JPH09292A - Glutathione-containing alga and production of glutathione - Google Patents

Abstract

PURPOSE: To increase glutathione content of an alga and produce a large amount of glutathione at a low cost by culturing a marine microalga. CONSTITUTION: A glutathione-containing alga is produced by culturing Crypthecodinium cohnii belonging to marine microalga and separating the alga after the accumulation of glutathione. Glutathione is produced by culturing Crypthecodinium cohnii belonging to marine microalga to effect the production and accumulation of glutathione and separating the produced glutathione from the cultured product. The cultivation is preferably carried out in a medium containing an organic nutrient source or inorganic nutrient source, more preferably in a medium containing sea water as the inorganic nutrient source and further preferably in a medium containing a surfactant.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inexpensively producing glutathione, which is useful as a health food, a medicine and a feed, by using algae.

[0002]

2. Description of the Related Art Glutathione, which is approved as a pharmaceutical in Japan, is contained in many foods, and its utility and application are expected from a nutritional and physiological viewpoint. Furthermore, the unique effects of glutathione, such as aging suppression, detoxification, cancer prevention and suppression of the harmful effects of alcohol, have been clarified, and the development of new applications for the aquaculture industry has progressed.
There is a demand for an inexpensive mass production method of glutathione, which is widely used for the treatment of feed-induced liver damage in cultured fish such as yellowtail.

Conventional production of glutathione is carried out by an extraction method from yeast or cyanobacteria and an organic synthesis method (Fermentation and Industry 40, 631 (1982)), and a microbial method is also carried out, but it is expensive. That is a drawback. Therefore, in order to eliminate these points, as a production method from green algae, selenium, a production method using a medium containing arsenic has been proposed, but selenium and arsenic are both toxic substances However, there is a problem in that the purification method is complicated for use as a pharmaceutical, food, or feed (Japanese Patent Laid-Open No. 2-234691).

Known glutathione-producing microorganisms are improved and bred yeast such as Escherichia coli, Saccharomyces, Candida, Pseudomonas, Arthrobacter, Brevibacterium and the like. The method using immobilized Escherichia coli into which a gene has already been introduced (Agricultural Chemistry Conference, 1983 summary p.53), continuous production by bioreactors using immobilized enzymes or immobilized microorganisms are also well known (fermentation and industrial 39, 900
(1981)), and a method using immobilized cells in which a gene for an enzyme involved in glutathione synthesis is incorporated into Escherichia coli (JP-A-59-192088 and JP-A-60-180592).
And a method using a bioreactor (Japanese Patent Publication No. 7-34754) have been proposed. However, it is difficult to stably and inexpensively produce glutathione industrially by these methods.

[0005]

The object of the present invention is to increase the glutathione content in algal cells by culturing marine microalgae that have never been used for the production of glutathione, and to produce large amounts of glutathione at low cost. It is to provide a manufacturing method.

[0006]

Means for Solving the Problems As a result of earnest research for the above purpose, the present inventors have found that a large amount of glutathione can be produced by purely culturing the marine microalga Crypthecodinium connie. In addition, it is particularly preferable to add a surfactant to the medium, and further,
The inventors have found that it is preferable to use seawater as an inorganic nutrient source for the medium, and completed the present invention.

[0007] The present invention cultivates the marine microalga Crypthecodinium connie in a medium rich in organic and inorganic nutrients, thereby producing Crypthecodinium connie containing a high content of glutathione. The present invention relates to a method for growing a glutathione-containing algal cell, and a method for manufacturing glutathione by extracting and collecting glutathione from the obtained culture.

[0008] That is, the present invention is characterized in that the marine microalga Crypthecodinium cohnii algal cells are cultured to produce and accumulate glutathione, and then glutathione is collected from the obtained culture. It is a method for producing glutathione, and the present invention is preferably carried out in a culture medium containing an organic nutrient source and an inorganic nutrient source, more preferably the medium contains seawater as an inorganic nutrient source. Preferably, more preferably, the medium contains a surfactant.

[0009] Further, the present invention further comprises culturing a marine microalga Crypthecodinium cohnii alga in a medium containing an organic nutrient and an inorganic nutrient, after producing and accumulating glutathione. The method for producing a glutathione-containing alga body, which comprises separating the alga body, more preferably the medium contains seawater as an inorganic nutrient source, and more preferably the medium contains a surfactant. Preferably.

[0010]

[Operation] The present invention will be described in detail below. The present inventors conducted a culturing experiment under the culturing conditions described below, such as using Crypthecodinium connie as a marine microalgae, an organic nutrient source and an inorganic nutrient source as a nutrient source, and adding a surfactant. As a result of analyzing the obtained algal components, it was newly found that Crypthecodinium cony contained a large amount of glutathione.

The marine microalga Crypthecodinium connie used in the present invention is a well-known alga and widely inhabits the ocean. For example, ATCC (American Typ)
e Culture Collection), Crypthecodinium cohnii ATCC 30021,30543,305
56,30571,30572,30775,50051,50053,50055,50056,5005
8,50060 can be illustrated.

In the present invention, cryptecodinium
In order to increase the glutathione content in connie, both organic and inorganic nutrients are used as nutrient sources for the medium,
Further, it is preferable to add a surfactant and aerobically perform agitation culture. Further, as a method of culturing glutathione and collecting it from the alga after separation, an extraction method with an aqueous perchlorate solution, or a conventional high-temperature short-time extraction method with hot water,
In the presence of high-concentration sulfuric acid (0.2 to 2.0 N sulfuric acid), a low-temperature and long-time sulfuric acid extraction method (see Japanese Patent Publication No. 22-1586) and in the presence of low-concentration sulfuric acid (0.02-0.2 N sulfuric acid) at high temperature for a short time Sulfuric acid extraction method (see JP-A-48-600), high temperature extraction method in the presence of low-concentration acetic acid (N / 120 to N / 30 acetic acid) (Journal of
biological chemistry, 84, 269 (1929)), a short-time heat treatment extraction method in the presence of acetic acid (0.1 to 0.5 N acetic acid), and the like.

As a medium for culturing algae used in the present invention, a medium of any composition can be used as long as this algae can grow well, but Crypthecodinium connie metabolizes organic matter to heterotrophs. However, since it is used, it is difficult to culture only with inorganic salts. As the nutrient source of the medium in the present invention, both an organic nutrient source and an inorganic nutrient source are used.

Specific examples of the organic nutrient source include organic compounds such as glycerin as a carbon source, carbohydrates such as glucose and galactose, and organic acids such as acetic acid, malonic acid and citric acid, and peptone as a nitrogen source. Examples thereof include yeast extract, casein hydrolyzate, glutamic acid, corn steep liquor and the like, and a mixture of these organic nutrient sources may be used.

As the inorganic nutrient source, it is preferable to use an inorganic salt. As the inorganic salt, natural seawater is most preferable, but various artificially prepared known artificial seawater can also be preferably used, and further various kinds can be used. Sodium salt, phosphate salt, magnesium salt, potassium salt, borate salt, carbonate salt, etc. can also be used. Further, as the inorganic nutrient source, a trace amount of a metal salt, for example, an iron salt, a manganese salt, a cobalt salt, a zinc salt, a chlorine compound, a bromine compound, or an inorganic nitrogen compound such as ammonium sulfate or ammonium nitrate as a nitrogen source can be used. .

Further, as the inorganic nutrient source, a mixture of the various inorganic nutrient sources described above may be used. In addition, as the nutrient source of the medium in the present invention, it is more preferable to use seawater as the organic nutrient source and the inorganic nutrient source from the viewpoints of glutathione production rate during culture and safety of the obtained glutathione.

Further, in the present invention, it is preferable that the medium contains a surfactant from the viewpoint of glutathione production rate. As the surfactant to be used, it is preferable to use a defoaming surfactant, more preferably to use a surfactant containing a fatty acid and a sugar ester as a main component. The pH during alga body culture is preferably 6 to 8.5, and the culture temperature is preferably 32 ° C or lower, particularly 25 to 30 ° C. Further, the culture is performed under aeration and agitation conditions, and the culture method may be any culture method such as static culture, liquid shaking culture, liquid aeration / agitation tank culture, batch culture, fed-batch culture, continuous culture, but the like. From the viewpoint of productivity, it is preferable to use liquid aeration / agitation tank culture or continuous culture.

Further, the aeration amount of air at the time of liquid aeration / agitation tank culture or continuous culture is preferably in the range of 0.5 to 2.0 VVM. If it is less than 0.5 VVM, the glutathione production rate will decrease, and if it exceeds 2.0 VVM, the production rate will not increase any more.If a surfactant is used, the amount of surfactant used will increase and it will not be economical. . As a method for separating and recovering algal cells from the culture (culture solution) after the completion of the culture, a general filtration method, a centrifugation method, or the like is used.

In order to efficiently extract glutathione from the glutathione-containing Crypthecodinium connie algal cells obtained by separation and recovery, an aqueous perchlorate solution is used. In this case, glutathione is 1
It is separated by 00% and the solution is 100% when developed with 0.1% trifluoroacetic acid solution on a column packed with ion exchange resin.
Glutathione can be recovered with a yield of. Normal,
As this perchlorate, sodium perchlorate or potassium perchlorate is used. The appropriate concentration of these perchlorates in the aqueous solution is 0.15 to 0.6 N. The glutathione extraction may be carried out directly from the culture.

The alga bodies isolated from the culture obtained according to the present invention, or the cultures and the extracts from the alga bodies can be used as they are as health foods, pharmaceuticals, feeds and the like. For example, a direct extract from the culture medium after completion of the culture,
Wet algal cells, algal cell pastes recovered from the culture solution by filtration, centrifugation, etc., or heat-dried products, freeze-dried products, spray-dried products, and extracts thereof are all used as health foods, pharmaceuticals, or feeds. Can be used as

According to the present invention, it is possible to use seawater and a harmless organic nutrient source as the nutrient source of the medium, and the surfactant may be a food additive, Therefore, no special purification treatment is required.

[0022]

EXAMPLES The present invention will be described in more detail below with reference to examples. (Example 1) The medium used was the medium (1) having the composition shown in Table 1,
All of them were sterilized for 15 minutes under the conditions of a temperature of 120 ° C. and a pressure of 1.2 kg / cm ² . The medium (1) having the composition shown in Table 1 was prepared by mixing artificial seawater (Aquamarine manufactured by Yasu Pharmaceutical Co., Ltd.) with glucose, a casein hydrolyzate, corn steep liquor, and distilled water in the proportions shown in Table 1. PH 7 with normal hydrochloric acid.
It is adjusted to 0.

One platinum loop of Crypthecodinium connie [ATCC30021] was ingested in 100 ml of the medium (1) shown in Table 1,
Static culture was carried out at 28 ° C for 7 days. 5 ml of the obtained culture solution was inoculated into 10 300 ml Erlenmeyer flasks charged with 100 ml of the same medium (1) (5% by weight seed seed inoculated to 100 ml of the initial medium), and the temperature was kept for 5 days using a rotary shaker. 28
Shaking culture was carried out under conditions of ℃, pH 6.8 and rotation speed of 180 rpm.

After completion of the culturing, combine 10 culture solutions,
After centrifugation for 10 minutes under conditions of a temperature of 5 ° C. and 8000 × g, the alga bodies were collected and then freeze-dried to obtain alga bodies. 12 g of the freeze-dried alga thus obtained was extracted with a 0.6 N potassium perchlorate aqueous solution. Then, the extract is adsorbed on ODS resin to 0.1%
As a result of development with a trifluoroacetic acid solution, glutathione 12
mg was obtained.

(Example 2) The result of culturing, extracting and purifying as in Example 1 except that the medium for Crypthecodinium cohnii algae was the medium (2) (sterilized) having the composition shown in Table 2. , Glutathione 11 mg was obtained. (Example 3) The defoaming surfactant KM was added to the medium (1) of Example 1.
As a result of adding 500 ppm of -70 (ester of fatty acid and sugar) and culturing, extracting and purifying in the same manner as in Example 1, 18 mg of glutathione was obtained.

Example 4 Crypthecodinium connie was cultured in the same manner as in Example 2 to obtain 6 300 ml Erlenmeyer flask culture solutions. Using this as a seed mother, 10
6 L of the medium (2) was charged into an L-volume jar fermenter, 0.1% of antifoaming surfactant Span 80 was added to this medium and autoclave sterilized, inoculated into the medium cooled to 28 ° C, and the culture temperature was 28 ° C. Airflow rate 1.0VVM (air 6L / min), 300r
Culture was performed at pm for 3 days. After completion of the culture, algal cells were collected, extracted and purified in the same manner as in Example 1 to give glutathione 92.
mg was obtained.

[0027]

[Table 1]

[0028]

[Table 2]

[0029]

INDUSTRIAL APPLICABILITY According to the present invention, the marine microalga Crypthecodinium connie itself, which contains a high content of glutathione, can be cultivated in a large amount, and the process for extracting and separating glutathione from the obtained culture is simple and high. Since glutathione having a high purity can be recovered in a high yield, it is suitable as a method for producing glutathione.

Further, according to the present invention, since seawater and harmless organic nutrient sources can be used as nutrient sources for the medium, the obtained algal cells themselves or the extracts as they are are used as health foods, pharmaceuticals, feeds, etc. It also has an excellent effect that can be used as.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:89)

Claims (4)

[Claims] 1. A method for culturing glutathione, which comprises culturing an alga of a marine microalga Crypthecodinium cohnii to allow glutathione to be produced and accumulated and then collecting glutathione from the obtained culture. Production method. 2. The method for producing glutathione according to claim 1, which comprises culturing in a medium containing an organic nutrient source and an inorganic nutrient source. 3. The method for producing glutathione according to claim 2, wherein the medium contains seawater as an inorganic nutrient source. 4. A marine microalga Crypthecodinium cohnii alga is cultured in a medium containing an organic nutrient and an inorganic nutrient, and glutathione is produced and accumulated. A method for producing a glutathione-containing algal body, which comprises separating.