JP4068103B2 - Production method of vitamin K by culture of Yunnan SL-001 - Google Patents

Description

The present invention relates to a method for producing vitamin K by culturing Bacillus subtilis.

  Vitamin K is conventionally known as a factor necessary for blood coagulation, and since this vitamin deficiency causes a decrease in blood coagulation ability, it is a kind of fat-soluble vitamin also called anti-hemorrhagic vitamin. In recent years, it was suggested that vitamin K is essential for the biosynthesis of several blood coagulation factors including prothrombin as a cause of the decrease in blood coagulation ability due to the lack of vitamin K. However, the amount of vitamin K required to prevent a decrease in blood coagulation ability is extremely small, on the order of μg, and is generally supplied from intestinal bacteria in adults, resulting in vitamin K deficiency. This is rare, and synthetic vitamins K1 and K2 are used as medicines for the treatment of vitamin K deficiency, and so far, natural vitamin K1 concentrate has been used as a food for the purpose of this prevention. Vitamin K has not received much attention so far.

  However, in recent years, vitamin K has a bone formation promoting action and a bone resorption suppressing action, and it has been clarified that administration of vitamin K increases bone density. On the other hand, osteoporosis is a fragile condition caused by aging, illness, etc., and is a disease that causes fractures and severe pain, and is becoming a major social problem in terms of geriatric medicine. When the blood concentration of K was examined, it was reported that it was about 1/2 that of healthy people. For this reason, clinical trials have been conducted for synthetic vitamin K as a therapeutic agent for osteoporosis. Unlike the case of treatment / prevention of blood outbreak, the treatment / prevention of osteoporosis has a large amount of vitamin K of 45 mg or more per day. Clinical trials have shown that administration has the effect of improving bone mass. In addition, osteoporosis is more important to prevent than treatment after its onset. For this purpose, it is desirable to take vitamin K on a daily basis from foods. It is not clear whether ingestion increases bone mass and helps prevent osteoporosis, and it is difficult to consume the above-mentioned amounts with existing foods.

  As described above, it is desirable to take vitamin K through daily foods. In fact, vitamin K1 can be taken from green-yellow vegetables and seaweeds, and vitamin K2 can be taken from fermented foods such as natto. However, when trying to ingest 45 mg of vitamin K, which is reported to be effective in improving osteoporosis from commercially available foods, for example, in a food containing 1 ppm of vitamin K, a large amount of food of 45 kg per day This is actually very difficult. Of the foods, natto has the highest vitamin K content of over a dozen ppm. Even if you eat such natto, you do not eat several hundred grams to several kilograms a day. In addition, it is difficult to eat this amount every day for preference, and the half-life of ingested vitamin K is short. The problem of side effects remained when taking large amounts at once. For this reason, it is desirable to ingest concentrated vitamin K. However, commercially available natural vitamin K concentrate added to formula milk for the prevention of blood discharge is expensive, and synthetic vitamin K as a pharmaceutical is a food. There was a problem that could not be used.

  By the way, among vitamin Ks, only the vitamin K1 and vitamin K2 groups exist in nature. Vitamin K1 is abundant in foods, especially in green vegetables, vegetable oils, seaweeds, etc., for example, several tens of ppm in seaweed, laver, tea leaves, and several ppm in soybean oil, spinach and broccoli. Further, it is synthesized by condensing 2-methyl-1,4-naphthoquinone and phytyl acetate. Further, in the vitamin K2 group, homologues of menaquinone-1 to 14 (MK-1 to 14) are known due to the difference in side chain length. Among these, menaquinone-7 (also simply referred to as “MK-7” in the present specification) is a typical substance of vitamin K2, which is synthesized mainly by Bacillus natto, but in the natural world, natto is also a few. Isolation is extremely difficult because it is present in a relatively small amount of ˜10 and several ppm and the half-life is short, and examples of preparing lipids with a high content of MK-7 until now are disclosed in JP-A-8-73396. Only one example of the publication is known.

  For this reason, it has been attempted to produce vitamin K2 in large quantities using microorganisms such as Bacillus natto, and many studies are known as methods for obtaining natural vitamin K2. For example, microorganisms belonging to the genus Flavobacterium A method of collecting vitamin K2 from the culture solution (Japanese Patent Publication No. 7-28748 and Japanese Patent Publication No. 7-51070), a method of producing vitamin K by inoculating soybean fermented soybeans or tofu cake with natto bacteria and fermenting them ( JP-A-10-295393, JP-A-8-19378, JP-A-8-9916, and JP-A-8-173078). In addition to these methods, a method for obtaining concentrated lipids containing a large amount of natural vitamin K2, particularly natural MK-7, by subjecting fermented natto to solvent extraction with an organic solvent such as alcohol, ether, ester or ketone. (Japanese Patent Laid-Open No. 8-73396) has been proposed. However, the method using a vitamin K-producing bacterium such as Flavobacterium has a problem that Flavobacterium cannot be immediately used for food because its safety as a food has not been proved. In addition, in the method of preparing vitamin K using Bacillus natto, a culture with a relatively high vitamin K content of up to about 40 mg / liter culture was obtained, but the water-soluble product is produced here. Since it is a fat-soluble vitamin K, its usable applications are very limited. Furthermore, the natural menaquinone-7 high-content lipid prepared by extracting fermented natto bacteria with an organic solvent is prepared from raw materials that can be used for food such as soybeans, but the organic solvent In order to use it in food, this organic solvent must be completely removed, and more equipment and time are required for this purpose. In addition, as in the above, the obtained high content lipid of MK-7 is its name. As it is clear from the above, since it is fat-soluble, its usable applications are limited.

As described above, vitamin K and MK-7 are produced from natto, fermented byproducts such as natto produced in the process of natto and boiled juice using microorganisms such as natto (ie, vitamin K from outside the cell). Many methods have been reported.
Japanese Patent Publication No.7-28748 Japanese Patent Publication No. 7-51070 JP-A-10-295393 JP-A-8-19378 JP-A-8-9916 JP-A-8-173078 JP-A-8-73396

The object of the present invention is to provide the most amount of vitamin K derivatives, particularly menaquinone-7 (vitamin K2) derivatives, in Bacillus.
The present invention provides a method for culturing the microorganism so as to be stored in the subtilis) .

Another object of the present invention is to enable a simple daily intake of natural vitamin K, particularly natural MK-7, which cannot be consumed in a sufficient amount or difficult from normal foods. The present invention provides a method for culturing a microorganism such that vitamin K derivative, particularly menaquinone-7 (vitamin K2) derivative, is stored in the largest amount in Bacillus subtilis .

In view of the above circumstances, the inventor of the present invention (Experientia,
43: 1110, 1987; Acta Haematol.,
84: 139, 1990; Fibrinolysis,
6: 86, 1992; Journal of the Japanese Pharmacists Association, 30: 73, 1994; Bioindustry, 14: 47, 1996), or analysis of vitamin K in natto and blood (Japan Thrombosis and Hemorrhage, 8: 287, 1997; Fibrinolysis & Proteolysis, Vol.12,
Supplement 1, 205, p.75, 1998) As a result of intensive research, high levels of plasma were obtained not by ingestion of fermented natto bacteria such as natto and vitamin K contained therein, but by ingestion of live Bacillus subtilis itself. Of vitamin K, especially MK-7 concentration, and especially in the case of Bacillus natto, it has been found that it exhibits a sustained effect of extremely high plasma concentration compared to others.

  In addition, the present inventor has accumulated a large amount of MK-7 in the microbial cells of natto cultured until a specific growth stage, and is high by ingesting the natto bacteria recovered at this stage or the culture itself. It has also been found that plasma vitamin K, especially MK-7 levels are observed and plasma vitamin K levels when taken in this way last for a long time. In addition to the above findings, the present inventor has also discovered that vitamin K, particularly MK-7, accumulated in the cells of Bacillus natto collected at the above stage is water-soluble. With the use of vitamin K, especially MK-7, it was expected that its application would be greatly expanded.

  The production method of vitamin K according to the first aspect of the present invention includes a step of culturing Yunnan SL-001 bacteria and obtaining a vitamin K derivative from the cultured cells and / or culture solution.

  Yunnan SL-001 bacteria is a kind of natto bacteria, and its safety is ensured. A food, beverage or feed containing a culture cultured by the above method or a water-soluble vitamin K derivative derived from such a culture contains a larger amount of a water-soluble vitamin K derivative, particularly menaquinone-7 derivative, compared to the prior art and Since it has high safety, it is possible to take vitamin K efficiently and easily on a daily basis by eating such foods, beverages or feeds, and further improvement of osteoporosis can be expected.

  In addition, the water-soluble vitamin K derivative derived from the culture cultured by the culture method of the present invention is a revolutionary invention in which vitamin K, which has been conventionally fat-soluble, can be changed to water-soluble. It is included. Furthermore, the water-soluble vitamin K derivative according to the present invention has excellent light stability. It goes without saying that the use of vitamin K can be considerably expanded by the present invention as compared with the prior art, and therefore the present invention can be expected to have a very high value from an industrial point of view.

Furthermore, by ingesting the culture or the food containing the water-soluble vitamin K derivative according to the present invention, the blood vitamin K concentration can be increased efficiently, and the vitamin K itself consumed by conventional medicines and foods can be increased. Compared with this, since the increase in blood concentration can be sustained for a long time, a higher effect of preventing osteoporosis can be expected.

  As of May 7, 1999, Yunnan SL-001 bacteria used in the present invention were deposited internationally at the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry under the accession number FERM BP-6713.

  In the present invention, it is preferable to terminate and collect Yunnan SL-001 bacterium before the vitamin K produced in the microbial cell is released outside the microbial cell. And culture conditions), the same medium and culture conditions as those used for general culture of Bacillus subtilis can be used. As a result, vitamin K is selectively released in a large amount in the microbial cell without being almost released outside the microbial cell. Therefore, the simple operation of recovering the microbial cell in such a specific state. A cell containing a large amount of vitamin K is obtained, and the cell recovered in this way is a fungus that has been confirmed to be safe, without the need to extract vitamin K from the cell. It is possible to eat the cells as they are, or to include a culture of such cells in a general food.

  In the present invention, as described above, the recovery time of the cells of Yunnan SL-001 is preferably before the vitamin K produced in the cells is released to the outside of the cells, more preferably This is the time when the number of bodies enters the stationary phase from the logarithmic phase. In addition, it is preferable to recover the bacterial cells at the time of entering the stationary phase from the logarithmic phase and before nattokinase is produced. More specifically, although the culture time varies depending on the culture conditions and culture methods such as culture temperature and pH and initial cell concentration, for example, a 300 ml culture solution (composition: 1.5) using a 500 ml Erlenmeyer flask is used. % Polypeptone-S, 1% glucose, 0.1% yeast extract, pH 7.2) inoculated with Yunnan SL-001 strain at an initial concentration of 5 × 10 6 cells / ml and cultured at 37 ° C. with shaking (100 rpm) Is performed for 0.5 to 4 days, preferably immediately after exhibiting the maximum value in absorbance at 660 nm (1 to 2 days).

  In the present invention, in the culture of Yunnan SL-001 bacteria, the same medium and culture conditions as those used for general culture of Bacillus subtilis are used.For example, the medium used for the culture according to the present invention is: A medium comprising known ingredients can be used by those skilled in the art and is not particularly limited. The medium may be prepared by mixing various culture components as appropriate, or a commercially available medium may be used as it is, or the above known ingredients may be used in a commercially available medium. A medium supplemented with as an auxiliary component may be used. At this time, the medium may be either a solid or liquid medium, and is appropriately selected depending on the purpose of use, and contains a carbon source that can be assimilated by the microorganism to be used, an appropriate amount of nitrogen source, an inorganic salt, and other nutrients. As long as it is a medium to be used, either a synthetic medium or a natural medium may be used.

  The carbon source that can be used in the culture of Yunnan SL-001 according to the present invention is not particularly limited as long as the strain grows well and can produce vitamin K efficiently. Specifically, starch or a fraction thereof, roasted dextrin, modified starch, starch derivative, physically treated starch, α-starch, soluble starch, amylose, amylopectin, maltooligosaccharide, cyclodextrin, pullulan, corn starch, potato starch , Carbohydrates such as sweet potato starch and dextrin, glycerin, sorbitol, wort and glucose. Of these carbon sources, glucose and starch are preferably used from the viewpoint of vitamin K production. These carbon sources can be used alone or in the form of a mixture of two or more.

  The nitrogen source that can be used in the culture of Yunnan SL-001 according to the present invention is not particularly limited as long as the strain grows well and can produce vitamin K efficiently. Specifically, meat extract, malt extract, peptone, soybean-derived polypeptone (for example, polypeptone-S), yeast extract, taste liquid (soy protein acid hydrolyzate), soybean powder, milk casein, casamino acid, various amino acids And organic nitrogen compounds such as corn steep liquor, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, nitrates such as sodium nitrate, inorganic nitrogen compounds such as urea, and the like. Among these nitrogen sources, from the viewpoint of production of vitamin K, soybean-derived polypeptone (for example, polypeptone-S) and soybean powder are preferably used. These nitrogen sources can also be used alone or in the form of a mixture of two or more.

  The inorganic salt that can be used in the culture according to the present invention is also not particularly limited as long as it varies depending on the species used, and the strain can grow well and can produce vitamin K well. One, two or more selected from phosphates, hydrochlorides, sulfates, acetates, etc., such as calcium, sodium, potassium, copper, iron and zinc can be used.

  Moreover, as a commercially available culture medium when using a commercially available culture medium for culture | cultivation by this invention, a nutrient broth (dry broth) (made by Nissui Pharmaceutical Co., Ltd. or Nippon Pharmaceutical Co., Ltd.), polypeptone-S, for example. (Made by Wako Pure Chemical Industries).

  Or, in the present invention, as a medium used for culturing Yunnan SL-001 bacteria, raw materials such as okara, soybeans, soybean soup produced as a by-product during production of miso and natto, tofu cake produced as a by-product during production of tofu and fried chicken, and soybean Materials that can be fermented by Bacillus natto, such as soybean meal produced as a by-product during oil production and soybean seed coat as a by-product during production of miso, may be used. At this time, if necessary, a carbon source, a nitrogen source and an inorganic salt as described above may be appropriately added to this material.

  In the present invention, the culture of Yunnan SL-001 is carried out in the same manner as a conventionally known method, and the culture conditions at that time are appropriately selected according to the composition of the medium and the culture method, and the strain grows and vitamin K is efficiently produced. There is no particular limitation as long as it can be produced well. The culture temperature is usually 20 to 45 ° C., preferably 37 to 42 ° C., and the pH of the medium suitable for the culture is usually 6.0 to 9.5, preferably 7.0 to 8.5. It is.

  In the present invention, the term “culture of Yunnan SL-001 bacterium” includes both microbial cells of Yunnan SL-001 cultivated by the above method and products produced outside the microbial cells. In the latter case, Yunnan SL-001 is preferable because it shows a higher yield outside the cell than other Bacillus subtilis.

  As described above, the culture of Yunnan SL-001 cultivated by the method of the present invention accumulates a large amount of vitamin K, particularly vitamin K2, and more particularly menaquinone-7 (MK-7) in the microbial cell. Yes. Specifically, the amount of vitamin K, vitamin K2 and MK-7 accumulated in the culture varies depending on the type of culture medium and the culture conditions, but is usually 10 to 200 mg / 100 g vacuum heat-dried cells. is there. In the present specification, the amounts of MK-7, MK-4 and vitamin K1 accumulated in the microbial cells are values measured by the method described in the Examples section below.

  A culture of Bacillus subtilis containing Yunnan SL-001 cultivated by the method of the present invention is collected by a known method such as filtration or centrifugation, and then freeze-dried, air-dried, vacuum-heat dried When the dried cells are dissolved in water, vitamin K is dissolved in water. That is, taking into account the fact that a large amount of vitamin K (especially MK-7) is contained in the microbial cells, vitamin K (especially MK-7) accumulated in the microbial cells is subject to some changes in the microbial cells. In this specification, “water-soluble vitamin K” is considered to be vitamin K, vitamin K 2 and menaquinone-7 (MK-7), which are considered to be water-soluble. Derivatives "(or simply referred to as" vitamin K derivatives ")," water-soluble vitamin K2 derivatives "(or simply referred to as" vitamin K2 derivatives ") and" water-soluble menaquinone-7 derivatives (water-soluble MK-7 derivatives) ". At this time, the water-soluble menaquinone-7 derivative shows a single band having a molecular weight of about 100,000 when measured by SDS-polyacrylamide electrophoresis, and 100,000 or more when measured by gel filtration. In view of the fact that the molecular weight of menaquinone-7 and the molecular weight of menaquinone-7 are about 649, some substances (for example, glycoproteins) bind to MK-7 and are stabilized and water-soluble vitamin K It is presumed that derivatives (including vitamin K2 derivatives and menaquinone-7 derivatives; hereinafter omitted) may be formed in the microbial cells. However, it goes without saying that the concept of the present invention is not limited by the above hypothesis.

  In the present invention, the solubility of vitamin K derivatives, vitamin K2 derivatives, and menaquinone-7 derivatives in water varies depending on the type of cells used, the type of culture medium, the culture conditions, and the culture treatment (extraction) method. For example, the solubility of vitamin K derivative is about 105 μg / 100 ml water (20 ° C.) in the case of water extraction operation of cultured cells, and about 300 μg / 100 ml water (20 ° C.) in the case of liquid culture supernatant, Furthermore, in the case of water extraction operation of natto, it is about 1,500 μg / 100 ml water (20 ° C.).

  The food, beverage or feed according to the present invention uses a microorganism that has been confirmed to be safe, Yunnan SL-001 (natto), so that it may be composed only of a culture and / or a water-soluble vitamin K derivative. Good.

  Alternatively, the food, beverage or feed according to the present invention may be a food, beverage or feed that can normally be fed with the culture according to the present invention and / or the water-soluble vitamin K derivative according to the present invention. Good. At this time, foods, beverages or feeds that can be usually eaten include, for example, okara, okara natto, natto, soybeans, soybean soup produced as a by-product during production of miso and natto, tofu cake produced as a by-product during production of tofu and fried chicken, Soybean koji produced as a by-product during soy production from soybeans, soy seed coat as a by-product during the production of miso, yogurt, dairy products such as cheese, kamaboko, chikuwa, hanpen, fried fish, and so on; Processed seafood products, sausages, frankfurters, liver paste and other processed meat products, tofu, grilled tofu, freshly fried, deep-fried tofu, gourd, okara, frozen tofu, bean products such as yuba, processed vegetables such as puree, mashed potatoes, Koji, Harusame, Konnyaku, Shirataki and other processed rice cakes, rice cakes, white balls, white rice, rice cakes, rice noodles, macaroni, spaghetti, somen noodles, Baked noodles, Chinese noodles, instant noodles, processed bread products such as bread, dried bread, and anpan, frozen sweets, nutraceuticals, sweets such as jams, butters, margarines, mayonnaise, dressings and other fats and oils, candy, rak cancer, Rice crackers, hail, castella, yokan, monaka, manju, daifukumochi, dango, uirou, chocolate, biscuits, cookies, donuts, cakes, pie, ice cream, pudding, bavaria, gum and other sweets, tofu, jelly, konjac, All foods including gel foods such as agar or tokoroten, seaweeds such as kombu, wakame, seaweed, and prickly pears; various juices (including orange, pineapple, apple, grape, melon and strawberry juice), various carbonated drinks, Tea (including Sencha, Oolong tea, etc.), drinking yogurt, cow , Soy milk, formula, mineral water, soft drinks, coffee, tea, cocoa, etc .; and livestock such as pigs, cows, horses, sheep and goats, pets such as dogs, cats, rabbits and hamsters, poultry and Examples thereof include food (feed) for animals usually raised such as fish.

  In the present invention, when the food, beverage or feed contains the culture according to the present invention, the amount of the culture added in the food is the type of cells used, the culture conditions and the content of the water-soluble vitamin K derivative. However, it is usually an amount containing 0.001 to 20% by weight of dried microbial cells, preferably 0.1 to 5% by weight of dried microbial cells. 0.0001 to 5 (w / v)% of dried microbial cells, preferably 0.01 to 5 (w / v)% of dried microbial cells. It is an amount containing -5% by weight of dried microbial cells, preferably 0.001-1% by weight of dried microbial cells. In addition, the amount of the water-soluble vitamin K derivative in the food when the food, beverage or feed contains the water-soluble vitamin K derivative according to the present invention varies depending on the cells used, the type of culture medium, the culture conditions, and the like. In food, it is usually an amount containing 0.00001 to 10% by weight of water-soluble vitamin K derivative, preferably 0.0001 to 0.1% by weight of water-soluble vitamin K derivative. In an amount containing 00001 to 0.1 (w / v)% water-soluble vitamin K derivative, preferably 0.0001 to 0.01 (w / v)% water-soluble vitamin K derivative, Usually, the amount contains 0.00001 to 10% by weight of water-soluble vitamin K derivative, preferably 0.0001 to 1% by weight of water-soluble vitamin K derivative.

  Therefore, ingesting a cell culture containing a large amount of the water-soluble vitamin K (especially MK-7) derivative of the present invention and / or a food containing a water-soluble vitamin K (particularly MK-7) derivative derived from such a culture. By doing so, vitamin K, especially MK-7, can be ingested efficiently and in a large amount at once without excessive effort (without excessive discomfort).

  In the production of vitamin K according to the present invention, first, the cultured Yunnan SL-001 cells are used at the boiling point of an organic solvent used in an organic solvent such as hexane, diethyl ether, acetone, ethanol and isopropanol using a Soxhlet extractor. Soxhlet extraction can be performed to obtain a fat-soluble fraction. Next, this fraction is extracted with an organic solvent such as hexane, diethyl ether, acetone, ethanol and isopropanol at 30 to 100 ° C. for 0.1 to 20 hours. The obtained extract is diluted with an organic solvent similar to the above to a predetermined total volume. A part of the diluted extract is mixed with water and isopropanol, and further mixed with the same organic solvent as described above by using a touch mixer. The obtained liquid mixture is centrifuged, the supernatant is dried, and then the residue is dissolved in ethanol to obtain higher-purity fat-soluble vitamins such as vitamin K2 (MK-7, MK-4) and vitamin K1. K is prepared.

  By the above method, fat-soluble vitamin K is extracted more efficiently than known methods, so that fat-soluble vitamin K such as vitamin K2 (MK-7, MK-4) and vitamin K1 is higher. It can be recovered from cultured cells in a yield.

  In the production of vitamin K according to the present invention, Yunnan SL-001 is cultured by a known method or the above-described method to obtain a culture solution. Next, a precipitate is obtained by lowering the pH of this culture solution, preferably by adjusting the pH to 1 to 3. This precipitate contains water-soluble substances accumulated in the cells and released outside the cells. Vitamin K derivatives are included. Therefore, the water-soluble vitamin K derivative can be separated by separating the precipitate by centrifugation or the like.

Alternatively, the Yunnan SL-001 bacterium is cultured by a known method or the above-described method, and the obtained culture solution is centrifuged to obtain a culture supernatant from which the microbial cells have been removed. Next, a precipitate is obtained by lowering the pH of this culture supernatant, preferably by adjusting the pH to 1 to 3, and this precipitate contains a water-soluble vitamin K derivative released outside the cells. . Therefore, the water-soluble vitamin K derivative can be separated by separating the precipitate by centrifugation or the like.

[Examples] The present invention will be described more specifically below with reference to examples.

  In the following examples, the amounts of MK-7, MK-4 and vitamin K1 were measured according to the following method.

  <Method for Measuring Various Vitamin K Amounts> First, a method for preparing a cell sample in the case of fat-soluble MK-7, MK-4 and vitamin K1 will be described below: 1 g of cultured microbial cells (dry weight) As a sample, extract the fat-soluble fraction with a Soxhlet extractor (SIBATA SPC 34, WATER BATH SIBATA WB-6C, filter paper: ADVANTEC 84 24 × 100 mm). After extracting this with 100 ml hexane at 80 ° C. for 6 hours, the total liquid volume is adjusted to 100 ml with hexane. 100 μl of this extract is mixed with 1.0 ml of distilled water and 1.5 ml of isopropanol, and 4.9 ml of hexane is further mixed with a touch mixer for about 10 seconds. The mixture is centrifuged (3,000 rpm × 10 minutes, 20 ° C.). After 4.0 ml of the obtained supernatant (organic layer: aqueous layer = 5.8: 1.7) is concentrated and dried with an evaporator, this is dissolved in 100 μl of ethanol. In this manner, an HPLC sample (hereinafter referred to as “Soxhlet-HPLC sample”) for measuring the amount of fat-soluble MK-7, MK-4 and vitamin K1 accumulated in the microbial cells is prepared. A method for measuring the amount of MK-7 by the following HPLC using this Soxhlet-HPLC sample is referred to as “Soxhlet-HPLC method”.

  Next, a sample preparation method in the case of a water-soluble vitamin K derivative (including MK-7) will be described below: 45 ml of distilled water is used for 5 g obtained after the culture is kneaded well with a spatula. Add. This mixed solution is centrifuged at 3,000 rpm for 10 minutes at 20 ° C., and 0.5 ml of the obtained supernatant is used as an extraction sample. This extracted sample is mixed with 0.5 ml of distilled water and 1.5 ml of isopropanol, further added with 5.0 ml of hexane and stirred, and then centrifuged at 20 ° C. and 3,000 rpm for 10 minutes. 4.0 ml of the obtained supernatant is concentrated and dried with an evaporator, and dissolved with 100 μl of ethanol. In this manner, an HPLC sample (hereinafter referred to as “HPLC sample”) for measuring the amount of the water-soluble MK-7 derivative is prepared. A method of measuring the amount of MK-7 by the following HPLC using this HPLC sample is referred to as “HPLC method”.

  Further, the culture solution is used as it is as an HPLC sample for measuring the amounts of MK-7, MK-4 and vitamin K1 in the culture solution.

  Furthermore, the measurement of MK-7, MK-4 and vitamin K1 amounts by high performance liquid chromatography (HPLC) utilizes the fact that vitamin K is reduced to a hydroquinone by a platinum-alumina catalyst and becomes a fluorescent substance. . Specifically, the measurement is performed under the following conditions.

Equipment Pump: PU-980 (manufactured by JASCO)
Injector: 7125 (manufactured by JASCO)
Column oven: CO-965 (manufactured by JASCO)
Detector: Fluorescence detector 821-FP (manufactured by JASCO)
Data processing device: C-R5A (manufactured by Shimadzu Corporation)
Conditions Column: ODS-II column (4.6 × 250 mm) (manufactured by Shimadzu Corporation)
Reduction column: Platinum-alumina catalyst column (manufactured by Wako Pure Chemical, primary platinum-alumina, packed with about 0.2 g of Pt = 5%; 4.0φ × 10 mm)
Mobile phase: 97% ethanol (flow rate; 0.7 ml / min)
Separation / reduction temperature: 40 ° C
Measurement wavelength: Excitation 320 nm
Fluorescence 430nm
Injection volume: 10 μl

  When calibration curves were prepared for MK-7, menaquinone-4 and vitamin K1 by such a measuring method, the amount of MK-7 was within the range of 0.05 to 50 ng, and the formula [-0.89661 + 1.9933 × 10 −6 × (area of HPLC corresponding to vitamin K (μV, sec))], and the amounts of menaquinone-4 and vitamin K 1 are in the range of 0.01 to 10 ng, respectively, in the formula [−0.58657 + 4. 8030 × 10 −9 × (HPLC area corresponding to vitamin K (μV, sec))] and formula [−0.44381 + 4.0626 × 10 −7 × (HPLC area corresponding to vitamin K (μV, sec)) )], Measurement was possible.

  A standard product of MK-7 for preparing a calibration curve was prepared as follows. That is, 1 liter of 75% isopropanol and 1 liter of n-hexane were added to 600 g of natto, and the mixture was slowly stirred and allowed to stand for 1 hour. The upper layer of the two separated layers was taken, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain about 20 g of extract. This was mixed with 10 ml of n-hexane, adsorbed through 400 ml of silica gel for chromatography, and eluted and fractionated with 2 liters of n-hexane / toluene (1: 1). The fraction containing MK-7 was evaporated to dryness under reduced pressure, and the resulting silica gel concentrate was dissolved in 5 ml of n-hexane, fractionated again on a silica gel column in the same manner as above, and evaporated under reduced pressure. About 350 mg was obtained by drying. 50 mg of this was dissolved in a small amount of acetone, passed through 60 ml of chromatography ODS-silica gel packed with acetonitrile / methanol (1: 1), and developed with acetonitrile / methanol (1: 1). While monitoring the eluate by HPLC, a fraction in which a substance considered to be MK-7 was eluted alone was collected and dried under reduced pressure. About the obtained substance, the infrared absorption spectrum and the mass spectrum were obtained, and it confirmed that it was MK-7. When the purity of this substance was tested, it was 99.8%. Moreover, as standard products of phylloquinone (vitamin K1) and menaquinone-4 (MK-4), special grades manufactured by Sigma were used, respectively. The measurement was determined by the average value extracted and analyzed three times.

Example 1
Each of the following 6 types of natto bacteria is placed in a 500 ml Erlenmeyer flask with 300 ml of 3% nutrient broth (nutrient).
broth) (dried broth) (manufactured by Nippon Pharmaceutical Co., Ltd.) in culture at 30 ° C. with shaking (100 rpm). The growth of the bacteria at this time was measured as absorbance at 660 nm, and the amount of MK-7 in the culture solution and in the cells was measured over time.

For culture time, the growth of various Bacillus natto, MK-7 in the culture solution (μg / ml) and MK-7 per cell 1g of various Bacillus natto ([mu] g / g), respectively, FIG. 1, FIG. It is shown in 2 and 3.

<Used natto bacteria>
Yunnan SL-001 (or “Yunnan”)
Miyagino fungus (Miyagino Natto Factory, Sendai)
Meguro fungus (Meguro Institute, Osaka)
Nitto Bacteria (Nitto Pharmaceutical Co., Ltd., Kyoto)
Takahashi fungus (Yamagata, manufactured by Yuzo Takahashi Laboratory)
Naruse bacteria (Naruse Fermentation Chemistry Laboratory, Tokyo)
Hereinafter, only the name of the natto is described.

As shown in FIG. 1 , the growth of the fungus showed similar behavior with all Bacillus natto, expressed as the average absorbance at 660 nm (n = 6), 0.583 on day 0.5, 1 day 1.011 on the second day, 0.948 on the second day, 0.854 on the third day, 0.592 on the fifth day, and 0.552 on the seventh day. It was found that a steady state was reached in ~ 2 days. In addition, as shown in FIG. 2 , the amount of MK-7 released into the culture broth also showed similar behavior with all Bacillus natto, 0.5 days in terms of average value with 6 kinds of Bacillus natto. 0.563 μg / ml on day 1, 1.592 μg / ml on day 3, 3.867 μg / ml on day 3, 4.317 μg / ml on day 5, 4.784 μg / ml on day 7, From this, the amount of MK-7 released into the medium increases rapidly after the first rather than the first or second day when the growth of the bacterium becomes steady state, and a large amount of MK-7 is especially generated after the fourth day. Released into the medium. In proportion to this result, MK-7 amount when converted to MK-7 per cell 1 g (again on all natto showed similar behavior), as shown in Figure 3, It was found that the maximum was reached on the second day (average of about 300 μg / g dry bacteria), and rather decreased after the fourth day when a large amount started to be released into the medium.

Example 2
Okara (Asahimatsu Food Co., Ltd., Iida City) was stored frozen at −25 ° C. and thawed for use when necessary. The thawed okara was sterilized in an autoclave at 120 ° C. for 30 minutes, and then placed in a polystyrene paper (PSP) container of about 120 ml, which was used as a culture medium for Bacillus natto.

  Separately, four natto strains (Takahashi, Naruse, Miyagi and Asahi) used in commercial natto, two natto strains (Nitto and Meguro) used as pharmaceuticals, and China A total of seven natto strains of the natto strain (Yunnan fungus SL-001) isolated from Yunnan natto, each in a 500 ml Erlenmeyer flask, 150 ml of 3% nutrient broth (dried bouillon) By shaking culture (100 rpm) in a medium at 37 ° C. for 3 days, a preculture solution of the above 7 types of Bacillus natto was prepared.

  To the okara prepared above (wet weight 50 g), 0.5 ml of each of these 7 kinds of Bacillus natto precultures (viable cell count: 2 × 10 8 cells / ml) was added, respectively, at 8 ° C. at 8 ° C. It was left still for days and continued to ferment.

After a predetermined number of days (1, 2, 4, 6, 8 days) from the start of fermentation, natto bacteria are suspended in water, filtered through a metal tea strainer, and then centrifuged (3,000 rpm × 10 minutes), a culture of each bacterial cell was prepared, and this was used as a Soxhlet-HPLC sample for MK-7 measurement. Thus the Soxhlet -HPLC samples prepared to measure the MK-7 content and fibrinolysis (thrombolysis) activity, and the results are shown in Figure 4.

As is clear from FIG. 4, the concentration of MK-7 in ¹⁰ cells per 10 ¹⁰ cells was high on the first and second days, decreased thereafter, and MK-7 was in the medium in proportion to this. The amount of MK-7 released in the medium is determined at the time when the thrombolytic activity by nattokinase (NK), which peaks at 2 days after the fermentation, is increased (fermentation is started in both the standard fibrin plate method and the synthetic substrate decomposition method). It was found that the peak was on the fourth day later than the maximum value was reached within two days.

In addition, for any natto bacteria, high MK-7 productivity of 1.9 μg or more per 1 g of Okara wet weight (the amount dissolved in water is 36.6 μg / g Okara wet weight on average; 1.9 μg / g Okara wet weight; Naruse bacteria 14.2 μg / g Okara wet weight; Takahashi bacteria 6.8 μg / g Okara wet weight; Asahi bacteria 11.9 μg / g Okara wet weight; Meguro 1.9 μg / g Okara Wet weight; and Nitto bacteria 5.2 μg / g okara wet weight). In particular, Yunnan fungus (SL-001) shows a high MK-7 production amount of 36.6 μg / g okara wet weight, and this value is calculated to be 2 to 20 times that of other Natto bacteria. In addition, a previous report (supervised by Ikuo Yamaguchi, Japanese Food Ingredients Table, Ishiyaku Shuppan, Tokyo, 1997, pp.52-53; Toshiyuki Sakano et al., Vitamin, 62, 393-398 (1988) H. Ikeda and Y. Doi, Eur. J. Biochem., 192,
219-223 (1990); and Hiroshi Ikeda, Home Economics Journal, 43, 643-648 (1992)), which is four times higher than the analytical value of natto (6.2 to 8.7 μg / g okara wet weight) It was a thing.

Comparative Example 1
The cells were cultured with shaking at 41 ° C. for 2 days, and the culture was centrifuged to separate into supernatant and cells. 2 g of this microbial cell was placed in a mortar, and 3 ml of sea sand and 3 ml of water were added to the mortar. Next, this mortar was ground and extracted with acetone (20 ml × 4), and suction filtered through a glass filter. The total amount of the filtrate was transferred to a separatory funnel and extracted by adding 100 ml of diethyl ether and 50 ml of water, and the ether layer was separated. An additional 100 ml of diethyl ether was added to the aqueous layer, and the ether layer was separated. These ether layers were combined, the solvent was distilled off, and this was further subjected to silica gel chromatography (hexane 10 ml, silica gel 5 g) and eluted with 30 ml of hexane / diethyl ether (85/15 (v / v)). Furthermore, after distilling off the solvent from the eluate, it was dissolved in 2-propanol to prepare fat-soluble MK-7.

The fat-soluble MK-7 thus prepared was dried to prepare a dried MK-7 product. After this MK-7 dried product was irradiated with a fluorescent lamp at room temperature for 5 hours, fat-soluble MK-7 was measured, and the results are shown in FIG. 5 (black squares). As shown in FIG. 5 , the amount of MK-7 becomes impossible to measure when irradiated with a fluorescent lamp for about 3 hours, and thus MK-7 prepared in this way is very unstable to light. It is shown that.

Example 3
Okara was provided by Asahimatsu Foods Co., Ltd. (Iida City), frozen at -25 ° C., and thawed when necessary.

  Separately, a natto strain (Yunnan SL-001) isolated from Yunnan, China, in a 500 ml Erlenmeyer flask, 150 ml of 3% nutrient broth (dried broth) (manufactured by Nissui Pharmaceutical Co., Ltd.) A preculture of Bacillus natto was prepared by shaking culture (100 rpm) at 37 ° C. for 3 days.

1 kg (wet weight) of thawed okara was sterilized by autoclave at 120 ° C. for 30 minutes, and then placed in a polystyrene paper (PSP) container having a capacity of about 120 ml. The pre-cultured solution of Bacillus natto prepared above was added to this container and fermented at 37 ° C. for 4 days. To 1 kg (wet weight) of Okara natto fermented in this manner, 5 liters of water was added, stirred at room temperature for 1 hour, and centrifuged (3,000 rpm, 10 minutes). To the obtained supernatant, 620 g of an ion exchange resin (DEAE-Sepharose CL-613) was added and stirred, and then allowed to stand at room temperature for 30 minutes. Next, this was filled in a glass column (7.5 cmφ × 100 cm), washed with distilled water and 0.05 M phosphate buffer (pH 7.0), and then 0.1 to 0.8 M.
Gradient elution was performed with 0.05M phosphate buffer (pH 7.0) containing NaCl. For each fraction, the amount of vitamin K was measured by HPLC, the fraction containing vitamin K was concentrated with a membrane filter (Millipore, molecular weight 10,000), dialyzed with distilled water, freeze-dried, and light yellow powder. As a result, a water-soluble vitamin K derivative was obtained.

Those the same okara as thus obtained water-soluble vitamin K derivative and the extracted with isopropanol, and analyzed by HPLC, and the results are shown in Figure 6. In FIG. 6 , the retention times of MK-4, vitamin K1, and MK-7 are approximately 8.2 minutes, approximately 11.3 minutes, and approximately 17.5 minutes, respectively, and the arrows indicate the retention of MK-7. Show time. From FIG. 6 , 60% or more of the isopropanol extract of okara was extracted into the water-soluble fraction, and as vitamin K, the amount of vitamins K1 and MK-4 was small, and 95% or more was always occupied by MK-7. I found out that

Moreover, when this water-soluble vitamin K derivative was analyzed by SDS-polyacrylamide gel electrophoresis, it showed a wide single band at a molecular weight of about 100,000. From this result, it is considered that the water-soluble vitamin K derivative may form a complex having a molecular weight of about 100,000 with other glycoproteins. Moreover, the yield of the water-soluble vitamin K derivative from 1 kg of natto at this time was about 5.3 g (830 μg) on average by three operations.
Water-soluble MK-7 derivative / g).

Example 4
In each of five 500 ml Erlenmeyer flasks, 300 ml of 3% dry broth (manufactured by Nissui Pharmaceutical Co., Ltd.) medium was placed, and autoclaved at 120 ° C. for 15 minutes. These media were inoculated with Yunnan SL-001 bacteria one by one, and cultured at 37 ° C. with shaking (100 rpm). Four days later, the combined culture was centrifuged (5,000 rpm × 10 minutes), and the resulting supernatant was divided into three 400 ml portions, and the pH of each supernatant was 1.02, with dilute hydrochloric acid. Adjusted to 07 and 3.01. After standing at room temperature for 3 hours, the supernatant was centrifuged (5,000 rpm × 10 minutes, 10 ° C.) to separate a white precipitate. This white precipitate was dissolved in a small amount of distilled water, adjusted to pH 7.0 with ammonium bicarbonate powder, and then lyophilized. As a result, the weight of the lyophilized product was 0.52 g, 0.28 g, and 0.31 g at pH values of 1.02, 2.07, and 3.01, respectively. Next, the MK-7 content (μg / g dried product) of this lyophilized product was examined by the Soxhlet-HPLC method. When the pH was 1.02, 2.07 and 3.01, 2, respectively. They were 800 μg / g dried product, 2,200 μg / g dried product and 2,000 μg / g dried product. Further, a solution obtained by adding 15 mg of each lyophilized product to 5 ml of distilled water was centrifuged (10,000 rpm × 10 minutes). The obtained supernatant (water-soluble fraction) was similarly examined for MK-7 content. When the pH was 1.02, 2.07, and 3.01, 1,500 μg / g dry product, respectively. , 1,800 μg / g dried product and 1,800 μg / g dried product, it was found that the solubilization rate of each water-soluble fraction was about 54%, about 82% and about 90%.

  From these results, it was difficult to extract natto bacteria efficiently by the conventional method, and the amount of MK-7 in the culture could not be measured well, whereas the Soxhlet- The HPLC method showed that the extraction efficiency of MK-7 was improved and the amount of MK-7 in the culture could be measured better than the conventional method.

These are graphs showing the growth of various natto bacteria with respect to the culture time in Example 1 . These are graphs which show the amount of MK-7 in the culture solution of various Bacillus natto versus the culture time in Example 1 . These are graphs showing the amount of MK-7 accumulated in the microbial cells of various Bacillus natto versus the culture time in Example 1 . These are the graphs which show the amount of MK-7 accumulated in the microbial cells in Example 2 , the fibrinolytic activity and the amount of MK-7 released outside the microbial cells. These are the graphs which show MK-7 quantity with respect to the irradiation time by a fluorescent lamp in the comparative example 1. FIG. These are the graphs which show the HPLC analysis result of the water-soluble vitamin K derivative and the isopropanol extract of Okara in Example 3 .

Claims (5)

Yunnan SL-001 (International Deposited with Biotechnology Institute of Industrial Science and Technology, Ministry of International Trade and Industry, International Deposit No. FERM BP-6713) is solid- cultured, and vitamin K derivative from the cultured cells and / or culture broth A method for producing vitamin K, comprising the step of:   The method for producing vitamin K according to claim 1, wherein the vitamin K derivative is a vitamin K2 derivative.   The method for producing vitamin K according to claim 2, wherein the vitamin K2 derivative is a menaquinone-7 derivative.   The method for producing vitamin K according to claim 1, 2 or 3, wherein vitamin K is obtained by Soxhlet extraction of the cultured cells.   The method for producing vitamin K according to claim 1, 2, 3, or 4, further comprising a step of acidifying the culture solution to obtain a precipitate.