JPH0757183B2 - Selenium-containing microbial cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing selenium-containing microbial cells. More specifically, the present invention relates to a method for producing organic selenium, which is expected to be used as a drug, for example, an anticancer agent, an antidote for heavy metal poisoning, an anti-inflammatory agent and the like.

Conventional Technology Selenium (Se) is an element with an atomic number of 34 and an atomic weight of about 79, which has properties very similar to sulfur. This Se is widely distributed in the natural world, but it is the 40th most abundant resource in the crust, and it is also produced as Clausthal ore, selenium copper silver mine, and krug ore, but generally copper and lead. , As a by-product of nickel ore, and SeO ₂ , Se, Na ₂ SeO ₃ as by-products during the smelting of these metals.
It has been collected in the form of.

Conventionally, selenium has been mainly used in the glass industry in addition to decolorizing agents, colorants, photoconductive materials for xerography, catalysts, etc., higher pigments, auxiliary materials for photovoltaic cells, or lubricating oil additives, rubber vulcanization accelerators, or special Insulating oil for use
(SeF ₆ ) plays an important role industrially.

On the other hand, selenium compounds are toxic and often cause dermatitis when contacted, and acute poisoning of livestock due to excessive intake of selenium has long been known. However,
K. Schwarz et al., Journal of American Chemical Society (J.Amer.Chem.Soc.), 1957, 79 , 3292-3293,
Since selenium was proven to be a biotrace element, it has been shown to be an essential trace element for mammals.

That is, selenium has adverse effects such as poisoning when it is collected in a large amount, but when it is deficient, it causes various diseases such as liver necrosis of mice, rats and monkeys, and whiteness of sheep, cattle, horses, etc. It is known that myopathy, cardiomyopathy, etc. cause human endemic diseases such as Keshan disease and Kashinbeck's disease in northeastern China.

Currently, in these various diseases, it has been confirmed that the preventive and therapeutic effects are achieved by the administration of selenium,
The compound form at the time of administration has also been investigated.
For example, Richard A. Passwater (Richard A.
Passwater's "Selen as feed and medicine
ium as Food and Medicine) ”184-188 (1980
Karts Publishing Inc. (Keat
s Publishing Inc.), it has been proved that organic compound type selenium exhibits a biological effect 10 to 20 times as high as that of inorganic compound type selenium, and its toxicity is extremely low.

The physiological action of selenium is proved by the action of an enzyme (animal glutathione peroxidase) containing selenium as an essential component and the reactivity of the selenium compound itself. It is based on its existence as a form, directly or indirectly entering the enzyme protein, and exhibiting a unique reactivity. Furthermore, this organic selenium has the effect of increasing physiological effects when used in combination with vitamin E, plays an important role in mitochondrial respiratory function and drug metabolism in microsomes, and is also an antidote for heavy metal poisoning and an anticancer agent. There are also great expectations for its use as a drug such as an anti-inflammatory agent.

For example, the anticancer activity of selenium has been gradually elucidated in recent years, and the evidence that supports the anticancer activity of selenium in animal experiments and mutagenicity tests originated from epidemiological studies on selenium content in food and cancer. Has been submitted. Furthermore, Jansson et al. [Cancer,
1977, 36 , 2373] pointed out that the incidence of colon cancer, rectal cancer, breast cancer, gastric cancer, etc. is high in low selenium soil areas in the United States.

As a method for producing such organic selenium, there is a method of enzymatically synthesizing (for example, “Chemistry and Biology” No. 20 of Esaki, Soda, etc.).
Vol. 425, 1982; JP-A-54-5 using methionase.
Invention of 2033; See invention of 58-146286 using tryptophan synthetase), a method of absorbing and accumulating in microorganisms such as brewer's yeast (Kenhiko Shino, Nu Food)
Industry (New Food Industry), Volume 27, 65-7
6,1985; JP-B-55-36314, invention, JP-A-54-46881
However, since the organic selenium production rate and the content rate are low and the operation is complicated in any of the methods, the organic type selenium is highly efficient and inexpensive. It has been difficult to produce type selenium. By the way,
For example, brewer's yeast is at most about 800 μg selenium / g dry cells, and even when using other bacteria, radioactive bacteria, mold, etc., the limit is several thousand μg selenium / g dry cells.

Problems to be Solved by the Invention As described above, selenium is an essential trace element for mammals, and its deficiency causes various diseases. These diseases can be prevented and treated by administration of selenium, and since organic selenium has high efficacy and toxicity is extremely low, various manufacturing methods have been proposed. However, in the conventional method, the yield is poor, and the operation is complicated in terms of method, and it has been desired to develop a method that can be mass-produced at low cost.

Therefore, it is an object of the present invention to provide a method for producing organic selenium, which has great expectations for its use as a drug, with high efficiency and good mass productivity, and which is easy to operate.

Means for Solving Problems The present inventors have conducted various studies and researches to solve various drawbacks of the conventional methods as described above, and as a result, absorb and accumulate selenium in microbial cells having a remarkable growth rate. Based on the idea that a large amount of organic selenium could be produced at low cost, we conducted a diligent search for microorganisms that can grow in high-concentration selenium-containing medium and that can accumulate high-concentration organic selenium in cells. . As a result, they have found that certain microorganisms belonging to the genus Bacillus show high resistance to high-concentration selenium and can accumulate a large amount of organic selenium in their cells, and completed the present invention.

That is, the method for producing a selenium-containing microbial cell of the present invention belongs to the genus Bacillus and is optimal for alkaline growth.
Alkali microbial having pH and selenium accumulating ability,
It is characterized in that it is grown in an alkaline medium containing a selenium compound.

The alkalophilic microorganism used in the method of the present invention is newly isolated from nature by the present inventors, and its mycological properties are as shown in Table 1 below. Since these cells are typical alkalophilic bacteria, add 1% sodium carbonate to the medium used to adjust the pH of the medium.
Tested in the alkaline condition of 10.5.

From the results shown in Table 1 above, this strain is an aerobic spore bacillus, has motility, and is positive for Gram's stain, so it is clear that it belongs to the genus Bacillus, but it is neutral. It is difficult to grow at a pH in the vicinity, so it is different from the conventionally known bacteria of the genus Bacillus.

In addition, these strains are
FERM P-8488 (SY-133 strain) and FERM P-8487 (SR-1
38 bacteria).

The method of the present invention, a predetermined medium, after adding an aqueous solution of a predetermined amount of selenium compound sterilized by a membrane filter, inoculate the production strain as described above, in the range of 30 ~ 37 ℃. It can be carried out by aerobically culturing at a temperature for 24 to 96 hours, whereby a target bacterial cell containing a large amount of organic selenium can be advantageously produced.

The medium for culturing the above-mentioned producing bacteria contains a carbon source, a nitrogen source, a selenium source, and, if necessary, known micronutrients, and also the bacterium used to carry out the method of the present invention. Since the optimum pH is in the alkaline range, it is necessary to adjust the pH of the medium.

First, various conventionally known materials can be used as the carbon source, and typical examples thereof include glucose, starch syrup, and soluble starch. The nitrogen source is not particularly limited, and yeast nitrogen, peptone, meat extract, corn steep liquor, amino acid solution, organic nitrogen such as soybean meal (of course, these are also effective as a carbon source), Alternatively, ammonium sulfate, urea, inorganic nitrogen such as ammonium nitrate, ammonium chloride and the like can be cited as an example which is particularly inexpensive and easily available.

On the other hand, as the pH adjustor, any one can be used as long as it can adjust the pH of the medium to be alkaline, such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide and calcium hydroxide. The pH of the preferred medium in the method of the present invention is in the range of 8-12. Actually, it is carried out under the optimum pH of each microorganism.

Further, a selenium source is added to the medium, and the selenium source is preferably a water-soluble selenium compound such as sodium selenite, sodium selenate, selenious acid, selenate, and selenium dioxide. In particular, sodium selenite (Na ₂ SeO ₃ ) is particularly preferable. The concentration of this selenium compound in the medium is preferably within the range of 0.1 to 500 mmol. That is, it is difficult to obtain high selenium-containing cells under a concentration of less than 0.1 mmol, or it takes a long time. On the other hand, even if the concentration is 500 mmol or more, it is not possible to increase the selenium content of the cells so much. This is not preferable because it may not be possible and the growth of microorganisms may be hindered.

In addition to the above components, various commonly used salts, for example, inorganic salts such as magnesium salts, potassium salts, phosphates, iron salts, vitamins and the like can be used.

Thus, the medium used in the present invention may be, for example, 0.5% yeast extract, 0.5% peptone, 0.5% glucose, 0.1%.
% K ₂ HPO ₄ , 0.02% MgCl ₂ · 7H ₂ O, 1% separately sterilized sodium carbonate in a medium of pH 10.5, which was also sterilized with a membrane filter, etc. Can be added, and the microorganisms can be grown in a batch system or a continuous system.

The microorganisms cultivated as described above can be collected and purified as follows depending on the type of use. First, when adding and mixing as it is to an animal sample, etc., the culture solution is collected by filtration and centrifugation,
It can be used as it is. Further, when used as a drug or the like, various conventionally known extraction methods can be applied, for example, ultrasonic disruption of a culture solution containing cells, disruption of cells by French press treatment, etc., centrifugation, filtration. The desired organic selenium can be separated and purified by obtaining a supernatant with a method such as salting out and, if necessary, purifying by a method such as salting out, dialysis, gel filtration and the like.

In the method of the present invention, the content of organic selenium in the cells was measured by the following method. That is, the microbial cells that have absorbed and accumulated selenium in the microbial cells are separated by filtration, centrifugation, or the like, and 0.5 l of 0.05-0.1N alkaline aqueous solution and 1
Thoroughly washed with water etc. to remove impurities. Then, after thoroughly washing with 0.5 l of acetone for the purpose of dehydration, the acetone is removed under reduced pressure, and the mixture is heated and dried at 105 ° C. for 24 hours to obtain dried bacterial cells.

Then, the dried bacterial cells (0 to 100 mg) thus obtained were wet-ashed with hydrofluoric acid and nitric acid, then oxidatively decomposed with nitric acid and perchloric acid, and fluorinated with 2,3-diaminonaphthalene. Luminous intensity method (eg “Fluorescence / phosphorescence analysis method”, Taiji Nishikawa,
The selenium concentration can be measured by Keizo Hiraki, edited by The Japan Society for Analytical Chemistry, P150-156, published by Kyoritsu Publisher. According to this method, the selenium content can be accurately measured in the range of 1 mg to 1 μg.

Examples Hereinafter, the method of the present invention will be described in more detail with reference to Examples, and the effect of the method of the present invention will be demonstrated, but the scope of the present invention is not limited by these Examples.

Example 1 5 g of yeast extract, 5 g of peptone, 5 g of glucose, 1 g of K ₂ HPO ₄ and 0.2 g of MgCl ₂ / 7H ₂ O were dissolved in 0.9 liter of tap water at 121 ° C.
After adjusting the concentration to 200 mM and adding 50 ml of sodium selenite sterilized with a membrane filter to the medium (A medium) sterilized for 15 minutes, the pH was adjusted to 9.0-each with 1N sodium hydroxide sterilized separately. Adjust to 12.0, 1 for the whole
And 150 ml of the thus-prepared medium was dispensed into a 500 ml Erlenmeyer flask sterilized by dry heat, and the agar was added to the same medium.
Platinum loops were inoculated with SY-133 or SR-138 cultivated at 37 ° C for 2 days on agar slants containing 0 g / l, and the conditions were 220 rpm on a rotary shaker at 37 ° C. And cultured for 48 hours. The amount of dried cells and the selenium content in the cells at this time are shown in Table 2.

Example 2 To the A medium used in Example 1, 50 ml of 10% sodium carbonate separately sterilized as a pH adjuster and a predetermined amount of sterilized sodium selenite aqueous solution were added to give a selenium concentration of 0.
The medium was prepared to be ˜500 mM.

Using these media adjusted to pH 10.5, SY-13
Three bacteria and SR-138 were cultured under the same conditions as in Example 1. The amount of dried cells and the selenium content in the cells at this time are shown in Table 3.

Example 3 To the A medium used in Example 1, 50 ml of 10% sodium carbonate separately sterilized as a pH adjusting agent and a predetermined amount of various sterilized water-soluble selenium compounds were added to adjust the selenium concentration to 10 mM. The medium was prepared.

Using these media adjusted to pH 10.5, SY-13
Three bacteria and SR-138 were cultured under the same conditions as in Example 1. Table 4 shows the amount of dried cells and the selenium content in the cells at this time.

Effects of the Invention As described in detail above, according to the method of the present invention, based on the use of a novel alkalophilic bacterium belonging to a specific Bacillus genus, drugs, such as anti-cancer agents, anti-inflammatory agents, heavy metal poisoning A large amount of organic selenium, which is expected as an antidote, can be obtained.

According to the method of the present invention, according to the method of the present invention, although the conventional method was able to achieve only a few mg selenium content per 1 g of dry cells at best.
It has become possible to increase the selenium content by about one digit.
Further, the method is simple, and the usefulness of the present invention can be said to be extremely great.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuyuki Nakamura 1-4-23, Kunitachi, Tokyo (72) Inventor Hiroki Horikoshi 4-39-8 Sakuradai, Nerima-ku, Tokyo (56) References JP-A-60 -180582 (JP, A)

Claims (1)

[Claims] 1. A SY133 bacterium (FERM P-8488) or an SR138 bacterium (FERM P-8487), which is an alkaliphilic microorganism belonging to the genus Bacillus that has the ability to accumulate selenium-containing compounds and has an optimum pH for alkaline growth.