JP3896606B2 - Production method of yeast extract - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a yeast having resistance to a drug that impairs the structure and function of cell membranes and having an improved ability to accumulate L-glutamic acid in the microbial cells, and a method for producing a yeast extract using the yeast.
[0002]
[Prior art]
The demand for natural seasonings continues to expand in the current trends that can be expressed by keywords such as natural, natural, healthy, full-scale, luxury, and gourmet. Yeast extract has also expanded its market scale with the development of natural seasonings. ing. In general, beer yeast, baker's yeast, Torula yeast, etc. are used for the production of yeast extract. When using beer yeast produced as a by-product in beer brewing, selection and improvement of yeast within the beer quality design range is performed. It is pointed out that it is not necessarily satisfactory from the viewpoint of quality such as unpleasant odor and lack of umami taste because it is not a yeast only for the purpose of producing the extract. Has been.
[0003]
As a method for solving these problems, there is a method in which yeast is bred and improved to produce a yeast having a preferable taste and flavor, and a yeast extract is produced using the yeast. In terms of flavor, there is a known example in which aroma components such as esters and alcohols are increased by using yeast imparted with norvaline resistance (Journal of the Institute of Brewing, Vol. 76). 843 to 847, 1981) The production of alcoholic beverages using this has been reported. However, an example in which the taste such as umami is changed is not known.
[0004]
L-glutamate sodium, 5′-sodium inosinate, and 5′-sodium guanylate are known as substances that enhance the taste of umami and the like, and are added to foods as seasonings according to their use.
[0005]
[Problems to be solved by the present invention]
The object of the present invention is to develop a method for efficiently breeding yeast having a high ability to accumulate L-glutamic acid, which is a substance that enhances umami taste, in the microbial cells, and yeast having a higher L-glutamic acid content using the bred yeast. It is to find a method for producing an extract. The above problem has never existed before, that is, the problem itself is new.
[0006]
[Means for Solving the Problems]
The present inventors have found that a yeast mutant imparted resistance to a glutamine antimetabolite such as 6-diazo-5-oxo-norlosin accumulates L-glutamic acid at a high concentration in the microbial cell, When a yeast extract was prepared, the L-glutamic acid concentration in the extract was high, and as a result of sensory evaluation, it was found that the yeast extract has an increased umami taste and excellent taste such as flavor, hot taste, and body.
[0007]
However, among the yeast mutants imparted with 6-diazo-5-oxo-norleucine resistance, the intracellular L-glutamic acid concentration was not different from that of the parent strain, but rather, the extracellular L-glutamic acid accumulation amount was improved. . Moreover, even if it is a yeast mutant which gave 6-diazo-5-oxo-norleucine resistance and the mutant L-glutamic acid concentration increased, it has accumulated a large amount of L-glutamic acid outside the cell. It was.
[0008]
The present inventors can grow a yeast having a high intracellular L-glutamic acid concentration if L-glutamic acid biosynthesized by a yeast mutant that produces a significant amount of L-glutamic acid can be specifically retained in the bacterial body. As a result, it was considered possible to prepare a yeast extract having a high L-glutamic acid content and excellent taste.
[0009]
Therefore, as a result of further investigations to solve such a new problem, the present inventors confer L-glutamic acid in the bacterial body by imparting resistance to a drug that impairs the structure and function of the cell membrane of yeast. The inventors have found that yeast having a high ability to accumulate can be produced efficiently, and have completed the present invention.
[0010]
The present invention is as follows.
(1) Yeast which has resistance to a drug that impairs the structure and function of cell membranes and has an improved ability to accumulate L-glutamic acid in the microbial 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, wherein the yeast according to (1) to (3) is used.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
[0012]
The yeast used in the present invention is a yeast belonging to the genus Saccharomyces (Saccharomyces cerevisiae, Saccharomyces ubalum, Saccharomyces chevalier, Saccharomyces roseii, etc.) Examples include yeasts (for example, Kleiberomyces thermotolerance), yeasts belonging to the genus Pichia (for example, Pichia membranefaciens), yeasts belonging to the genus Yarrowia (for example, Yarrowia lipolytica), and the like.
[0013]
Examples of drugs that impair the structure and function of the cell membrane used in the present invention include polyene antibiotics such as amphotericin B and nystatin, and antibiotics that inhibit ergosterol synthesis such as econazole, miconazole, and ketoconazole. Commercially available antibiotics can be used. A particularly preferred drug is nystatin.
[0014]
The yeast of the present invention is a yeast that has resistance to a drug that impairs the structure and function of cell membranes and has an improved L-glutamic acid concentration in the cell, and if it has such properties, the L-glutamic acid concentration in the cell It may have resistance to other drugs that are effective in improving the resistance, such as 6-diazo-5-oxo-norleucine and bromopyruvic acid used in the present invention.
[0015]
In order to confer resistance to a parent strain on a drug that impairs the structure and function of the cell membrane, the parent strain is irradiated with ultraviolet light, or a mutagenic agent (for example, N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfone). After treatment with acid or the like, 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 such that the parent strain cannot grow may be collected.
[0016]
A mutant resistant to a drug that impairs the structure and function of the cell membrane is a strain that is more resistant to the drug when compared to the parent strain.
[0017]
The yeast of the present invention is a yeast that is resistant to drugs that impair the structure and function of cell membranes and that has an improved L-glutamic acid concentration in the cells. For example:
Yarrowia Lipitica AJ14718
[0018]
The medium used for culturing yeast in the present invention to obtain microbial cells is a normal nutrient medium containing a carbon source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary. Either synthetic or natural media can be used. The carbon source and nitrogen source used in the medium may be any one that can be used by the yeast to be cultured.
[0019]
As the carbon source, glucose, glycerol, mannitol, ethanol, n-paraffin and the like are used. Organic acids such as lactic acid and citric acid can also be used alone or in combination with other carbon sources.
[0020]
As the nitrogen source, urea, ammonia, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, ammonium acetate and the like are used.
[0021]
As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, and peptone, casamino acids, yeast extracts, soy protein digests, etc. containing these are used, and wild strains that require amino acids, vitamins, etc. for growth In addition, when cultivating auxotrophic mutants, it is necessary to supplement the required nutrients.
[0022]
As the inorganic salts, phosphates, magnesium salts, iron salts, zinc salts, calcium salts and the like are used.
[0023]
As the culture method, aeration culture is performed at a culture temperature of 20 to 37 ° C. and a pH of 3 to 8. When the pH falls during the cultivation, calcium carbonate is added or neutralized with an alkali such as ammonia water or ammonia gas. Thus, yeast cells can be obtained by culturing for about 10 hours to 4 days.
[0024]
In order to prepare a yeast extract from the obtained microbial cells, the microbial cells may be collected from the culture broth by centrifugation and performed according to a known method. For example, toluene or ethyl acetate is added at 1 to 2% per cell, self-digestion is performed at a temperature of 45 to 50 ° C., the extract is concentrated under reduced pressure, and a powdered yeast extract is obtained by spray drying.
[0025]
【Example】
Next, the present invention will be described in more detail with reference to examples.
[0026]
[Example 1]
Isolation of Bromopyruvic Acid-Resistant Strain from Yarrowia lipolytica AJ5004 Yarrowia lipolytica AJ5004 (Agricultural and Biological Chemistry, Vol. 33, pages 158-167, 1969) isolated from nature was sporulated medium (1% acetic acid Potassium, 0.1% yeast extract, 0.05% glucose) for 1 to 2 days at 30 ° C., washed with 0.85% physiological saline, and finally containing 0.1 mg / ml zymolyase 5000. It was suspended in 85% physiological saline and treated at 30 ° C. for 120 minutes. Further, the mixture was filtered through a nylon membrane having a pore size of 60 μm to collect spores. Suspension in 0.1 M phosphate buffer (containing 2.5% final concentration of glucose) to which ethyl methanesulfonic acid was added to a final concentration of 30 μl / ml was performed, and mutation treatment was performed at 30 ° C. for 60 minutes. . After centrifugation, the spores are washed with 0.1 M phosphate buffer and minimal medium containing 0.5 g / l bromopyruvic acid (Difco East Nitrogen Base Without Amino Acid 0.67%, glucose 2%, agar 2%) and cultured at 30 ° C. for 3 to 7 days.
[0027]
Bromopyruvic acid, known as an antimetabolite of pyruvic acid, inhibited the growth of Yarrowia lipolytica AJ5004, and no growth of Yarrowia lipolytica AJ5004 was observed on a minimal medium containing 0.5 g / L of bromopyruvic acid.
[0028]
A colony that appeared on a minimal medium containing bromopyruvic acid was picked up, and a single colony was isolated on the minimal medium having the same composition, and then a medium for cell preparation (n-paraffin 80 g / l, ammonium nitrate 20 g / l, phosphoric acid) Potassium dihydrogen 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, calcium carbonate 20 g / l, Inoculated in a 500 ml Sakaguchi flask into which 20 ml of pH 5.0) was injected, seeded at 30 ° C. for 2 days, then inoculated into a 500 ml Sakaguchi flask into which 20 ml of the same composition was injected, and cultured at 30 ° C. for 5 days. After that, the culture broth was centrifuged, and the L-glutamic acid concentration in the supernatant was measured with Biotech Analyzer AS200 (Asahi Kasei Co., Ltd.). N46 strain (AJ14717) that accumulates 230 mg / l of L-glutamic acid in the cell was found.
[0029]
[Example 2]
Isolation of nystatin resistant strain from bromopyruvate resistant strain N46 (AJ14717)
Spores of bromopyruvate resistant strain N46 (AJ14717) were prepared by the method used in Example 1, and suspended in a 1 M sorbitol solution to which N-methyl-N′-nitro-N-nitrosoguanidine was added to a final concentration of 10 mM. It became cloudy and was subjected to a mutation treatment at 30 ° C. for 20 minutes. After centrifugation, the mutated spore was washed with 1 M sorbitol solution and applied to a minimal medium containing 1 g / l nystatin. The bromopyruvate resistant strain N46 (AJ14717) could not grow at all on a minimal medium containing 1 g / l nystatin.
[0031]
After culturing at 30 ° C. for 3 to 7 days, colonies appearing in a minimal medium containing 1 g / l nystatin were picked up and separated into single colonies with a medium having the same composition, and then 5 ml of the cell preparation medium used in Example 1 was added. The inoculated 50 ml large test tube was inoculated and cultured at 30 ° C. for 5 days. After completion of the culture, 1 ml of culture broth was taken 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 the extraction residue was removed by centrifugation, the L-glutamic acid concentration in the supernatant was measured with Biotech Analyzer AS200, and the measured value was taken as the L-glutamic acid concentration in which the inside and outside of the cells were combined. On the other hand, the extracellular glutamic acid concentration was obtained 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 L-glutamic acid concentration in the microbial cell can be expressed by a value obtained by subtracting the extracellular L-glutamic acid concentration from the L-glutamic acid concentration in the microbial cell.
[0032]
Of the 13 nystatin resistant strains that were isolated and evaluated for culture, there were 9 strains having a higher L-glutamic acid concentration than the parent strain N46 (AJ14717), both inside and outside the cell (FIG. 1). Among the 13 strains, only one strain had a significantly improved extracellular L-glutamic acid concentration compared to the parent strain. Nine strains with improved L-glutamic acid concentration, both inside and outside the cells, had no significant increase in the extracellular L-glutamic acid concentration compared to the parent strain, and were equivalent to or decreased from the parent strain. It was revealed that the L-glutamic acid concentration in the body was significantly improved.
[0033]
In the nystatin resistant strain AJ14718 having the highest L-glutamic acid concentration outside and inside the cells, 700 mg / l (3.5% per cell dry weight) of the cells and 20 mg / l of L-glutamic acid were accumulated outside the cells.
[0034]
Only the carbon source of the cell preparation medium of Example 1 was changed from n-paraffin to glucose (8%), and culture evaluation was performed. The cultured strains were cultured in YPD agar medium (Difco Bacto yeast extract 1%, Difco Bacto peptone 2%, glucose 2%, agar 2%) at 30 ° C. for 2 minutes. 16 plates were inoculated into a 50 ml large test tube into which 5 ml of cell preparation medium (carbon source glucose) was injected, cultured at 30 ° C. for 5 days, and the L-glutamic acid concentration inside and outside the cells and in the cells was determined. When measured, AJ14718 accumulates 530 mg / l L-glutamic acid inside the cell and 42 mg / l outside the cell, while bromopyruvic acid resistant strain N46 (AJ14717), which is the parent strain, contains 198 mg / l outside the cell. 49 mg / l was accumulated. It was revealed that even when the carbon source was changed to glucose, the concentration of L-glutamic acid in the cells was significantly improved in the nystatin-resistant strain as in the case where the carbon source was n-paraffin.
[0035]
【The invention's effect】
The L-glutamic acid concentration in the yeast cells can be increased by the method of the present invention, and a yeast extract having a high L-glutamic acid content can be produced by using the yeast bred by the method of the present invention.
[0036]
Yarrowia Lipolytica AJ14717 was deposited on May 29, 1996, at the Biotechnology Institute of Industrial Technology (Postal code 305 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan). -15653.
Yarrowia Lipolytica AJ14718 was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Postal Code 305 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan) on May 29, 1996. -15654 is given.
[Brief description of the drawings]
FIG. 1 shows the results of evaluation of L-glutamic acid concentration of nystatin-resistant strains 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 a parent strain.

Claims (1)

A method for producing a yeast extract, characterized by using Yarrowia lipolytica yeast that has resistance to the drug nystatin, which impairs the structure and function of cell membranes, and has the ability to accumulate 530 mg / l or more of L-glutamic acid in the cells.

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