JP2001333790A - Method for producing menaquinone-7 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a composition with a high content of menaquinone-7 by culturing a bacterium producing the menaquinone-7 and by using a culture medium made from a distillation residue formed as a by- product in the production of a distilled split (SHOCHU) using barley. SOLUTION: This method for producing the menoquinone-7 is characterized by subjecting a distillation residual liquor formed as a by-product in the production of the distilled liquor using the barley to solid-liquid separation, providing a liquid portion containing an amino acid, a polyphenol, an organic acid and glycerol substantially carrying out a neutralizing treatment of acids contained in the liquid portion, affording a prepared liquid, subjecting the resultant prepared liquid to a sterilizing treatment, using the obtained culture medium and culturing the bacterium belonging to Bacillus subtilis and capable of producing the menaquinone-7.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a distilled liquor using barley, using a medium having a liquid component as a main component, which is obtained by solid-liquid separation of a distillation residue produced as a by-product.
The present invention relates to a method for producing menaquinone-7 in high yield by culturing menaquinone-7 producing bacteria belonging to Bacillus subtilis. In particular, the present invention uses a medium containing as a main component a liquid component obtained by solid-liquid separation of barley shochu distillation residue by-produced in the production of shochu using barley, Baci
The present invention relates to a method for producing menaquinone-7 in high yield by culturing menaquinone-7-producing bacteria belonging to llus subtilis.

[0002]

2. Description of the Related Art Vitamin K is an essential nutrient for maintaining normal blood clotting ability. What is generally called vitamin K includes vitamin K1 including synthetic ones.
~ Vitamin K7 is known. Of these, vitamin K1 (phyloquinone) and vitamin K2 (menaquinone) are naturally occurring. Vitamin K1 is mainly synthesized in plant chloroplasts, so it is abundant in green and yellow vegetables,
In addition, it is relatively high in vegetable oils, legumes, seaweeds and seafood. On the other hand, vitamin K2 is produced by general bacteria and intestinal bacteria. Fermented foods such as natto, blue seaweed, chicken eggs,
Relatively high in meat and dairy products. Vitamin K2 is classified into menaquinone-1 to menaquinone-14 depending on the side chain structure. Among these, vitamin K2, which is particularly contained in natto, is mainly menaquinone-7 (hereinafter referred to as "menaquinone-7").
"MK-7").
Is known to be produced by Bacillus natto. MK−
7 is not only involved in the maintenance of blood coagulation ability, but also promotes bone formation by osteoblasts, and at the same time, suppresses the elution of calcium from bone, and is known to have the effect of suppressing the decrease in calcium content in bone. I have. For these reasons, MK-7 has already been used as a therapeutic agent for osteoporosis, and its demand is increasing.

[0003] In the industrial production of MK-7, as described below, (1) using soybean-derived materials such as soybean broth or tofu cake as a medium, or (2) soybean broth or soy sauce A method of producing MK-7 by mixing a specific substance with a soybean-derived material such as a fired boiler and using it as a culture medium and culturing Bacillus natto has been proposed.

[0004] The method (1) is disclosed in
No. 378, JP-A-8-73396, and JP-A-11-73396
-196820. That is, JP-A-8-1937
In No. 8 publication, soy broth produced as a by-product during natto production is adjusted to pH 7 and sterilized, inoculated with natto bacteria, and cultured at a temperature of 48 ° C.
A method for producing MK-7 by culturing with shaking at 120 rpm for 4 days is described. The publication also describes a method for producing MK-7 by sterilizing tofu cake by-produced during the production of tofu, inoculating this with natto bacteria, and culturing the culture at 48 ° C. for 5 days. Have been. JP 8-73
Japanese Patent Publication No. 396 describes a method for producing MK-7 by inoculating a soybean broth produced as a by-product during natto production with natto bacteria and culturing it. Japanese Patent Application Laid-Open No. 11-196820 discloses that tofu cake (okara) generated during the production of tofu is sterilized, inoculated with Bacillussp TT-52 natto, and formed as a layer having a thickness of about 1 cm on a stainless steel wire mesh. Place and ferment at 37 ° C for 48 hours, then ferment at 25 ° C for 17 hours and culture
A method for producing MK-7 is described.

[0005] The method (2) is disclosed in
No. 95393 and JP-A-11-32787. That is,
JP 10-295393 A, the Brix concentration is 5-10%
By adding 3 to 10% by weight of glycerol to the soy broth adjusted to be sterilized, inoculating this with natto bacteria, culturing at 40 ° C., aeration 0.5 L / min, and stirring at 500 rpm for 4 days by culturing. A method for producing MK-7 is described. JP 11
The -32787 discloses, sauce burning cage 7% (W / V), glucose 5% (W / V), K 2 HPO 4 0.25% (W / V), MgSO 4 · 7H 2 O 0.05% (W
/ V), NaCl 2% (W / V) was adjusted to pH 8.0 and sterilized, and inoculated with MK-7 producing bacteria belonging to Bacillus subtilis, and cultured in a jar fermenter. Temperature 40
A method for producing MK-7 by culturing for 48 hours while maintaining the pH value of the culture solution at 7.0 with 2N NaOH under the conditions of ° C, a stirring speed of 400 rpm, and an aeration rate of 1 vvm is described.

[0006]

However, in any of the above-mentioned conventional methods for producing MK-7, the MK-7 concentration in the obtained culture solution cannot be said to be sufficiently high. That is, the content of MK-7 contained in ordinary commercial natto is about 10 to 20 ppm. On the other hand, in the methods described in the above-mentioned publications, in any case, the MK-7 content in the obtained culture is not much different from the MK-7 content in the commercial natto. That is, in the method described in JP-A-8-73396, the concentration of MK-7 in the culture product is 17.2 mg / kg, and in the method described in JP-A-11-32787, The MK-7 concentration is 29.8 mg / kg. In addition, JP 10
In the method described in JP-295393, MK-
7 is 40.6 mg / L, and according to the method described in JP-A-11-196820, the MK-7 concentration in the culture product is 29636 ng / g of tofu cake (okara) fermented product (that is, 29.6 mg / L). kg)
It is. JP-A-8-19378 describes that the concentration of MK-7 in the culture product is 11.7 mg / 100 ml (that is, 117 mg / L), but this value is a tremendously high concentration. Therefore, it cannot be considered that it can be achieved by the method described in the publication. By the way, the present inventors performed a supplementary test described in Comparative Example 3 described below according to the method described in the publication, and as a result, the MK-7 concentration in the obtained culture solution was 10.3 mg / L.
Met. From this, it was found that the method described in this publication could not achieve the MK-7 concentration of 117 mg / L described therein. Therefore, these conventional methods for producing MK-7 have a low MK-7 concentration in the culture solution obtained in any case, and cannot be said to be sufficient as an industrial production method.

Further, in the method for producing MK-7 described in Japanese Patent Application Laid-Open No. H11-32787, a soy sauce burner containing protein dissolved in soy sauce as a main component is used.
It requires a medium to which glucose, K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, and NaCl are added as nutrients. Further, the method for producing MK-7 described in JP-A-10-295393 described above requires a medium in which 1% by weight or more of glycerol is added to soybean broth whose Brix concentration is adjusted to 5 to 10%. .
The use of a soybean-derived material in addition to a specific chemical increases the production cost of the product.
Therefore, as described above, these methods for producing MK-7 have low concentrations of MK-7 in the culture solution and have problems in terms of economy. For this reason, high yields of MK-7 can be produced using only a single material that is readily available and relatively inexpensive and uses no additional nutrients in the culture medium. There is an urgent need for an early provision of a method for producing MK-7 that enables it. In view of the above-mentioned problems, the present invention has been completed as a result of intensive studies by the present inventors. A main object of the present invention is to provide a method for efficiently and with high yield of MK-7, which solves the above-mentioned problems in the conventional method for manufacturing MK-7 and fulfills the above-mentioned needs. Another object of the present invention is to use only a medium mainly composed of a liquid component obtained by solid-liquid separation of a distillation residue produced as a by-product in the production of distilled liquor using barley, and to use other nutrient components. An object of the present invention is to provide a simple and efficient method for producing MK-7 which does not require any method. MK- produced by the method of the present invention
The 7-containing substance has a sufficiently high MK-7 concentration and can be used as it is as a food or as a vitamin preparation. The MK-7-containing substance produced by the method of the present invention can be purified to a high-purity MK-7-containing substance by a known purification method.

[0008]

SUMMARY OF THE INVENTION The present inventors have found that in many cases the use of barley shochu distillation bottoms, which is subject to disposal, is a conventional method.
Based on the assumption that the above-mentioned problems in the method of manufacturing MK-7 could be solved, the inventor conducted extensive research through experiments. That is, the present inventors solid-liquid separated a shochu distillation residue produced as a by-product in the production of shochu using barley to obtain a liquid component containing an amino acid, a polyphenol, an organic acid, and glycerol. Is used as a medium, and Ba
Bacillus natto, an MK-7 producing bacterium belonging to cillus subtilis, was cultured. As a result, without adding any other nutrients to the liquid, soybean broth conventionally used in culturing natto bacteria, soybean broth with glycerol added, tofu cake, steamed Compared with the case of using a soybean or a synthetic medium to which soy sauce fired rice was added as a medium, not only the amount of the proliferating cells significantly increased, but also the concentration of MK-7 produced by Bacillus natto in the culture broth. Was also found to increase dramatically.

[0009] The inventors of the present invention suppose that similar effects can be obtained in a medium obtained from a whiskey distillation residue produced as a by-product in the production of whiskey using barley as a raw material, similarly to barley shochu. The following experiment was performed. That is, a whiskey distillation residue produced as a by-product in the production of whiskey using barley is solid-liquid separated to obtain a liquid component, and the acid contained in the liquid component is substantially neutralized to obtain a prepared liquid. Using a medium obtained by sterilizing the preparation, natto bacteria, which are MK-7 producing bacteria belonging to Bacillus subtilis, were cultured. As a result, without adding any other nutrients to the liquid, soybean broth conventionally used in culturing natto bacteria, soybean broth with glycerol added, tofu cake, steamed Compared with the case of using a soybean or a synthetic medium to which soy sauce fired rice was added as a medium, not only the amount of the proliferating cells significantly increased, but also the concentration of MK-7 produced by Bacillus natto in the culture broth. Was also found to increase dramatically.

[0010] From the above results, the distillation residue produced as a by-product in the production of distilled liquor using barley is subjected to solid-liquid separation to obtain a liquid component containing amino acids, polyphenols, organic acids and glycerol. Is substantially neutralized to obtain a prepared solution, and the prepared solution is used as a culture medium to prepare Bacill.
By culturing MK-7-producing bacteria belonging to us subtilis to obtain a culture solution, the MK-7 concentration in the culture solution is extremely high.
As a result, it was found that the above object of the present invention could be achieved.

The present invention has been completed based on the above-mentioned facts. Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited thereto. The method for producing MK-7 of the present invention is a method for producing a liquid component containing amino acids, polyphenols, organic acids, and glycerol by solid-liquid separation of a distillation residue produced as a by-product in the production of distilled liquor using barley. Step 1, a second step of obtaining a preparation by substantially neutralizing the acid contained in the liquid component to obtain a preparation, and culturing MK-7 producing bacteria belonging to Bacillus subtilis using the preparation as a medium. It consists of three steps. Hereinafter, the distillation residue used as a raw material when performing the method for producing MK-7 of the present invention and each step will be described in detail.

The distillation residue used in the present invention is obtained by producing (a) barley koji and steamed barley using barley or refined barley as a raw material, and obtaining the obtained barley koji and starch contained in the steamed barley with koji; And / or saccharification using an enzyme agent, followed by alcohol fermentation with yeast to obtain an aged moromi, and when the obtained aged moromi is distilled using a distillation apparatus such as a vacuum distillation or a normal pressure distillation, a residue is obtained as a distillation residue. The raw malt and (ii) malt are produced from barley as a raw material, and the starch contained in the obtained malt is saccharified using malt and / or an enzyme agent, and further subjected to alcohol fermentation by yeast and aged moromi. And a by-product as a distillation residue when the obtained ripened mash is distilled using a distillation apparatus such as a pot still or a patent still. Typical examples of such a distillation residue include a distillation residue in the production of barley shochu or whiskey. In the case of distillation residue in the production of whiskey, malt whiskey using only barley malt as a raw material, as well as grain whiskey using ungerminated grains as a main raw material, is by-produced when barley malt is partially used. Whiskey distillation bottoms are also included in the distillation bottoms referred to in the present invention. In the case of distilled liquor in the production of shochu, in the production of rice shochu, sweet potato shochu, and buckwheat shochu, shochu distillation residual liquid by-produced when barley is used as a part of the raw material in the production of these shochus is also used. It is included in the distillation residue as referred to in the invention.

As described above, the medium obtained from the distillation residue used in the method for producing MK-7 of the present invention contains components derived from barley, barley koji, malt and yeast. That is, the medium obtained from the distillation residue used in the present invention is a liquid medium of a material derived from soybeans such as soybean broth, steamed soybean, tofu lees, and soy sauce fired ori used for conventional MK-7 production. Or, it is completely different from a solid medium. Specifically, as shown in Table 1,
Barley shochu distillation residue by-produced in the production of shochu using barley used in the present invention, crude protein (when measuring the protein amount of food, measure the nitrogen content, multiply the value by nitrogen-protein conversion count Calculated amount)
4%, and the main amino acids are proline, leucine,
Contains arginine, alanine, and glutamic acid.
And the barley shochu distillation residue is glycerol, citric acid, acetic acid produced in the fermentation process in shochu production.
And lactic acid, or polyphenols derived from barley and the like, which are preferable for cultivation of Bacillus natto. Therefore, the medium used in the method for producing MK-7 of the present invention is clearly different from the medium used in the conventional method for producing MK-7, which is objectively distinguished.

By the way, in the method for producing MK-7 described in JP-A-10-295393, a medium in which 1% by weight or more of glycerol is added to soybean broth whose Brix concentration is adjusted to 5 to 10% is used. It is described that the use increases the content of vitamin K (MK-7) in a culture solution. On the other hand, as is clear from Table 1, barley shochu distillation residue produced as a by-product in the production of shochu using barley used in the present invention,
As such it already contains 1.3% glycerol. Conventional MK
The soybean-derived material described above used in the culture medium in the production method of -7 does not contain such a compound, and the compound is added separately to the soybean-derived material.
Therefore, in the present invention, it is not necessary to add glycerol as in the prior art at all to increase the amount of MK-7 produced in the medium. That is, in the present invention, an increase in the production of MK-7 is achieved without separately adding glycerol.

[0015] In the present invention, the distillation residue obtained in the distillation step in the production of distilled liquor is subjected to solid-liquid separation to obtain amino acids,
The first step of obtaining a liquid component containing a polyphenol, an organic acid, and glycerol is performed for the purpose of removing a water-insoluble fermentation residue derived from raw barley, koji or malt from the distillation residue to obtain a liquid component. Things. The solid-liquid separation in the first step can be performed via a solid-liquid separation method such as a screw press method or a roller press method, or using a filtration-pressing type solid-liquid separator. First
In the second step of obtaining a preparation by substantially neutralizing the acid contained in the liquid component obtained in the step, a neutralization treatment can be performed using a suitable neutralizing agent. As the neutralizing agent, sodium hydroxide, potassium hydroxide and the like can be used.

In the third step of culturing an MK-7 producing bacterium belonging to Bacillus subtilis using the above-prepared solution obtained in the second step as a medium, the MK-7 producing bacterium may be Bacillus subtilis.
Any strain belonging to illussubtilis and capable of producing MK-7 can be used. Particularly preferably, Bacillus subtilis, a typical MK-7 producing bacterium belonging to Bacillus subtilis, can be used. Specific examples of the Bacillus natto include commercially available Bacillus natto, Naruse, Miyagi, Takahashi, and the like. In addition to these, MK-
7A natto strain having a high productivity can also be used. The cultivation of the Bacillus natto using the medium can be performed by a known liquid culture method.
It is desirable to carry out in a temperature range of 50 ° C. At this time, the pH value of the culture solution during the culture is preferably maintained at about 7.0 using sodium hydroxide or the like.

In the present invention, the culture solution of Bacillus natto obtained in the third step, that is, the culture solution containing MK-7, can be directly used as an MK-7-containing product. However, when it is necessary to separate and purify only MK-7 from the culture solution, MK-7 can be extracted from the culture solution containing the MK-7. Methods for extracting MK-7 in that case include, for example, a solvent extraction method using an organic solvent such as alcohol or ether, an adsorption fractionation method using activated carbon, and molecular distillation described in JP-A-8-73396. , A distillation method using high vacuum distillation such as steam distillation, a chromatography method using a synthetic adsorbent or the like can be used. In addition, a separation and concentration method using an ultrafiltration membrane or a reverse osmosis membrane and a method of dehydration by a drying operation described in JP-A-11-32787 can be used.
In this case, by performing dehydration treatment in the absence of an organic solvent, an MK-7-containing material having excellent stability to light can be obtained.

[0018]

EXAMPLES The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0019]

[Example 1] 1. Preparation of Medium from Barley Shochu Distillation Residue 1 kiloliter of shochu distillation residue obtained in the distillation process of barley shochu production is subjected to solid-liquid separation using a screw press type solid-liquid separator manufactured by Shinwa Engineering Co., Ltd. 0.8 Kl of the liquid was obtained, and the pH was adjusted to 7.0 by adding sodium hydroxide to the liquid to obtain about 0.9 Kl of the preparation. The obtained preparation was sterilized to obtain a culture medium for natto culture. 2． Preculture of Bacillus natto Dissolve 10 g of meat extract and 10 g of peptone in 1 L of distilled water, add sodium hydroxide to the resulting solution, adjust the pH value to 7.0, and sterilize at 121 ° C for 15 minutes To obtain a meat extract medium. 5 ml of the meat extract medium and one platinum loop of Bacillus natto commercially available Bacillus natto were introduced into a test tube, stirred, and shake-cultured at 42 ° C for 15 hours to obtain a Bacillus natto preculture solution. 3． Main culture of Bacillus natto 2 L jar fermenter, 1 L of the medium obtained in the above 1 and 5 ml of the Bacillus natto preculture obtained in the above 2 were introduced, and aeration 0.2 vvm,
Culturing was performed for 14 days at a stirring speed of 300 rpm and a culture temperature of 42 ° C. Thus, a natto culture containing MK-7 was obtained. Since MK-7 (vitamin K) produced by culturing may be decomposed by light, the jar fermenter was covered with aluminum foil before culturing.

[0020]

[Comparative Example 1] In this comparative example, a soybean soup waste liquid produced as a by-product during natto production was used in place of the liquid obtained from the distillation residue of barley shochu used in Example 1.
A natto culture solution containing MK-7 was obtained in the same manner as in 1. 1. Preparation of culture medium from soybean broth waste liquid Sodium broth waste liquid by-produced during natto production was adjusted to pH 7.0 by adding sodium hydroxide to obtain a control liquid, and the resulting control liquid was sterilized to natto A culture medium for fungal culture was obtained. 2. Preculture of Bacillus natto Dissolve 10 g of meat extract and 10 g of peptone in 1 L of distilled water, add sodium hydroxide to the resulting solution, adjust the pH value to 7.0, and sterilize at 121 ° C for 15 minutes To obtain a meat extract medium. 5 ml of the meat extract medium and one platinum loop of Bacillus natto commercially available Bacillus natto were introduced into a test tube, stirred, and shake-cultured at 42 ° C for 15 hours to obtain a Bacillus natto preculture solution. 3. Main culture of Bacillus natto Into a 2 L jar fermenter, 1 L of the medium obtained in the above 1 and 5 ml of the Bacillus natto preculture obtained in the above 2 were introduced, and the aeration rate was 0.2 vv.
The culture was performed for 14 days under the conditions of m, stirring speed of 300 rpm, and culture temperature of 42 ° C. Thus, a natto culture containing MK-7 was obtained. Since MK-7 produced by culturing may be decomposed by light, the jar fermenter was previously covered with aluminum foil for culturing.

[0021]

Comparative Example 2 In this comparative example, the conventional MK-7 described above was used.
In the production method, by adding glycerol to the soybean broth waste liquid, according to the method described in JP-A-10-295393, which describes that the concentration of MK-7 in the obtained culture solution can be increased. Thus, a natto culture solution was obtained. That is, natto containing MK-7 in the same manner as in Example 1 except that glycerol was added to the soybean broth waste liquid instead of the liquid component obtained from the barley shochu distillation residue used in Example 1. A bacterial culture was obtained. 1. Preparation of culture medium from soybean broth wastewater 5% by weight of glycerol was added to soybean broth wastewater adjusted to a Brix concentration of 10%, and sodium hydroxide was added to the obtained liquid to adjust its pH value to 7.0. An adjustment liquid was obtained. The obtained adjusted solution was sterilized to obtain a culture medium for natto culture. 2. Preculture of Bacillus natto Dissolve 10 g of meat extract and 10 g of peptone in 1 L of distilled water, add sodium hydroxide to the resulting liquid, adjust the pH value to 7.0, and sterilize at 121 ° C for 15 minutes To obtain a meat extract medium. 5 ml of the meat extract medium and one platinum loop of Bacillus natto commercially available Bacillus natto were introduced into a test tube, stirred, and shake-cultured at 42 ° C for 15 hours to obtain a Bacillus natto preculture solution. 3. Main culture of Bacillus natto Into a 2 L jar fermenter, 1 L of the medium obtained in the above 1 and 5 ml of the Bacillus natto preculture obtained in the above 2 were introduced, and the aeration rate was 0.2 vv.
The culture was performed for 14 days under the conditions of m, stirring speed of 300 rpm, and culture temperature of 42 ° C. Thus, a natto culture containing MK-7 was obtained. Since MK-7 produced by culturing may be decomposed by light, the jar fermenter was previously covered with aluminum foil for culturing.

[0022]

Example 2 Japanese Patent Application Laid-Open No. H8-19378 states that the highest MK-7 concentration can be achieved among the conventional MK-7 production methods described above.
In accordance with the method described in Japanese Patent Application Publication No. H11-107, a culture solution of Bacillus natto was obtained. However, in this example, instead of the soybean broth waste liquid by-produced during the production of natto described in the publication, a medium obtained from the distillation residue of barley shochu used in the present invention was used. That is, 1 kiloliter of the shochu distillation residue obtained in the distillation step in the production of barley shochu is solid-liquid separated by a screw press type solid-liquid separator manufactured by Shinwa Engineering Co., Ltd. to about 0.
Eight kiloliters of a liquid component was obtained, and sodium hydroxide was added to the liquid component to adjust the pH value to 7.0 to obtain about 0.9 kiloliter of a control liquid. After introducing 100 ml of the prepared solution into a 500-ml Erlenmeyer flask, the cotton stopper was placed, and the autoclave was used for 12 hours.
Steam pressure sterilization was performed at 1 ° C. × 20 min to obtain a culture medium. 10 μl of Saccharomyces natto having an OD of 660 nm of 10.0 was aseptically inoculated into the medium, and cultured with shaking at 120 rpm at 48 ° C. for 4 days to obtain a natto culture containing MK-7. In addition, since the MK-7 produced by the culture may be decomposed by light, the periphery of the Erlenmeyer flask was covered with aluminum foil in advance, and the culture was performed.

[0023]

Comparative Example 3 In this comparative example, a Bacillus natto culture was obtained according to the method described in JP-A-8-19378. That is, sodium hydroxide was added to the soybean soup waste liquid by-produced during the production of natto to adjust the pH value thereof to 7, thereby obtaining an adjusted liquid. 5
After introducing 100 ml into a 00 ml Erlenmeyer flask, stopper the cotton,
Steam pressure sterilization was performed in an autoclave at 121 ° C. for 20 minutes to obtain a culture medium. 10 μl of Saccharomyces natto having an OD of 660 nm of 10.0 was aseptically inoculated into the medium, and cultured at 48 ° C. with shaking at 120 rpm for 4 days to obtain a Bacillus natto culture containing MK-7. Since MK-7 obtained by the culture might be decomposed by light, the culture was performed by covering the periphery of the Erlenmeyer flask with aluminum foil in advance.

[0024]

[Quantification of MK-7] Example 1, Example 2, Comparative Example 1, Comparative Example 2,
And MK in each of the natto cultures obtained in Comparative Example 3
The -7 concentration was determined by the following method. That is, Example 1,
For each natto culture solution obtained in Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3, 1.5 ml was added to 1.5 ml thereof.
Of isopropyl alcohol and 5 ml of hexane,
After shaking, centrifugation was performed under the conditions of 1710 g × 10 min to obtain an organic layer and an aqueous layer.The organic layer 4 ml was recovered, and hexane contained in the organic layer 4 ml was completely removed using an evaporator. The remaining tan oil was dissolved in 500 μl of 97% ethanol, and the MK-7 concentration was quantified using high performance liquid chromatography under the following conditions. That is, Inertsil OD is added to the column.
S 4.6 x 250 mm (GL Science), 97% using a platinum-alumina catalyst (beads) column 4.6 x 50 mm (platinum-alumina beads manufactured by Wako Pure Chemical Industries, Ltd.)
Using ethanol as the eluent, the flow rate was 0.7 ml / min, the column temperature was 40 ° C, and the sample injection volume was 10 μl.
(20 nm, fluorescence 430 nm). In addition, MK-7
In order to prepare a calibration curve, a 100 ppm MK-7 standard sample was used. The results obtained are shown in Tables 2 to 4.

[0025]

[Evaluation 1] Table 2 shows the quantification results of MK-7 in the culture solutions obtained in Example 1, Comparative Example 1 and Comparative Example 2. As is clear from the results shown in Table 2, the MK-7 concentration in the culture solution obtained in Example 1 was 83.1 mg / l, whereas the MK-7 concentration in the culture solution obtained in Comparative Example 1 was MK-7 concentration was 12.2 mg / l, and the MK-7 concentration in the culture solution obtained in Comparative Example 2 was 33.8 mg / l. That is, the concentration of MK-7 in the culture solution obtained in Example 1 was
It was found that the concentration of MK-7 in the culture solution obtained in 1 reached about 6.8 times and the concentration of MK-7 in the culture solution obtained in Comparative Example 2 reached about 2.5 times. That is, when the medium obtained from the shochu distillation residue obtained in the distillation step in barley shochu production is used, compared with the case where the medium obtained from the soybean broth waste liquid by-produced during natto production is used. MK- in the resulting culture solution
7 concentrations were found to increase significantly.

[0026]

[Evaluation 2] Table 3 shows the results of quantification of MK-7 in the culture solutions obtained in Example 2 and Comparative Example 3. As is evident from the results shown in Table 3, MK-7 in the culture solution obtained by using the medium obtained from the barley shochu distillation residue produced as a by-product in the distillation step in the barley shochu production of Example 2. Concentration 22.3mg / l
On the other hand, the MK-7 concentration in the culture solution obtained by the method described in JP-A-8-19378 of Comparative Example 3 was 10.
It was 3 mg / l. That is, according to the method described in JP-A-8-19378, the concentration of MK-7 in the culture product is 11.7 mg / 100m
1 (ie, 117 mg / L), but this value is a ridiculously high concentration, and it has been found that it is impossible at all to achieve the concentration by the method described in the publication.
In addition, when using a medium obtained from the shochu distillation residue obtained in the distillation step in barley shochu production, compared to using a medium obtained from soy broth waste liquid by-produced during natto production. It was found that the concentration of MK-7 in the obtained culture was significantly increased.

[0027]

Example 3 In this example, the whiskey distillation obtained in the distillation step in the production of malt whiskey was replaced with the liquid component of the shochu distillation residue produced as a by-product in the distillation step in the production of barley shochu used in Example 1. A Bacillus natto culture containing MK-7 was obtained in the same manner as in Example 1 except that the liquid obtained by solid-liquid separation of the residual liquid was used. 1． Preparation of Medium from Whiskey Distillation Residue The whiskey distillation residue obtained in the distillation step in the production of malt whiskey is solid-liquid separated to obtain a liquid component, and sodium hydroxide is added to the liquid component to adjust the pH value to 7.0. The resulting solution was sterilized to obtain a culture medium for natto culture. 2． Preculture of Bacillus natto Dissolve 10 g of meat extract and 10 g of peptone in 1 L of distilled water, add sodium hydroxide to the resulting solution, adjust the pH value to 7.0, and sterilize at 121 ° C for 15 minutes To obtain a meat extract medium. 5 ml of the meat extract medium and one platinum loop of Bacillus natto commercially available Bacillus natto were introduced into a test tube, stirred, and shake-cultured at 42 ° C for 15 hours to obtain a Bacillus natto preculture solution. 3． Main culture of Bacillus natto 2 L jar fermenter, 1 L of the medium obtained in the above 1 and 5 ml of the Bacillus natto preculture obtained in the above 2 were introduced, and aeration 0.2 vvm,
Culturing was performed for 14 days at a stirring speed of 300 rpm and a culture temperature of 42 ° C. Thus, a natto culture containing MK-7 was obtained. Since MK-7 produced by culturing may be decomposed by light, the jar fermenter was covered with aluminum foil before culturing.

[0028]

Comparative Example 4 In this comparative example, a soybean broth waste liquid was used in place of the liquid obtained by solid-liquid separation of the whiskey distillation residue obtained in the distillation step in the production of malt whiskey used in Example 4. A natto culture solution containing MK-7 was obtained in the same manner as in Example 3 except that the above procedure was repeated. 1． Preparation of culture medium from soybean broth wastewater Sodium broth wastewater by-produced during natto production was adjusted to pH 7.0 by adding sodium hydroxide, and the resulting adjusted solution was sterilized for natto culture. A medium was obtained. 2． Preculture of Bacillus natto Dissolve 10 g of meat extract and 10 g of peptone in 1 L of distilled water, add sodium hydroxide to the resulting solution, adjust the pH value to 7.0, and sterilize at 121 ° C for 15 minutes To obtain a meat extract medium. 5 ml of the meat extract medium and one platinum loop of Bacillus natto commercially available Bacillus natto were introduced into a test tube, stirred, and shake-cultured at 42 ° C for 15 hours to obtain a Bacillus natto preculture solution. 3． Main culture of Bacillus natto 2 L jar fermenter, 1 L of the medium obtained in the above 1 and 5 ml of the Bacillus natto preculture obtained in the above 2 were introduced, and the aeration rate 0.2 vvm,
Culturing was performed for 14 days at a stirring speed of 300 rpm and a culture temperature of 42 ° C. Thus, a natto culture containing MK-7 was obtained. Since MK-7 produced by culturing may be decomposed by light, the jar fermenter was covered with aluminum foil before culturing.

[0029]

[Evaluation 3] The MK-7 concentration of each of the culture solutions obtained in Example 3 and Comparative Example 4 was measured by the above-described method for determining MK-7. As a result, it was found that the MK-7 concentration in the culture solution obtained in Example 3 was significantly higher than the MK-7 concentration in the culture solution obtained in Comparative Example 4. That is, when the medium obtained from the whiskey distillation residue produced as a by-product in the distillation step of whiskey production is used, compared with the case where the medium obtained from the soybean broth waste liquid by-produced during the production of natto is used, It was found that the concentration of MK-7 in the obtained culture was significantly increased.

From the above results, according to the method for producing an MK-7-containing substance according to the present invention, wherein a medium obtained from a distillation residue produced as a by-product in the production of distilled liquor using barley is used. It was found that an MK-7-rich material having a significantly higher MK-7 concentration than the conventional MK-7 production method could be obtained.

[0031]

Example 4 An MK-7-containing oily substance was extracted from each of the Bacillus natto cultures obtained in Examples 1 and 2.
That is, the same amount of isopropyl alcohol as the Bacillus natto culture was introduced into each of the Bacillus natto cultures obtained in Example 1 and Example 2, and then 3 times the amount of hexane of the Bacillus natto culture was added. After introducing and shaking, centrifugation was performed under the conditions of 1710 g × 10 min to obtain an organic layer and an aqueous layer, and after collecting the organic layer, completely removing hexane contained in the organic layer using an evaporator. A tan oil was obtained which contained no ammonia.

[0032]

Comparative Example 5 An oily substance containing MK-7 was extracted from each of the natto cultures obtained in Comparative Example 1, Comparative Example 2 and Comparative Example 3. That is, the same amount of isopropyl alcohol as the Bacillus natto culture was introduced into each of the Bacillus natto cultures obtained in Comparative Examples 1, 2 and 3, and then 3 times the Bacillus natto cultures. After introducing the amount of hexane and shaking, 1710 g × 10 min
Centrifugation under the conditions described above to obtain an organic layer and an aqueous layer.After recovering the organic layer, hexane contained in the organic layer is completely removed using an evaporator, so that a yellow-brown oil containing no ammonia at all. Material was obtained.

[0033]

[Evaluation 4] Example obtained in Example 4 and Comparative Example 5
1, the weight of each oily substance extracted from the natto culture solution obtained in each of Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was measured, and further, by the above-described method for quantifying MK-7. MK-7 concentration was measured. Table 4 shows the obtained measurement results. The following facts were found from the results shown in Table 4. (1) As apparent from Table 4, the MK-7 concentration in the oil obtained from the culture of Example 1 was 19786 ppm, whereas the oil obtained from the culture of Comparative Example 1 was 19786 ppm. The MK-7 concentration in the medium was 1258 mg / l, and the MK-7 concentration in the oily substance obtained from the culture solution of Comparative Example 2 was 3414 mg / l. That is, the MK-7 concentration in the oily substance obtained from the culture solution of Example 1 was about 15.7 times the MK-7 concentration in the oily substance obtained from the culture solution of Comparative Example 1, and the culture of Comparative Example 2 was carried out. It was found to be about 5.8 times the concentration of MK-7 in the oil obtained from the liquid. (2) Similarly, M in the oily substance obtained from the culture solution of Example 2
While the K-7 concentration was 5186 ppm, the MK-7 concentration in the oil obtained from the culture solution of Comparative Example 3 was 1084 mg / l.
That is, it was found that the MK-7 concentration in the oily substance obtained from the culture solution of Example 2 was about 4.8 times the MK-7 concentration in the oily substance obtained from the culture solution of Comparative Example 3. From the above, when using the medium obtained from the shochu distillation residue by-produced in the distillation step in barley shochu production, by adding glycerol to the medium obtained from soybean broth waste liquid, or soybean broth waste liquid It was found that an oily substance containing a higher content of MK-7 was obtained as compared with the case where the obtained medium was used.

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

[0037]

[Table 4]

[0038]

As described in detail above, according to the method for producing an MK-7-containing material of the present invention, the following effects can be obtained. That is, the distillation residue produced as a by-product in the production of distilled liquor using barley is subjected to solid-liquid separation to obtain a liquid component containing an amino acid, a polyphenol, an organic acid, and glycerol, and the acid contained in the liquid component is substantially reduced. Neutralized to obtain a preparation, and using the medium obtained by sterilizing the preparation, Bacillus
By culturing MK-7 producing bacteria belonging to subtilis,
MK- without adding any other nutrients to the medium
An MK-7-containing substance having an extremely high concentration of 7 can be efficiently and stably obtained.

[Brief description of the drawings]

FIG. 1 is a manufacturing process diagram schematically showing a manufacturing process of MK-7 of the present invention.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kei Hayashi 223-1 Yamamoto, Usa-shi, Oita Sanwa Sake Co., Ltd. F-term (reference) 4B064 AD94 CA03 CE08 DA01 DA10

Claims (4)

[Claims] Claims 1. A liquid residue containing an amino acid, a polyphenol, an organic acid, and glycerol is obtained by solid-liquid separation of a distillation residue obtained as a by-product in the production of distilled liquor using barley, which is contained in the liquid component. A method for producing a menaquinone-7-containing substance, which comprises culturing a menaquinone-7-producing bacterium belonging to Bacillus subtilis using a preparation obtained by substantially neutralizing an acid as a medium. 2. The method for producing a menaquinone-7-containing substance according to claim 1, wherein the distillation residue is a distillation residue produced as a by-product in the production of shochu using barley. 3. The method for producing a menaquinone-7-containing substance according to claim 1, wherein the distillation residue is a distillation residue produced as a by-product in the production of whiskey using barley. 4. The method for producing a menaquinone-7-containing substance according to claim 1, further comprising a step of purifying the menaquinone-7-containing substance into a high-purity menaquinone-7-containing substance.