JPS59151894A - Production of yeast of high glutathione content - Google Patents

Abstract

PURPOSE:A yeast in Candida is subjected to mutagenic treatment and the resultant mutant resistant to sulfites and ethionine is cultured to produce yeasts of high glutathione content. CONSTITUTION:A yeast in Candida is subjected to mutagenic treatment with X-rays or nitrosoguanidine to obtain a yeast resistant to both sulfite and ethionine, for example, Candida utilis KJS-0571 strain (FERM-6907) from Candida utilis IAM4264. Then, the mutant is aerobically cultured in a medium containing ethionine and sulfite salt over the minimum inhibition concentrations, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention (1.
This is related to the beshi construction method.

More specifically, the present invention involves aerobically dissolving a mutant strain of yeast of the genus Canodida that can grow in a medium containing both Ethionia and sulfuric acid through mutation treatment. The present invention relates to a method for producing tfF mother in which a large amount of Gurkuchi is accumulated.

General 1) Glutathion is widely distributed in yeast and liver moths, and butide is involved in the redox system in living organisms, and plays important roles such as liver function recovery and detoxification. It is an extremely useful substance in medicine.

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

In recent years, most of glutathione has been produced by the extraction method from yeast cells, and many methods have been proposed and attempted to increase the glutathione content in yeast cells.

However, the amount of glutathiono accumulated by either method was limited to 't1', and none of the methods could be said to be particularly superior industrially.

The present inventors first developed a yeast containing a significant amount of glutathione by culturing a mutant strain of yeast of the genus Gitonodeida that has acquired methionine/analog resistance.
was successful (Special Publication No. 56-46826),
As a result of extensive research in order to increase the amount of glutathione accumulated, we were able to produce yeast with a higher accumulation amount in the present invention.

Inventors 1 and ? i4 In order to increase the amount of money that is circulating in the body, it is necessary to use one of the amino acids that make up glutathione.
RUSH - Cysteine inside the bacterial body - F [~ I thought that fighting is the essence of it. In order to increase the intracellular ζ~ degree of L-cysdine, the combination of L-cystine; r? At the same time as increasing the intracellular ν418 of 1 night, during other biosynthesis of L-7 sudein: 1
It is necessary to increase the 1.5 change in the bacterial body of Methioni, which is 11 bodies, and for that purpose, 1 degree of suffering L (41 drunkenness 1 student and Megu-237 prog Ethionia!li41 cow 1) [Under the assumption that the first thing to do is to change the ``needle 1y 144'', we carried out mutation JzJl of Kya/-j-Ida 11 yeast, and found that 4'O-44e 1trt and EchiA2 As a result of culturing this 2"L strain, we found that a significant amount of curtathione was accumulated, which led us to complete the present invention.1. Due to treatment 1, Canoteida 1 was able to grow in a medium containing Ethionii/ and →Tokoe at the same time.
A mutant strain of common yeast was cultured aerobically, and a significant amount of +
This is a method for producing a ferment with a high glutathione content, which is characterized by accumulating glutathione.

Spit used in this invention! The accumulation of glutathione by the mutant u strain that has both A acid resistance and ethionine production is extremely rapid, and 'f+-Of wax, f also has a uniquely rigid mutant strain or ethionine alone 4 sex mutant 1 (vermilion glutathione accumulation). Even compared to Qiqing, it's noticeable ((it's Q immediately).

In addition, since it has a strong anti-mountain activity against bacteria (11), by using a mutant strain that has 1 well of 11 bacteria, 12 people can be vomited underground.
< 1% The name is chosen because it can prevent bacterial contamination.

The present invention's Ethionii/ and A'I 14 were grown to a capacity of 6 plants on a tr-containing medium at 1 h).
The mutant strains include the well-known strain of W.
+6826 +, for example Kya/Deida Utilisg+t
o61. T" [LM-PNo. 3510 etc.' (Ultraviolet light.

Ethionine and hydrogen sulfate were added to J'
☆ It can be obtained by culturing in a medium containing a low initial concentration, and selecting bacteria with good growth and f in a synthetic medium. The ethionine used at this time may be D-type, L-type, or DL-type;
rllj sulfur exchange J graves use E (bootrium stirrupate, potassium sulfate, tunshin-asa, etc.).

One of the high glutathione/containing paternal strains prayed for in the misfired Uno is Cantida Utelus IAM li
There is Candida uchilus KJS-0571 strain obtained by different treatment of 264.
Agency of Technology Microbial Technology'jFJ (L'f Institute 1-"
Deposited as En, Ml'-69075.
4 Ko (・No use! Haru 4! I11'; Kan, 3 resistance 1 student and ethionine! 1 I11 and L is f no (Y-C Ru gear/Dida fg fermentation IU mutant strain is negative) The sex 'r9 shows the same sex glume, and then the 7, 1 ethioni /
Friend υ" = 11. Grows in jg soil containing uniformity with sulfur 1'1 2, -1i ?' ~ [ ; 5, grows well in ρ 1, Fi i
It is possible to make a "T" kurtachimen tip and a J pattern.

3. Y 'J '4J': Forms a soft colony with all 8-1 semi-lenticular ridges, coat + #j i'jj smooth and pure color, and cre-1 color on heavenly medium F. .

4. Glucose-Yeast Extract-Haft 7 Cloudy and precipitated in water, and formation of y1 membrane and Yu ring was not observed.

5. Forms pseudohyphae in the form of Darmo's plate.

6. Sodium acetate λP large slant, malt extract 45 slant...i.

When culturing on V8 juice agar slants and Co7 meal agar slants, formation of larvae and ia agars was not observed.

Z Glucose, sucrose, and raffinose are fermented to produce calactose, maltose, trehalose, and lactose.

8 glucose, 7 ucrose, maltose.

Cellobiose, trehalose, raffinose.

Grows well in a 9-vitamin-free medium that assimilates melezitose, xylose, ethanol, man/nitol, milk, quinol, and KNOl.

The present invention is carried out by 1Gjl'ζ1-i, and the tiζ change capsule strain obtained by the method described in the present is used to produce lutathione in the body,
Things that increase accumulation 'F <''r(・; 1 nra cold)
Without JQ, the present inventors have found that in order to increase the content of curtabio/ in bacteria, it is necessary to increase the intracellular jealousy of L-cysteine, which is one of the constituent amino acids of glutathione. I thought it was. and ■, -Cysti/'s intracellular ζ~; To increase W, raw metal of L-cystine; 1. Inside the fungal body of bamboo shoots and vinegar which are lIj bodies y$ii
At the same time, it is necessary to increase the intracellular content of methionine, which is another biosynthetic intermediate of L-containing protein. Based on the assumption that the first step is to obtain a mutant strain that is resistant to Ethionia, we performed mutation treatment on yeast of the genus Ca/Deida, and obtained a mutant strain that is resistant to both sulfur and roe and resistance to Ethionia. As a result of culturing this, it was discovered that glutathione was accumulated in the young leaves, leading to the completion of the present invention. Through mutation treatment, Candida became able to grow in a medium containing ethionine and Ameco at the same time. F.A.
This is a method for producing yeast with a high glutathione content, which is characterized by culturing a yeast mutant strain aerobically to produce and accumulate a significant amount of glutathione in the cells.

Ability 44 used in the present invention Accumulation of glutathione by the mutant strain that has both acidity and ethionine resistance is extremely rapid, and glutathione/accumulation in the mutant strain having both acidity and ethionine resistance is observed Even compared to the amount)I
It's as good as any name.

In addition, since it has strong antibacterial activity against A.
By increasing νl+1j, it is possible to prevent contamination of culture medium 4 with bacteria.

The Ca/Deida mutant strain that can grow in a medium containing both ethionine and immediate salt of the present invention is -46826,
l, such as Ca/Deida utilis E11061. FE
RM-PNo.3510 etc. under ultraviolet light.

The mutated bacteria were subjected to mutation treatment using X-rays, nitroligine, etc., and were cultured in a medium containing etionia and sulfur salts at a minimum inhibitory concentration or higher, at a culture temperature of 30°C.
a air volume 150/pan, stirring number 20 Orpm,
It was performed at f4.5. After culturing for 24 hours and harvesting, 2,950 g of dry cells were obtained, and the percentage of glutathione in the cells was 4.0% (ratio to dry cells). The cells were heated and extracted, and the cell residue was removed by centrifugation to obtain a glutathione extract.

Sulfuric acid was added to this extract at a concentration of 0°5N, and oxidized steel was added to precipitate glutathione as a copper salt. After washing the glutathione copper salt with water, hydrogen sulfide was aerated and (piL
950 g of glutathione crystals were obtained by removing the chemical steel and concentrating it under reduced pressure.The resulting crystals were confirmed to be glutathione by high-pressure filtration, high-speed body chromatography, and Ward method. Purity was 99.0%.

Example 2 Sudden change ¥4 stock Kya/Deida Utilus KJS-0571
The strain I''E I (P=6907) was cultivated in a flask as in Example 1 and 501% fermented in a fermenter.

1-1 The following composition was used as the ground. ``Reject', (
Il (+, am/monium phosphate o, 1ss, potassium tiride 0.06 in pulp waste liquid (5% as assimilable sugar)
Add %. Culture is carried out in a fermenter with a draft tube, with tank liquid 1101. Culture 1ijJj 30'C, aeration rate 10
/pm.

Stirring was performed at 7 Q Q rpm, and ammonia was pressurized to serve as pII co/trol and a nitrogen source for the culture. After 62 hours of culture, the cells were collected by centrifugation 1 (1), and 165!j of tooth cells (converted to dry cells) were obtained, and the glutathione content in the cells was 38% (ratio to dry cells).

Comparative Example 1 In exactly the same way as in Example 1, the parent strain Canyig utilis I
As a result of culturing and comparing A M 4264 strains, 5oso
After drying No. 9, the v1 body was dried, and the glutathionofin: i in this dried m body was 0.52% (ratio of dry bacterial cells). '8.1 by J41 in exactly the same way as in Example 1.
, 9 glutathione! Star. .

Nobuto Dai, patent attorney, 1) Parent, male

Claims (1)

[Claims] (Sudden change) (Due to treatment, the strain of Candida yeast became able to grow in a medium containing ethionic acid and geochloride at the same time. The strain was cultured aerobically, and a significant amount of glutathione A method for producing glutathiono-containing yeast, characterized by producing and storing M/.