JPH0318872B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing yeast containing significant amounts of glutathione.

More specifically, the present invention relates to culturing a mutant strain of glutathione-producing yeast belonging to the genus Candeida that can grow in a medium containing ethionine and sulfite at a temperature lower than the optimum growth temperature of the strain by 5°C or more. The present invention relates to a method for producing yeast in which a significant amount of glutathione is produced and accumulated in the bacterial cells by aerobically culturing at a high temperature.

In general, glutathione is widely distributed in yeast and animal livers, and is a tripeptide that is involved in the redox system in living organisms. It is a substance.

An object of the present invention is to provide a method for producing glutathione advantageously in the fermentation industry.

In recent years, most glutathione has been produced by extraction from yeast cells, and many methods have been proposed to increase the glutathione content in yeast cells. It is being carried out in

The present inventors have conducted intensive research to increase the amount of glutathione accumulated in the Candeida genus. As a result, when culturing yeast, glutathione can be released by culturing at a culture temperature 5°C or more lower than the optimum growth temperature of the strain. They learned that it is possible to significantly increase the amount of

Further research led them to discover that the optimum temperature for glutathione accumulation is considerably lower than the optimum culture temperature for yeast strains.

The present invention was completed based on these findings,
By culturing a mutant strain of glutathione-producing yeast belonging to the genus Candeida that can grow in a medium containing ethionine and sulfite at a culture temperature that is 5°C or more lower than the optimal growth temperature of the strain, This is a method for producing yeast with a high glutathione content, which is characterized by producing and accumulating a significant amount of glutathione in the body.

In the present invention, a strain belonging to the genus Candeida that can grow in a medium containing ethionine and sulfite through mutation treatment (patent application 1983-
24815). Examples of strains that can grow in a medium containing ethionine and sulfite through mutation treatment include Candeida uchilus KJS-0571 and Candeida uchilus KJS-0571.
0582 etc. are advantageously used. In addition, Candeida uchirus KJS-0571 and Candeida uchirus KJS-0582 were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology through FERM P-6907 and FERM P-7396.
It has been deposited as.

Carbon sources used for culture include glucose,
Sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, etc. are used, and as nitrogen sources, ammonia, ammonium sulfate, urea, nitrates, etc. are used. Furthermore, as inorganic salts, lime superphosphate, ammonium phosphate, potassium chloride, potassium phosphate, caustic potassium, magnesium sulfate, magnesium chloride, etc. are used as sources of phosphoric acid, potassium, and magnesium, and trace amounts of zinc, copper,
Inorganic salts such as manganese and iron ions are also used. Vitamins, amino acids, and nucleic acid-related substances are not particularly required, but of course they may be added, or cornstarch liquor, yeast extract, peptone, etc. may be added. PH is preferably 3.5 to 8.0, especially 4.0 to 6.0.

If the culture temperature is 5° C. or more lower than the optimal growth temperature of the glutathione-producing strain, more glutathione will be accumulated. Generally, a temperature that is 5°C or more lower than the optimum growth temperature of the strain is suitable for accumulating glutathione. If only the accumulation of glutathione is considered, the most preferable culture temperature is 6 to 12°C lower than the optimum growth temperature.

Since the culture temperature in the present invention is 5° C. or more lower than the optimum growth temperature of the strain, the growth of yeast tends to be slow. Therefore, when lowering the culture temperature, it is necessary to lengthen the culture time. Although it varies depending on the strain, generally it is about 25 to 50 hours.

The culture format at this time may be either batch culture or continuous culture as long as it is aerobic culture.

Next, test examples, examples, and comparative examples will be given and explained. The quantification of glutathione was carried out using the glyoxalase method (Method in Enzymology, Vol. 1, p. 540, Academic Press, 1955 edition) using the bacterial cell extract.

Further, the usefulness of the present invention is shown in the following examples, but the present invention is not limited thereto.

Test example 1 Kyandida uchirus KJS-0582 strain
Using FERM P-7396, the culture temperature was varied in nine stages from 18°C to 34°C, and the remaining conditions were the same as in Example 1 except that the scale was 1/10. Rate and glutathione content were measured. The growth rate is the specific growth rate (0.693/doubling time) in the logarithmic phase at 32°C by 100.
%, and the specific growth rate at each culture temperature was determined as a relative value. The glutathione content was determined by collecting the bacterial cells after culturing at each culture temperature and treating them in the same manner as in Example 1.

The results are shown in Figure 1.
In the case of the KJS-0582 strain, it can be seen that the optimum temperature for growth is 32°C, and the optimum temperature for glutathione accumulation is 22°C to 24°C.

Test example 2 Kyandida utilis KJS-0571 strain,
Using FERM P-6907, the relative specific growth rate (%) and glutathione content were determined in the same manner as in Test Example 1.

The results are shown in Figure 2.
In the case of KJS-0571 strain, the optimum growth temperature is 30-32℃
It can be seen that the optimal temperature for glutathione accumulation is 22°C.

Example 1 Kyandida uchilus KJS-0582 strain,
FERM P-7396 was cultured in a flask in a medium consisting of 2% glucose, 2% yeast extract, and 1% peptone, and 1% of this was inoculated into a 300 volume fermenter.

As a medium, glucose 3%, ammonium sulfate 0.8%,
Phosphate - potassium 0.2%, magnesium sulfate 0.03
%, ferrous sulfate 10ppm, zinc sulfate 3ppm, copper sulfate
Batch culture was performed using 1 ppm and 10 ppm of manganese sulfate. The culture conditions are: tank liquid volume 200, culture temperature 24℃, ventilation rate 150pm, stirring number 200rpm, PH
I went with 4.5. When the bacteria were collected after 28 hours, 2940 g of dry cells were obtained, and the glutathione content in the cells was 5.0% (ratio to dry cells). The cells were heated and extracted, and the cell residue was centrifuged to obtain a glutathione extract. Add sulfuric acid to this extract
The solution was added to a concentration of 0.5N, and cuprous oxide was added to precipitate glutathione as a copper salt. After washing the glutathione copper salt with water, hydrogen sulfide is aerated, the copper sulfide is removed, and the glutathione crystals are concentrated under reduced pressure.
Obtained 120g.

The obtained crystals were confirmed to be glutathione by high pressure filter paper electrophoresis and high performance liquid chromatography, and the purity by the iodine method was 99.0%.

Comparative example 1 Kyandida uchirus KJS-0582 strain,
FERM P-7396 strain was cultured in the same manner as in Example 1 except that the culture temperature was 30°C for comparison. After culturing for 22 hours, 2950 g of bacterial cells (calculated when dried) were obtained. The glutathione content in the bacterial cells was 3.7% (based on dry bacterial cells). 87 g of glutathione was obtained by the same treatment as in Example 1.

Example 2 Candeida uchilus KJS-0571 strain,
FERM P-6907 was cultured in a flask in the same manner as in Example 1, and 30 fermenters were inoculated at 5%.

A medium with the following composition was used. Add 0.15% ammonium phosphate and 0.06% potassium chloride to sulfite pulp waste liquid (3% as assimilable sugar). Culture is carried out in a fermenter with a draft tube, reducing the amount of liquid in the tank.
10.Culture temperature 22℃, aeration rate 10pm, stirring number
It was carried out at 700 rpm, and ammonia was added to control the pH and serve as a nitrogen source for the culture. After 42 hours of incubation, the bacteria were collected by centrifugation and yielded 160g of bacteria.
was obtained, and the glutathione content in the bacterial cells was 4.6% (ratio to dry bacterial cells).

Comparative Example 2 The Candeida uchilus KJS-0571 strain was cultured in the same manner as in Example 2 except for the culture temperature of 30° C. for comparison. 165g of bacterial cells after 32 hours of culture (dry equivalent)
was obtained, and the glutathione content in the bacterial cells was 3.8%.
(ratio to dry bacterial cells).

[Brief explanation of drawings]

Figure 1 shows the relative specific growth rate (%) and glutathione content (%) of Candeida utilis KJS-0582 strain in Test Example 1, and Figure 2 shows the relative growth rate (%) and glutathione content (%) of Candeida utilis KJS-0572 strain in Test Example 2.
It is a diagram showing the relative specific growth rate (%) and glutathione content (%) of the strain. A... Relative growth rate (%), B... Glutathione content (%).

Claims (1)

[Claims] 1. By culturing a mutant strain of glutathione-producing yeast belonging to the genus Candeida that can grow in a medium containing ethionine and sulfite at a culture temperature that is 5°C or more lower than the optimal growth temperature of the strain, A method for producing yeast with a high glutathione content, which is characterized by producing and accumulating a significant amount of glutathione in bacterial cells.