to 0.5 μg / ml aureobasidin on YPD agar medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar) 2-ketoglutarate dehydrogenase activity was measured by the method described in Ujigawa-Takeda, K., Agric. Biol. Chem., 44, 1897-1904 (1980).
[0033]
(2) Cloning and gene disruption of glutamate decarboxylase gene (gad1)
DNAs of SEQ ID NOs: 4 and 5 in the sequence listing were used as PCR primers. PCR was performed under the same conditions as described above using yeast genomic DNA as a template, and amplification of an approximately 1.0 kb fragment was confirmed. The amplified DNA was purified, digested with the restriction enzyme SacI, and inserted into the SacI cleavage point of pKF18. The DNA sequence of the inserted fragment is shown in SEQ ID NO: 6 in the sequence listing. This fragment was a portion corresponding to the 141st to 461st amino acid residues in the coding region of the Saccharomyces cerevisiae gad1 gene (encoding 586 amino acid residues).
[0034]
This insert fragment was incorporated into pI-RED1 (Toyobo Co., Ltd.), a yeast yIp type vector, to obtain pI-RED1-GAD1. Plasmid DNA was digested with a restriction enzyme cleavage point NruI which this plasmid has only on the inserted fragment, and yeast was transformed by electroporation using this. Using a method described in Okada, Y. and Shimada, C., Brain. Res., 98, 202-206 (1975) for a transformant resistant to 100 μg / ml cycloheximide on a YPD agar medium. Glutamate decarboxylase activity was measured. However, the measurement of the amount of γ-aminobutyric acid carried out during the activity measurement was quantified using an amino acid analyzer ALC-1000 manufactured by Shimadzu Corporation.
[0035]
(3) Cloning and gene disruption of glutamine synthetase gene (gln1)
DNAs of SEQ ID NOs: 7 and 8 in the sequence listing were used as PCR primers. PCR was performed under the same conditions as described above using yeast genomic DNA as a template, and amplification of a fragment of about 0.4 kb was confirmed. The amplified DNA was purified, digested with the restriction enzyme SacI, and inserted into the SacI cleavage point of pKF18. The DNA sequence of the inserted fragment is shown in SEQ ID NO: 9 in the sequence listing. This fragment was the portion corresponding to the 92nd to 232nd amino acid residues in the coding region of the gln1 gene of Saccharomyces cerevisiae (encoding 351 amino acid residues).
[0036]
This insert fragment was incorporated into pI-RED1, which is a yeast yIp type vector, to obtain pI-RED1-GLN1. Plasmid DNA was digested at the restriction enzyme cleavage point Eco0109I which this plasmid has only on the inserted fragment, and yeast was transformed by electrolysis using this plasmid DNA. A transformant resistant to 100 μg / ml cycloheximide on a YPD agar medium was analyzed for glutamine synthetase activity using the method described in Stadtman, ER et al, Anal. Biochem., 95, 275-285 (1979). Was measured.
[0037]
(4) Measurement of intracellular free glutamine and free glutamate concentration
500 ml baffle containing 50 ml of synthetic medium (6.7 g / LYeast Nitrogen Base (Difco), 20 g / L glucose, optionally supplemented with 0.5 μg / ml aureobasidin or 100 μg / ml cycloheximide) The flask was shaken and cultured for 18 hours on a rotary shaker at 30 ° C. and 120 rpm. The yeast cells were collected by centrifugation at 1500 × g at 4 ° C. for 5 minutes, washed twice with water, and then washed cells were collected. This was freeze-dried and the dry cell weight was measured. About 30 mg of dried cells are put in a 1.5 ml Eppendorf tube, 450 μl of distilled water and 480 mg of glass beads (BRAUN, 0.45-0.50 mm) are added to the tube, and a Tommy microtube mixer is used. And stirred for 2 minutes at the maximum rotational speed. This was cooled on ice for 2 minutes. By repeating this operation 4 times, yeast cells were disrupted. The supernatant was recovered by centrifugation (10000 × g, 4 ° C., 5 minutes), and this was heated at 95 ° C. for 15 minutes to precipitate the protein. A part of the reaction product was taken, and an equivalent amount of 10 mg / ml glutaminase Daiwa C-100 solution was added to the remainder, followed by reaction at 37 ° C. for 20 minutes. The precipitate was removed by centrifugation (10000 × g, 4 ° C., 5 minutes). The samples before and after the glutaminase reaction were subjected to amino acid analysis using an amino acid analyzer ALC-1000 manufactured by Shimadzu Corporation, and the amount of glutamine was determined from the difference between the two. The amount of intracellular free glutamine and free glutamic acid was determined from the amounts of glutamine and glutamic acid analyzed and the weight of the dry yeast cells used.
[0038]
(5) Yeast culture
After yeast is cultured overnight on a YPD plate medium, one platinum loop is synthesized on a synthetic medium (6.7 g / LYeast Nitrogen Base (Difco), 20 g / L glucose, 0.5 μg / ml aureobasidin or 100 μg as required. 10 ml (with / ml cycloheximide added), and cultured at 30 ° C. for 24 hours under the same conditions as in (4) above. OD660nm of this culture solution was measured with a spectrophotometer UV-160A (Shimadzu Corporation). Next, using this culture solution as a seed, 50 ml of a synthetic medium was inoculated so that the OD660nm value was 0.063 ± 0.002, and cultured at 30 ° C. for 18 hours. The obtained culture solution was centrifuged (1500 × g, 5 minutes), and the obtained bacterial cells were washed with physiological saline. After washing, the cells were centrifuged under the same conditions as above to collect the cells.
[0039]
Example 1
Saccharomyces cerevisiae IFO10150 was transformed with a NruI digest of the recombinant plasmid pAUR101-KGD1. After confirming that there was no 2-ketoglutarate dehydrogenase activity for colonies showing aureobasidin resistance (below 0.62 nmol / min / mg protein), intracellular free glutamine and free glutamate concentrations were measured. As a control, IFO10150 transformed with pAUR101 was used. Concentrations of intracellular free glutamine and free glutamic acid were 7.4 mg / g dry cells and 10.6 mg / g dry cells in the control strain, respectively, whereas 16.2 mg / g dry in the gene-disrupted strain, respectively. The cells were 21.5 mg / g dry cells.
[0040]
Example 2
Saccharomyces cerevisiae IFO10150 was transformed with the NruI digest of the recombinant plasmid pI-RED1-GAD1. After confirming that the colony showing resistance to cycloheximide has no glutamate decarboxylase activity (0.01 U / mg or less), the intracellular free glutamine and free glutamate concentrations were measured. As a control, a transformant with vector DNA was used. Concentrations of intracellular free glutamine and free glutamic acid were 6.8 mg / g dry cells and 10.3 mg / g dry cells in the control strain, respectively, while 21.2 mg / g dry in the gene-disrupted strain, respectively. The cell body was 27.1 mg / g dry cell body.
[0041]
Example 3
Using the same method as in Example 2, the GAD1 gene was further disrupted from the transformant obtained in Example 1 above. Gene disruption was confirmed by confirming enzyme activity, and intracellular glutamine and free glutamate concentrations were measured. In the strain in which the gene was double disrupted, the concentrations of intracellular free glutamine and free glutamic acid were 24.2 mg / g dry cells and 29.7 mg / g dry cells, respectively.
[0042]
Comparative Example 1
Saccharomyces cerevisiae YPH499 was transformed with the NruI digest of the recombinant plasmid pAUR101-KGD1. After confirming the absence of 2-ketoglutarate dehydrogenase activity for colonies showing aureobasidin resistance, intracellular free glutamine and free glutamate concentrations were measured. As a control, YPH499 transformed with pAUR101 was used. Concentrations of intracellular free glutamine and free glutamic acid were 4.6 mg / g dry cells and 7.4 mg / g dry cells in the control strain, respectively, whereas 4.4 mg / g dry in the gene-disrupted strain, respectively. The cells were 6.7 mg / g dry cells.
[0043]
Comparative Example 2
Saccharomyces cerevisiae YPH499 was transformed with the NruI digest of the recombinant plasmid pI-RED1-GAD1. After confirming that there was no glutamate decarboxylase activity for colonies showing cycloheximide resistance, intracellular free glutamine and free glutamate concentrations were measured. As a control, a transformant with vector DNA was used. Concentrations of intracellular free glutamine and free glutamic acid were 3.9 mg / g dry cells and 6.2 mg / g dry cells in the control strain, respectively, while 6.5 mg / g dry in the gene-disrupted strain, respectively. The cells were 9.9 mg / g dry cells.
[0044]
Comparative Example 3
Using the same method as in Comparative Example 2, the GAD1 gene was further disrupted from the transformant obtained in Comparative Example 1. By confirming the enzyme activity, gene disruption was confirmed, and intracellular free glutamine and free glutamate concentrations were measured. In the strain in which the gene was double disrupted, the concentrations of intracellular free glutamine and free glutamic acid were 7.8 mg / g dry cells and 11.1 mg / g dry cells, respectively.
[0045]
Example 4
20 g of each of the yeasts obtained in Examples 1, 2, and 3 was used per dry solid content, water was added so that the solid content would be 10%, and the mixture was sufficiently stirred and suspended. This suspension was adjusted to pH 5.7 with a 40% NaOH solution, and then cell wall lytic enzyme YL-15 (Amano Pharmaceutical) was added at 0.1% per dry solid and reacted at 45 ° C. for 8 hours. After the reaction, the enzyme was inactivated by heating at 70 ° C. for 30 minutes and cooled to 37 ° C. Glutaminase Daiwa C-100 (Daiwa Kasei) was added at 1% per solid and reacted at 37 ° C. for 1 hour. After completion of the reaction, the mixture was heated at 70 ° C. for 30 minutes, and then a supernatant was obtained by centrifugation. The obtained supernatant was freeze-dried to obtain a yeast extract powder. Extract solids recovery was 52% to 56%. The glutamic acid content was 5.9% for the extract using non-deficient yeast as a control, 12.6% for the extract using 2-ketoglutarate dehydrogenase-deficient yeast, and 15.1 for the extract using glutamate decarboxylase-deficient yeast. %, And the extract using yeast lacking both enzymes was 16.2%. The taste of the obtained extract was such that the umami of glutamic acid was felt very strongly in the extract using the deficient yeast, and a unique taste thickness was felt.
[0046]
Example 5
20 g of each of the yeasts obtained in Examples 1, 2, and 3 was used per dry solid content, water was added so that the solid content would be 10%, and the mixture was sufficiently stirred and suspended. The suspension was adjusted to pH 8.5 with 40% NaOH solution, heated at 65 ° C. for 2 hours, and heated at 90 ° C. for 30 minutes. This solution was adjusted to pH 5.8 with 75% phosphoric acid, 0.2% of protease N “Amano” was added per substrate protein, and reacted at 52 ° C. for 12 hours. After heating at 70 ° C. for 30 minutes to inactivate the enzyme, and adjusting the pH to 5.3 with 75% phosphoric acid, 0.04% of nuclease “Amano” (Amano Pharmaceutical) was added per dry solid content. , And reacted at 64 ° C. for 4 hours. After the reaction with nuclease, the pH was adjusted to 5.6 with 40% NaOH solution. Deamizyme (Amano Pharmaceutical) was added at 0.04% per dry solid and reacted at 45 ° C. for 2 hours. Thereafter, for enzyme inactivation, the mixture was heated at 70 ° C. for 30 minutes, and a supernatant was obtained by centrifugation. The obtained supernatant was freeze-dried to obtain a yeast extract powder. Extract solids recovery was 50% to 52%. The glutamic acid content and the (inosinic acid + guanylic acid) content were 4.2% and 1.8% for the extract using the non-deficient yeast as a control, and 14.0% for the extract using the 2-ketoglutarate dehydrogenase-deficient yeast. And 1.7%, 17.4% and 1.8% in the extract using glutamate decarboxylase-deficient yeast, and 19.1% and 1.8% in the extract using yeast deficient in both enzymes. As for the taste quality of the obtained extract, the synergistic effect of umami due to glutamic acid and (inosinic acid + guanylic acid) was strongly drawn in the extract using defective yeast, and the umami was felt very strongly. This yeast extract also had a unique taste.
[0047]
  Comparative Example 4 The kgd1-disrupted strain obtained in Example 1 was further transformed with an Eco0109I digest of the recombinant plasmid pI-RED1-GLN1. After confirming that there was no glutamine synthetase activity (0.01 U / mg or less) for colonies showing cycloheximide resistance, intracellular free glutamine and free glutamate concentrations were measured. In strains in which kgd1 and gln1 were destroyed at the same time, the concentrations of intracellular free glutamine and free glutamate were4.4mg / g dry cells,40.2It was mg / g dry cells.
[0048]
A yeast extract was produced from this yeast using substantially the same method as in Example 4. The extract solids recovery was 53% and the glutamic acid content was 15.0%. Although the umami of glutamic acid was strongly felt in the obtained extract, the thickness of the unique taste was not felt.
[0049]
Example 6
Regarding the effect of intracellular free glutamine concentration on the thickness of the taste of yeast extract, 25 skilled panelists (those whose threshold for identifying sodium glutamate is 100 ppm or less) compared the difference between umami strength and taste thickness. For sensory evaluation, four types of yeast extracts obtained in Example 4, ie, those derived from the control strain (Extract A), those derived from the kgd1 disruption strain (Extract B), and those derived from the gad1 disruption strain (Extract C) and The one derived from the simultaneous disruption strain of kgd1 and gad1 (extract D) and the one derived from the kgd1 and gln1 simultaneous disruption strain obtained in Comparative Example 4 (extract E) were used.
[0050]
The results of sensory evaluation are shown in Table 1. As for umami potency, extracts B, C, D, and E have a 1% risk rate compared to extract A. Between B, C, and E, There was no significant difference. Further, in the comparison between the extract B and the extract D, it was evaluated that the extract D was excellent at a risk rate of 5%. On the other hand, regarding the thickness of the taste, it was evaluated that B, C, and D were superior to the extract A at a risk rate of 5%, but the extract E was not significantly different from the extract A.
[0051]
[Table 1]

[0052]
From the above data, it was confirmed that the taste thickness of the yeast extract was expressed when the concentration of free glutamine in the raw yeast cells was at least 15 mg / g dry cells or more. This value of intracellular free glutamine corresponds to 3% glutamic acid per solid extract when the yeast extract is produced.
[0053]
【The invention's effect】
By mutating glutamate and genes involved in glutamine metabolism in yeast, mothers were able to breed fermentations that accumulate large amounts of free glutamine and free glutamate in the cells. Moreover, by digesting the yeast in combination with glutaminase, it is possible to produce a yeast extract with a high taste balance that has a high glutamic acid content, excellent taste titer, and has a taste thickness not found in conventional yeast extracts. Became.
[0054]
[Sequence Listing]

Claims (10)

A yeast containing 15 mg or more of free glutamine per gram of dry cells in a cell ,
Derived from a strain belonging to Saccharomyces cerevisiae,
Yeast in which one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity are reduced as compared to the strain derived from the above . A yeast containing 15 mg or more of free glutamine per gram of dry cells in a cell ,
Derived from a strain belonging to Saccharomyces cerevisiae,
Yeast in which one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity are reduced as compared to Saccharomyces cerevisiae IFO10150 . The yeast according to claim 1, wherein glutamine synthetase activity is not reduced as compared to the strain derived therefrom. The yeast according to claim 2, wherein the glutamine synthetase activity is not reduced as compared to Saccharomyces cerevisiae IFO10150. Derived from a strain belonging to Saccharomyces cerevisiae,
Yeast in which either one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity are reduced but glutamine synthetase activity is not reduced as compared to the strain derived from the above. Derived from a strain belonging to Saccharomyces cerevisiae,
As compared with Saccharomyces cerevisiae IFO10150, yeast in which one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity are reduced but glutamine synthetase activity is not reduced. The manufacturing method of the yeast extract which comprises the process of digesting the yeast of any one of Claims 1-6 . The method for producing a yeast extract according to claim 7, comprising a step of converting free glutamine contained in the cells into glutamic acid with glutaminase. The seasoning containing the yeast extract obtained from the yeast of any one of Claims 1-6. A method for improving a yeast extract, comprising a step of reducing one or both of 2-ketoglutarate dehydrogenase activity and glutamate decarboxylase activity in a raw yeast, thereby releasing 15 mg or more per gram of dried cells into the cell. A method characterized by containing glutamine.