JPH09313169A - Production method of yeast extract - Google Patents

Abstract

(57) Abstract: A method for breeding yeast having a high intracellular concentration of L-glutamic acid that enhances umami is developed, and a method for producing a yeast extract having a high L-glutamic acid content is provided. A yeast mutant strain that is resistant to a drug that impairs the structure and function of the yeast cell membrane and has an improved ability to accumulate L-glutamic acid in the cells has been obtained. This was used to produce a yeast extract.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a yeast which is resistant to drugs that impair the structure and function of cell membranes and which has an improved ability to accumulate L-glutamic acid in the cells. And a method for producing a yeast extract using the yeast. [0002] Demand for natural seasonings continues to grow in a time trend that can be expressed by keywords such as natural, natural, healthy, authentic, luxury, gourmet, and yeast extract is also one of the natural seasonings. The market scale is expanding with the development. Generally, brewer's yeast, baker's yeast, Torula yeast, etc. are used in the production of yeast extract.When using brewer's yeast, which is a by-product of beer brewing, selection and improvement of yeast are carried out within the design range of beer quality. Since it is not a yeast for the purpose of producing only extracts, it is pointed out that it is not always satisfactory in terms of quality such as an unpleasant odor when producing an extract, lack of umami, etc. Has been done. As a method for solving these problems, there is a method of breeding and improving yeast to produce a yeast having a preferable taste and flavor, and using the yeast to produce a yeast extract. In terms of flavor, by using a yeast with imparted norvaline resistance,
There are known examples of increasing aroma components such as esters and alcohols (Journal of the Institute of Brewing, Vol. 76, p. 843-).
(Pp. 847, 1981) The production of alcoholic beverages using this is reported. However, there is no known example in which the taste such as umami is changed. L-is used to enhance taste such as umami.
Sodium glutamate, 5'-sodium inosinate and 5'-sodium guanylate are known, and they are added to foods as seasonings according to the use. The object of the present invention is to develop and breed a method for efficiently breeding yeast having a high ability to accumulate L-glutamic acid, which is a substance that enhances umami, in cells. The present invention is to find a method for producing a yeast extract having a higher L-glutamic acid content by using the yeast.
The above challenges are new to us:
The issue itself is new. [0006] The present inventors have found that a yeast mutant strain that is resistant to a glutamine antimetabolite such as 6-diazo-5-oxo-norrosine has a high L-glutamic acid level in the cells. Accumulation at a concentration, when a yeast extract was prepared using this mutant strain, the concentration of L-glutamic acid in the extract was high, and the sensory evaluation showed that the taste was increased, and the flavor, hot taste, and rich taste were excellent. It was found to be yeast extract. However, 6-diazo-5-oxo-
Among the yeast mutant strains to which norleucine resistance was imparted, the intracellular L-glutamic acid concentration was the same as that of the parent strain, but rather the extracellular L-glutamic acid accumulation amount was improved. Further, even a yeast mutant strain imparted with 6-diazo-5-oxo-norleucine resistance and having a high intracellular L-glutamic acid concentration, accumulates a large amount of L-glutamic acid outside the bacterial cell. It was [0008] The present inventors have found that if L-glutamic acid biosynthesized by a yeast mutant strain that produces a large amount of L-glutamic acid can be specifically retained inside the cells,
Yeast having a high glutamic acid concentration can be grown, and as a result, L-
It was thought that it would be possible to prepare a yeast extract having a high glutamic acid content and a better taste. Therefore, the present inventors have further studied to solve such a new problem, and as a result, by imparting resistance to yeast to a drug that impairs the structure and function of its cell membrane, L -The present inventors have found that yeast having a high ability to accumulate glutamate can be efficiently produced, and have completed the present invention. The present invention is as follows. (1) A yeast that is resistant to a drug that impairs the structure and function of the cell membrane and has an improved ability to accumulate L-glutamic acid in the cells. (2) The yeast according to (1) above, wherein the drug that impairs the structure / function of the cell membrane is nystatin. (3) The yeast according to (1) or (2) above, wherein the yeast is Yarrowia lipolytica. (4) A method for producing a yeast extract, which comprises using the yeast according to (1) to (3) above. BEST MODE FOR CARRYING OUT THE INVENTION As yeasts used in the present invention, yeasts belonging to the genus Saccharomyces (Saccharomyces cerevisiae, Saccharomyces ubalm, Saccharomyces.
Chevallier, Saccharomyces rosei, etc.) Yeast belonging to the genus Torluspora (eg Torluspora delbrutki), yeast belonging to the genus Kleiberomyces (eg Kleivalomyces thermotolerance), yeast belonging to the genus Pichia (eg Pichia membranefaci) Ens), yeast belonging to the genus Yarrowia (for example, Yarrowia lipolytica), and the like. Examples of the drug used in the present invention for impairing the structure / function of the cell membrane include polyene antibiotics such as amphotericin B and nystatin, and econazole,
Examples include antibiotics that inhibit ergosterol synthesis, such as miconazole and ketoconazole. As these antibiotics, commercially available products can be used. A particularly preferred drug is nystatin. The yeast of the present invention is a yeast having resistance to a drug that impairs the structure and function of the cell membrane and having an increased L-glutamic acid concentration in the cells. -It may have resistance to other drugs that are effective in improving the concentration of glutamic acid, such as 6-diazo-5-oxo-norleucine and bromopyruvic acid used in the present invention. To impart resistance to a drug that impairs the structure and function of the cell membrane to the parent strain, the parent strain is irradiated with ultraviolet rays or a mutagenizing agent (eg N-methyl-N'-).
After treatment with nitro-N-nitrosoguanidine, ethyl methane sulfonic acid, etc.), a strain that can grow on an agar medium containing a drug that impairs the structure and function of the cell membrane at a concentration at which the parent strain cannot grow may be collected. . A mutant strain resistant to a drug that impairs cell membrane structure / function is a strain that is more resistant to the drug than the parent strain. The yeast of the present invention is a yeast that is resistant to a drug that impairs the structure and function of the cell membrane and has an improved intracellular L-glutamic acid concentration. For example: Yarrowia lipolytica AJ14718 The medium used for culturing the yeast in the present invention to obtain the cells is a usual medium containing a carbon source, inorganic salts and, if necessary, organic micronutrients such as amino acids and vitamins. It is a nutrient medium. Either a synthetic medium or a natural medium can be used. Carbon source used in the medium,
The nitrogen source may be any available source of yeast to be cultured. As the carbon source, glucose, glycerol, mannitol, ethanol, n-paraffin and the like are used. In addition, organic acids such as lactic acid and citric acid can be used alone or in combination with other carbon sources. As the nitrogen source, urea, ammonia, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate, etc. are used. As the organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acid, yeast extract, soybean protein hydrolyzate containing these substances are used, and amino acids and vitamins are required for growth. When culturing wild strains or auxotrophic mutant strains, it is necessary to supplement the required nutrients. As the inorganic salts, phosphates, magnesium salts, iron salts, zinc salts, calcium salts and the like are used. The culture method is a culture temperature of 20 to 37 ° C.
Then, aeration culture is performed while controlling the pH to 3 to 8. If the pH drops during the culturing, calcium carbonate is added or neutralized with an alkali such as aqueous ammonia or ammonia gas. Thus, yeast cells can be obtained by culturing for about 10 hours to 4 days. In order to prepare a yeast extract from the obtained bacterial cells, the bacterial cells may be recovered from the culture broth by centrifugation and the method may be carried out according to a known method. For example, 1 to 2% of toluene or ethyl acetate is added to the cells, autolysis is performed at a temperature of 45 to 50 ° C., the extract is concentrated under reduced pressure, and spray-dried to obtain a powdered yeast extract. EXAMPLES The present invention will be described in more detail by way of examples. Example 1 Isolation of a Bromopyruvate-Resistant Strain from Yarrowia lipolytica AJ5004 Yarrowia lipolytica AJ5 isolated from nature
004 (Agricultural and Biological Chemistry, Vol. 33, pp. 158-167, 19
1969) sporulation medium (1% potassium acetate, 0.1%
After culturing in yeast extract, 0.05% glucose) at 30 ° C. for 1 to 2 days, washed with 0.85% physiological saline.
Final 1 mg / ml Zymolyase 5000 containing 0.
The cells were suspended in 85% physiological saline and treated at 30 ° C for 120 minutes. Furthermore, spores were collected by filtering with a pore size 60 μm nylon membrane. Ethylmethanesulfonic acid was suspended in a 0.1 M phosphate buffer solution (containing glucose at a final concentration of 2.5%) added to a final concentration of 30 μl / ml, and mutagenized at 30 ° C. for 60 minutes. . After centrifugation, the spores were washed with 0.1 M phosphate buffer and the minimum medium containing 0.5 g / l of bromopyruvic acid (Difco yeast nitrogen base without amino acid 0.67%, glucose 2%, agar) was added. 2%) and cultured at 30 ° C. for 3 to 7 days. Bromopyruvic acid known as an antimetabolite of pyruvic acid is Yarrowia lipolytica AJ50.
04 growth was inhibited and Yarrowia lipolytica AJ5 was added to the minimal medium containing 0.5 g / L of bromopyruvic acid.
No growth of 004 was observed. The colonies appearing on the minimal medium containing bromopyruvic acid were picked up, and single colonies were separated on the minimal medium having the same composition. Then, a cell preparation medium (n-paraffin 80 g / l, ammonium nitrate 20 g / l) was prepared. , Potassium dihydrogen phosphate 2 g / l, magnesium sulfate heptahydrate 1 g / l, ferrous sulfate heptahydrate 10 mg / l, zinc sulfate heptahydrate 40 mg / l, thiamine 6 μg / l,
20 ml of calcium carbonate 20 g / l, pH 5.0)
Inoculate the injected 500 ml Sakaguchi flask and incubate for 3 days
After seed culture at 0 ° C, the cells were inoculated into a 500 ml Sakaguchi flask containing 20 ml of the medium of the same composition, and incubated at 30 ° C for 5
After culturing for one day, the culture broth was centrifuged and the L-glutamic acid concentration in the supernatant was measured by Biotech Analyzer AS200.
As a result of measurement with Asahi Kasei, 230 in the culture broth
N46 strain accumulating mg / l L-glutamic acid (AJ
14717). Example 2 Bromopyruvate-resistant strain N46 (AJ14)
Isolation of nystatin resistant strain from 717) Spores of bromopyruvic acid resistant strain N46 (AJ14717) were prepared by the method used in Example 1,
N-methyl-N'-nitro-so that the final 10 mM
The cells were suspended in a 1M sorbitol solution containing N-nitrosoguanidine and subjected to a mutation treatment at 30 ° C for 20 minutes. After centrifugation, the mutated spores were washed with a 1 M sorbitol solution and applied to a minimal medium containing 1 g / l of nystatin. Bromopyruvate resistant strain N46 strain (AJ1471
7) could not grow at all on the minimal medium containing 1 g / l of nystatin. After culturing at 30 ° C. for 3 to 7 days, the colonies appearing in the minimal medium containing 1 g / l nystatin were picked up, and single colonies were separated in the medium of the same composition, and then used for preparing the bacterial cells used in Example 1. The cells were inoculated into a 50 ml large-sized test tube into which 5 ml of the medium was injected, and cultured at 30 ° C for 5 days. After completion of the culture, 1 ml of the culture broth was placed in an Eppendorf tube, sealed, and immersed in a boiling water bath for 30 minutes to extract free L-glutamic acid in the cells. After removing the extraction residue by centrifugation, the concentration of L-glutamic acid in the supernatant was measured with Biotech Analyzer AS200, and the measured value was taken as the L-glutamic acid concentration in the inside and outside of the cells. On the other hand, the extracellular glutamic acid concentration was determined by centrifuging the culture broth, measuring the L-glutamic acid concentration in the obtained supernatant, and taking the measured value as the extracellular L-glutamic acid concentration.
The concentration of L-glutamic acid inside the cells was L-
It can be represented by a value obtained by subtracting the extracellular L-glutamic acid concentration from the glutamic acid concentration. Of the 13 nystatin-resistant strains which were isolated and cultured and evaluated, 9 strains having higher L-glutamic acid concentration in combination with the inside and outside of the bacterial cells were higher than the parent strain N46 (AJ14717) (FIG. 1). . Of the 13 strains, only one strain had a significantly improved L-glutamic acid concentration outside the cells compared to the parent strain. Among the 9 strains having an improved L-glutamic acid concentration in addition to those inside and outside the cells, the extracellular L-glutamic acid concentration did not significantly increase from the parent strain, and was equal to or lower than that of the parent strain. L in the body
-It was revealed that the concentration of glutamic acid was significantly improved. Nystatin-resistant strain AJ14718, which had the highest L-glutamic acid concentration inside and outside the cells, contained 700 mg / l (3.5% based on the dry weight of the cells) inside the cells and 20 mg / l L-glutamic acid outside the cells. Accumulated. Only the carbon source of the cell preparation medium of Example 1 was
-The culture was evaluated by changing from paraffin to glucose (8%). Cultivation strains were tested on YPD agar medium (1% Difcobacto yeast extract, 2% Difcobactopeptone, 2% glucose, 2% agar).
1 out of 2 grown at 0 ℃ and grown on the entire surface of the agar plate
The / 16 plate portion was inoculated into a 50 ml large-sized test tube in which 5 ml of a medium for cell preparation (carbon source glucose) was injected,
After culturing at 30 ° C for 5 days, L-
When the glutamic acid concentration was measured, AJ14718 was L
-Glutamic acid 530 mg / l inside the cells, 4 outside the cells
2 mg / l accumulated, while the parent strain, bromopyruvic acid resistant strain N46 (AJ14717), contained 198 mg in the cells.
/ L, 49 mg / l accumulated outside the cells. It was revealed that even when the carbon source was changed to glucose, the concentration of L-glutamic acid in the cells was remarkably improved in the nystatin resistant strain as in the case where the carbon source was n-paraffin. INDUSTRIAL APPLICABILITY The method of the present invention can increase the concentration of L-glutamic acid in yeast cells. By using the yeast bred by the method of the present invention, a yeast extract having a high L-glutamic acid content can be produced. It becomes possible to do. Yarrowia Repolitica AJ14717
Has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (Postal Code 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) on May 29, 1996, with the deposit number FERM P.
-15653 is added. Yarrowia Lipolitika AJ14718 was deposited on May 29, 1996 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Postal code 305, 1-3-1, Higashi, Tsukuba, Ibaraki, Japan) with deposit number FERM P -15654 is assigned.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of evaluating the L-glutamic acid concentration of the nystatin-resistant strain both inside and outside the cells. The vertical axis represents the L-glutamic acid concentration, and the horizontal axis represents the strain number of the nystatin resistant strain. N46 is the parent strain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/16 C12R 1: 645) (C12N 15/01 C12R 1: 645) (C12P 13/14 C12R 1: 645)

Claims (1)

[Claims] [Claim 1] A yeast having resistance to a drug that impairs the structure and function of a cell membrane and having an improved ability to accumulate L-glutamic acid in the cells. [Claim 2] The yeast according to claim 1, wherein the drug is nystatin. [Claim 3] The yeast according to claim 1 wherein the yeast is Yarrowia lipolytica. [Claim 4] A method for producing a yeast extract, which comprises using the yeast according to Claims 1 to 3.