JP2008072966A - Method for producing nattokinase and vitamin k2 - Google Patents

Method for producing nattokinase and vitamin k2 Download PDF

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JP2008072966A
JP2008072966A JP2006256322A JP2006256322A JP2008072966A JP 2008072966 A JP2008072966 A JP 2008072966A JP 2006256322 A JP2006256322 A JP 2006256322A JP 2006256322 A JP2006256322 A JP 2006256322A JP 2008072966 A JP2008072966 A JP 2008072966A
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vitamin
dipicolinic acid
nattokinase
natto
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Hiroyuki Sumi
洋行 須見
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Honda Trading Corp
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing Nattokinase and a method for efficiently producing vitamin K<SB>2</SB>. <P>SOLUTION: The method for producing the Nattokinase comprises adding dipicolinic acid to a culture solution of Bacillus natto. The method for producing the vitamin K<SB>2</SB>comprises adding the dipicolinic acid to the culture solution of the Bacillus natto. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、納豆菌を用いたナットウキナーゼ及びビタミンＫ２（メナキノン−７）の製造方法に関する。 The present invention relates to a method for producing nattokinase and vitamin K 2 (menaquinone-7) using Bacillus natto.

糸引き納豆は、日本の伝統的な大豆発酵食品として約１０００年の歴史を持ち、年間３０万トン以上生産されている。納豆生産菌はビオチン要求性であり、それがないと生育できない。発明者は、これまで納豆中の強力な血栓溶解酵素ナットウキナーゼ、ビタミンＫ２及び、抗菌物質として知られているジピコリン酸（dipicolic acid: 2、6-pyridinedicarboxylic
acid、DPA）の研究を行ってきた。 Itobiki Natto has a history of about 1000 years as a traditional Japanese fermented soybean fermented food and is produced more than 300,000 tons per year. Natto-producing bacteria are biotin-requiring and cannot grow without it. The inventor has found that nattokinase, vitamin K 2 and dipicolic acid (2, 6-pyridinedicarboxylic acid), known as antibacterial substances, in natto.
acid, DPA).

血栓溶解のメカニズムとしては、生体内ではプラスミンが直接フィブリンに働きかけて溶解現象を起こす。ナットウキナーゼは、それ自体が直接フィブリンを溶解するだけでなく、例えば、プロ−ウロキナーゼ分子に対する強力な活性化能を持ち、間接的にもフィブリン溶解に働くことが分かっている。このため、ナットウキナーゼは、血栓症予防薬として開発が進められている。また、ナットウキナーゼは、酵素の基質特異性として血栓性の成分であるフィブリンを切る他、Suc-Ala-Ala-Pro-Phe-pNA を切ることが知られている。   As a mechanism of thrombolysis, plasmin works directly on fibrin to cause a dissolution phenomenon in vivo. Nattokinase not only directly dissolves fibrin itself, but also has a strong activating ability to, for example, pro-urokinase molecules, and has been found to work indirectly on fibrin dissolution. Therefore, nattokinase is being developed as a thrombosis preventive drug. Nattokinase is known to cleave Suc-Ala-Ala-Pro-Phe-pNA in addition to cleaving fibrin, which is a thrombogenic component, as the substrate specificity of the enzyme.

納豆に含まれるビタミンＫ２には、骨粗鬆症発症予防の効果があることが確認されている。 Vitamin K 2 contained in natto, has been confirmed to be effective for developing osteoporosis prevention.

上記のように、ナットウキナーゼ及びビタミンＫ２は、健康にとって極めて有用であり、生産効率の向上及び現実的な普及が望まれている。 As described above, nattokinase and vitamin K 2 is extremely useful for health improvement and practical spread of production efficiency is desired.

本発明は、上記のような状況に鑑みてなされたものであり、ナットウキナーゼを効率よく生産可能な方法を提供することを目的とする。   The present invention has been made in view of the above situation, and an object thereof is to provide a method capable of efficiently producing nattokinase.

また、ビタミンＫ２を効率よく生産可能な方法を提供することを他の目的とする。 Further, to provide a vitamin K 2 efficiently producible manner as other purposes.

上記目的を達成するために、本発明に係るナットウキナーゼの製造方法においては、納豆菌の培養液中にジピコリン酸を添加する。   In order to achieve the above object, in the method for producing nattokinase according to the present invention, dipicolinic acid is added to the culture solution of Bacillus natto.

また、本発明に係るビタミンＫ２の製造方法においては、納豆菌の培養液中にジピコリン酸を添加する。 Further, in the method for manufacturing a vitamin K 2 according to the present invention, the addition of dipicolinic acid in the culture solution of Bacillus subtilis natto.

発明者は、納豆菌培養液にジピコリン酸を添加した場合、ジピコリン酸はナットウキナーゼあるいはビタミンＫ2（メナキノン−７）量の生産増強に働くことを見出した。ビタミンＫ２（ＭＫ−７）は、納豆以外に食品由来の原料はないため、納豆に含まれるビタミンＫ２は、特に骨粗鬆症発症予防面から世界中で期待されている。 The inventor has found that when dipicolinic acid is added to a culture solution of Bacillus natto, dipicolinic acid works to enhance production of nattokinase or vitamin K 2 (menaquinone-7). Since vitamin K 2 (MK-7) has no food-derived ingredients other than natto, vitamin K 2 contained in natto is expected all over the world, particularly in terms of preventing osteoporosis.

ところで、ジピコリン酸は放射能除去物質とも言われているが、元々は抗菌物質であり、強い酵母抑制そしてＯ−１５７などの生育を抑制する。また、胞子中に存在するキレート物質で菌の耐熱性と深く係わることは周知である。更に、最近では、血液の抗血小板凝集活性を抑える効果が注目されている。すなわち、ジピコリン酸は重量当りでアスピリン以上に強い抑制効果を持つわけである。   By the way, although dipicolinic acid is also said to be a radioactivity-removing substance, it is originally an antibacterial substance and suppresses strong yeast suppression and growth of O-157 and the like. Further, it is well known that a chelate substance existing in the spore is closely related to the heat resistance of the bacterium. Furthermore, recently, the effect of suppressing the antiplatelet aggregation activity of blood has attracted attention. That is, dipicolinic acid has a stronger inhibitory effect than aspirin per weight.

納豆菌の培養液中にジピコリン酸を添加した場合、ナットウキナーゼの標準フィブリン平板でみたフィブリン溶解面積、およびSuc-Ala-Ala-Pro-Phe-pNAに対するアミダーゼ活性が著しく上昇することが分かった。例えば、蒸煮大豆を用いて納豆を作成した場合、１０−３２ｍＭのジピコリン酸を添加することによりアミダーゼ活性が通常（ジピコリン酸を添加しない場合）の１０倍以上も高まることが分かった。   It was found that when dipicolinic acid was added to the culture solution of Bacillus natto, the fibrinolysis area on the standard fibrin plate of nattokinase and amidase activity against Suc-Ala-Ala-Pro-Phe-pNA increased remarkably. For example, when natto was prepared using steamed soybeans, it was found that the addition of 10-32 mM dipicolinic acid increased the amidase activity 10 times or more than usual (in the case where dipicolinic acid was not added).

また、ビタミンＫ2（メナキノン−７）濃度に関しても、１０ｍＭジピコリン酸を添加することにより、通常（ジピコリン酸を添加しない場合）約４倍に高まることが確認された。これは静置培養だけでなく振盪培養でも同じ結果であった。すなわち、培養物中に添加するジピコリン酸の量をコントロールすることにより、ナットウキナーゼ及びビタミンＫ2（メナキノン−７）の両活性の優れた製品を作ることが可能となる。 It was also confirmed that the vitamin K 2 (menaquinone-7) concentration was usually increased about 4 times (when dipicolinic acid was not added) by adding 10 mM dipicolinic acid. This was the same result not only in static culture but also in shaking culture. That is, by controlling the amount of dipicolinic acid added to the culture, it is possible to produce a product excellent in both activities of nattokinase and vitamin K 2 (menaquinone-7).

ナットウキナーゼは高純度精製品を用いて、フィブリン及び、Suc-Ala-Ala-Pro-Phe-pNAを切ることが知られている。よって、これら両基質を切ればナットウキナーゼといえる。ジピコリン酸は、両基質（フィブリン及び、Suc-Ala-Ala-Pro-Phe-pNA）でナットウキナーゼの賦活化を証明した。また、ビタミンＫ２についても、より低濃度のジピコリン酸で同時にビタミンＫ２の含量が高まった。 Nattokinase is known to cut fibrin and Suc-Ala-Ala-Pro-Phe-pNA using high purity purified products. Therefore, it can be said that nattokinase is obtained by cutting both these substrates. Dipicolinic acid demonstrated nattokinase activation with both substrates (fibrin and Suc-Ala-Ala-Pro-Phe-pNA). As for the vitamin K 2, it has increased content of vitamin K 2 simultaneously at lower concentrations of dipicolinic acid.

ジピコリン酸は、主に普通納豆の胞子内に存在するが、長時間の培養では少量はフリーの形でも存在する。納豆を食べた場合のその生理活性、基質や培養条件、また納豆菌によってジピコリン酸の至適濃度が異なる。   Dipicolinic acid is mainly present in the spore of ordinary natto, but a small amount is also present in a free form during long-term culture. The optimum concentration of dipicolinic acid varies depending on its physiological activity when natto is eaten, its substrate and culture conditions, and natto bacteria.

以下、本発明を実施するための最良の形態について、実施例を用いて詳細に説明する。
まず、本発明の実施例（実験）に使用される材料及び培養方法について説明する。 Hereinafter, the best mode for carrying out the present invention will be described in detail using embodiments.
First, materials and culture methods used in the examples (experiment) of the present invention will be described.

（材料）
１．ジピコリン酸は、ナカライテスク株式会社のものを使用した。使用に当たり、水溶液を水酸化ナトリウムでｐＨ７．４に調整した。
２．人工血栓に用いる牛製フィブリノーゲン、合成基質であるSuc-Ala-Ala-Pro-Phe-pNAは、シグマ（Sigma）社のものを用いた。
３．牛製トロンビンは持田製薬株式会社のものを使用した。
４．培地には日水製薬株式会社の乾燥ブイヨンを用いた。
５．納豆菌は、食品用の代表株である宮城野株、成瀬株、さらに胃腸薬として用いられる目黒株を使用した。また、Bacillus属で枯草菌の代表株Bacillus
subtilis IAM 12118 (type Marburg)は東京大学分子細胞生物学研究所から入手した。 (material)
1. Dipicolinic acid was used from Nacalai Tesque. In use, the aqueous solution was adjusted to pH 7.4 with sodium hydroxide.
2. Cow made fibrinogen for use in artificial thrombus, a synthetic substrate Suc-Ala-Ala-Pro- Phe- p NA used was a Sigma (Sigma) Corporation.
3. The bovine thrombin used was from Mochida Pharmaceutical Co., Ltd.
4). Dry broth from Nissui Pharmaceutical Co., Ltd. was used as the medium.
5. As the Bacillus natto, Miyagino strain and Naruse strain, which are representative stocks for food, and Meguro strain used as a gastrointestinal drug were used. Bacillus, a representative strain of Bacillus subtilis
subtilis IAM 12118 (type Marburg) was obtained from the Institute for Molecular Cell Biology, the University of Tokyo.

（培養方法）
納豆菌の培養は次の３種の方法を用いた。
１．試験管（18mm×105mm）内で、培地0.5mlにジピコリン酸を終濃度０〜４ｍＭになるよう0.5ml添加し、これに納豆菌１白金耳／10ml（宮城野菌、約1.0×108個/ml）を50μl接種した。その後、37℃で24時間静置培養を行なった。
２．90mm角のＰＳＰ（ポリスチレンペーパー）内に蒸煮大豆、並びにジピコリン酸を終濃度０〜１００ｍＭになるよう加え（湿重量50ｇ）、これに納豆菌0.1g/10ml（目黒菌、約1.0×109個/ml）を0.5ml接種した。その後、このＰＳＰ内で37℃24時間静置培養を行った。
３．500mlの三角フラスコに１％グリセリン−３％ポリペプトン−Ｓ、ならびにジピコリン酸を終濃度０〜２０ｍＭになるよう加え（湿重量150ｇ）、これに納豆菌１白金耳／10ml（宮城野菌、約0.5×107個/ml）を1.0ml接種した。その後、37℃で72時間100rpmで振盪培養を行なった。 (Culture method)
The following three methods were used for culture of Bacillus natto.
1. In a test tube (18 mm × 105 mm), add 0.5 ml of dipicolinic acid to 0.5 ml of medium to a final concentration of 0 to 4 mM, and add 1 ml of natto 1 platinum ear / 10 ml (Miyagino, approximately 1.0 × 10 8 cells / ml) was inoculated. Thereafter, static culture was performed at 37 ° C. for 24 hours.
2. Steamed soybeans and dipicolinic acid are added in 90 mm square PSP (polystyrene paper) to a final concentration of 0 to 100 mM (wet weight 50 g), and 0.1 g / 10 ml of natto (Meguro fungus, approximately 1.0 × 10) 9 ml / ml) was inoculated. Thereafter, static culture was performed in this PSP at 37 ° C. for 24 hours.
3. To a 500 ml Erlenmeyer flask, add 1% glycerin-3% polypeptone-S and dipicolinic acid to a final concentration of 0-20 mM (wet weight 150 g). 0.5 × 10 7 cells / ml) was inoculated. Thereafter, shaking culture was performed at 37 ° C. for 72 hours at 100 rpm.

次に、ナットウキナーゼ活性の測定方法について説明する。
（標準フィブリン平板法）
144mm×104mmの角型シャーレ内に終濃度0.5%フィブリノーゲン20mlと50U/mlのトロンビン100μlを加えることによりフィブリン平板を作製した。試料１検体につき30μlを平板上にのせ37℃、4時間インキュベーション後に生じる溶解面積（mm2）を測定した。 Next, a method for measuring nattokinase activity will be described.
(Standard fibrin plate method)
A fibrin plate was prepared by adding 20 ml of final concentration 0.5% fibrinogen and 100 μl of 50 U / ml thrombin in a 144 mm × 104 mm square petri dish. 30 μl of each sample was placed on a plate and the dissolution area (mm 2 ) generated after incubation at 37 ° C. for 4 hours was measured.

（合成アミド基質分解法）
試料0.1ml及び0.1Mリン酸緩衝液−生理的食塩水（pH7.8）を加えた反応系に、終濃度5×10-4Mになるように調整したSuc-Ala-Ala-Pro-Phe-pNA を0.1ml添加し、37℃で5分間インキュベーション後遊離して生じるpNA量の405nmにおける吸光度を測定した。なお、基質添加前に試料及び0.1Mリン酸緩衝液‐生理的食塩水を37℃で2分間プレインキュベーションした。標準曲線を基に反応系1mlに対し1分間当たりの遊離pNA量(nmol)を算出した。 (Synthetic amide substrate decomposition method)
Suc-Ala-Ala-Pro-Phe adjusted to a final concentration of 5 × 10 −4 M in a reaction system in which 0.1 ml of a sample and 0.1 M phosphate buffer-physiological saline (pH 7.8) were added -0.1 ml of -pNA was added, and the absorbance at 405 nm of the amount of pNA produced after incubation at 37 ° C for 5 minutes was measured. The sample and 0.1 M phosphate buffer-saline were preincubated at 37 ° C. for 2 minutes before adding the substrate. Based on the standard curve, the amount of free pNA (nmol) per minute was calculated for 1 ml of the reaction system.

次に、ビタミンＫ２の測定方法について説明する。
メナキノン−７（MK-7）が白金−アルミナ触媒でヒドロキノン体に還元され、蛍光化することを利用して、すでに報告されているＨＰＬＣ法で行った8）9）。すなわち、逆相分配型カラムとして固定相にオクタデシル（C18）基を結合させたODS-II（島津製作所；φ4.6×250mm）、触媒カラムとして白金-アルミナ（和光純薬；φ4.0×10mm）、および展開液として97％エタノールを用いて40℃、0.7ml／minで操作した。試料0.1ml及び蒸留水0.9ml、イソプロパノール1.5mlを混和し、ヘキサン5.0mlを加え撹拌した後、遠心分離し、20℃で10分間、1,710×ｇで遠心分離し、上清のヘキサン画分4mlをエバポレーターで濃縮し、100μlのエタノールで溶解したものをＨＰＬＣで分析した。 Next, a description method for measuring vitamin K 2.
Menaquinone-7 (MK-7) was reduced to a hydroquinone form with a platinum-alumina catalyst and fluorescentized, and this was performed by the previously reported HPLC method8 ) 9) . That is, ODS-II (Shimadzu Corporation; φ4.6 × 250 mm) in which octadecyl (C 18 ) group is bonded to the stationary phase as a reverse phase distribution type column, and platinum-alumina (Wako Pure Chemicals; φ4.0 ×) as a catalyst column. 10 mm), and 97% ethanol as a developing solution, and operated at 40 ° C. and 0.7 ml / min. Mix 0.1 ml of sample, 0.9 ml of distilled water and 1.5 ml of isopropanol, add 5.0 ml of hexane, stir, centrifuge, centrifuge at 1,710 xg for 10 minutes at 20 ° C, 4 ml of supernatant hexane fraction The solution was concentrated with an evaporator, and dissolved in 100 μl of ethanol and analyzed by HPLC.

次に、実験の結果（成績）について説明する。
図１は、一定量（２ｍＭ）のジピコリン酸を添加して納豆菌を培養した結果である。宮城野菌、成瀬菌など納豆菌の代表的なものがジピコリン酸の濃度でナットウキナーゼ生産量が大きく変わることが分かる。それに比べて、コントロールである枯草菌（B. Subtilis）では、ジピコリン酸の濃度でナットウキナーゼ生産量がほとんど変化しないことを確認した。 Next, the results (results) of the experiment will be described.
FIG. 1 shows the result of culturing Bacillus natto after adding a certain amount (2 mM) of dipicolinic acid. It can be seen that nattokinase production varies greatly with the concentration of dipicolinic acid in representative natto bacteria such as Miyagino and Naruse. In contrast, it was confirmed that the amount of nattokinase produced in the control Bacillus subtilis (B. Subtilis) hardly changed with the dipicolinic acid concentration.

図２は、大豆を基質として用い納豆製造に近い条件とした。蒸煮大豆に０−１００ｍＭのジピコリン酸を添加し、納豆菌を接種し、３７℃、２４時間培養した結果である。発酵終了後、当倍量の生食と共にミキサーで粉砕し、遠心後の上清を試料とした。ジピコリン酸の添加量が１０−３２ｍＭの時に、ナットウキナーゼの活性が特に高いことが分かる。   In FIG. 2, soybean was used as a substrate and the conditions were close to those of natto production. It is the result of adding 0-100 mM dipicolinic acid to steamed soybean, inoculating natto bacteria, and culturing at 37 ° C. for 24 hours. After completion of the fermentation, the mixture was ground with a mixer together with an equivalent amount of raw food, and the supernatant after centrifugation was used as a sample. It can be seen that nattokinase activity is particularly high when the amount of dipicolinic acid added is 10-32 mM.

図２と同条件でナットウキナーゼのアミダーゼ活性をSuc-Ala-Ala-Pro-Phe-pNA分解能で観察した。ジピコリン酸の添加量が１０−３２ｍＭの時に強い値を示し、controlに比べると、高いものは１０倍以上になっていることが分かる（図３）。   Under the same conditions as in FIG. 2, nattokinase amidase activity was observed with Suc-Ala-Ala-Pro-Phe-pNA resolution. It shows a strong value when the added amount of dipicolinic acid is 10-32 mM, and it can be seen that the higher one is 10 times or more compared to control (FIG. 3).

図４は、図２と同条件下で作られた水溶性ビタミンＫ２量を調べたものである。すなわち、図２の実験で試料とした上清を使用し、公知のＨＰＬＣ法で発酵物中のビタミンＫ２量の測定を行った。control（１．２２μｇ／ｍｌ）に比べて、例えば、ジピコリン酸の添加量が１０ｍＭ（４．６０μｇ／ｍｌ）の時に約４倍に高まっていることが分かる。 Figure 4 is an examined soluble vitamin K 2 amount made under the same conditions as FIG. That is, using the supernatant as a sample in the experiment 2 was measured of vitamin K 2 of fermentations in a known HPLC methods. Compared to control (1.22 μg / ml), for example, it can be seen that when the amount of dipicolinic acid added is 10 mM (4.60 μg / ml), it increases about four times.

次に、１％（重量％）グリセリンを含む３％ポリペプトン−Ｓを基質に用い、納豆菌（宮城野菌）を３７℃、３日間振盪培養した（図５）。ナットウキナーゼは、ジピコリン酸添加量が４ｍＭの時に最高値７２７を示した（図１）。また、ビタミンＫ２はcontrolが１．１７μｇ／ｍｌであったのに対し、ジピコリン酸添加量が１０ｍＭの時に３．２９μｇ／ｍｌと約３倍になった。 Next, 3% polypeptone-S containing 1% (weight%) glycerin was used as a substrate, and Bacillus natto (Miyagino) was cultured with shaking at 37 ° C. for 3 days (FIG. 5). Nattokinase showed a maximum value of 727 when the amount of dipicolinic acid added was 4 mM (FIG. 1). Vitamin K 2 had a control of 1.17 μg / ml, whereas when the amount of dipicolinic acid added was 10 mM, it increased to about 3.29 μg / ml.

普通、ジピコリン酸含量は、その菌の胞子形成による熱安定性と深く関係することが知られている。   Usually, it is known that the content of dipicolinic acid is closely related to the thermal stability due to sporulation of the fungus.

本発明に係わる方法によって製造されるナットウキナーゼの抽出に際しては，メンブランフィルターで分子量2万〜5万のものを集め、一定濃度の硫酸アンモニウムで平衡化したButyl-Toyopearlカラムにかけてナットウキナーゼを吸着させ、水-硫酸アンモニウムのグラジェントで溶出する方法などを採用することができる。   For extraction of nattokinase produced by the method according to the present invention, those having a molecular weight of 20,000 to 50,000 were collected by a membrane filter, adsorbed on a Butyl-Toyopearl column equilibrated with a constant concentration of ammonium sulfate, and water-ammonium sulfate was adsorbed. For example, a method of eluting with a gradient of

また、本発明に係る方法によって製造されるビタミンＫ２の抽出に際しては、ビタミンＫ２が水溶性であるという性質を利用し、菌と培養液に分離した後、ゲル濾過により分子量200,000、等電点4.2という形式で取り出すことができる。また、分離した培養液をｐＨ２．０に下げることにより、ビタミンＫ２を等電点沈殿させる。その後、ヘキサンなどの有機溶媒処理を行うことにより、ビタミンＫ２を抽出（分離）することができる。 In addition, when extracting vitamin K 2 produced by the method according to the present invention, utilizing the property that vitamin K 2 is water-soluble, it is separated into bacteria and a culture solution and then gel filtered to obtain a molecular weight of 200,000. Can be extracted in the form of point 4.2. In addition, vitamin K 2 is isoelectrically precipitated by lowering the separated culture solution to pH 2.0. Thereafter, by performing the organic solvent treatment, such as hexane, it can be extracted (separated) vitamin K 2.

以上、本発明の実施例について説明したが、本発明はこれらの実施例に何ら限定されるものではなく、特許請求の範囲に示された技術的思想の範疇において変更可能なものである。   As mentioned above, although the Example of this invention was described, this invention is not limited to these Examples at all, It can change in the category of the technical idea shown by the claim.

図１は、本発明に係る方法によりジピコリン酸を添加して納豆菌を培養した場合のナットウキナーゼ活性を示すグラフであり、（Ａ）が標準フィブリン平板法で測った結果（溶解面積）を示し、（Ｂ）が各菌に対するアミド分解能を示す。FIG. 1 is a graph showing nattokinase activity when natto bacteria are cultured by adding dipicolinic acid by the method according to the present invention, (A) shows the results (dissolution area) measured by the standard fibrin plate method, (B) shows the amide resolution for each bacterium. 図２は、大豆を基質として用い納豆製造に近い条件とした場合における標準フィブリン平板法で測った結果を示す写真であり、目黒菌を用いて蒸煮大豆に０−１００ｍＭのジピコリン酸を添加し、３７℃、２４時間培養した結果である。FIG. 2 is a photograph showing the result of measurement by the standard fibrin plate method when soybean is used as a substrate and the conditions are similar to those of natto production, and 0-100 mM dipicolinic acid is added to steamed soybean using Meguro. It is the result of culturing at 37 ° C. for 24 hours. 図３は、納豆中のジピコリン酸添加量とナットウキナーゼ量（アミド分解能）との関係を示すグラフである。FIG. 3 is a graph showing the relationship between the amount of dipicolinic acid added in natto and the amount of nattokinase (amide resolution). 図４は、本発明に係る方法によって納豆菌培養中のビタミンＫ２の変化を示すグラフである。Figure 4 is a graph showing changes of vitamin K 2 in natto cultured by the method according to the present invention. 図５は、本発明に係る方法によって納豆菌振盪培養中のビタミンＫ２の変化を示すグラフである。Figure 5 is a graph showing changes of vitamin K 2 in natto shaking culture by the method according to the present invention.

Claims (2)

納豆菌の培養液中にジピコリン酸を添加することを特徴とするナットウキナーゼの製造方法。   A method for producing nattokinase, comprising adding dipicolinic acid to a culture solution of Bacillus natto. 納豆菌の培養液中にジピコリン酸を添加することを特徴とするビタミンＫ２の製造方法。 Method for producing vitamin K 2, characterized by the addition of dipicolinic acid in the culture of Bacillus natto.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108047313A (en) * 2017-12-29 2018-05-18 中国农业大学 Chicken blood ball anti-oxidation peptide and its fermentation preparation
CN112812986A (en) * 2020-12-21 2021-05-18 苏州微克生活科技有限公司 Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof

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JP2001204400A (en) * 2000-01-19 2001-07-31 Hiroyuki Sumi Method for producing menaquinone-7
JP2006061114A (en) * 2004-08-30 2006-03-09 Chisso Corp Method for isolating vitamin k2 from cultured product of bacillus bacterium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001204400A (en) * 2000-01-19 2001-07-31 Hiroyuki Sumi Method for producing menaquinone-7
JP2006061114A (en) * 2004-08-30 2006-03-09 Chisso Corp Method for isolating vitamin k2 from cultured product of bacillus bacterium

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108047313A (en) * 2017-12-29 2018-05-18 中国农业大学 Chicken blood ball anti-oxidation peptide and its fermentation preparation
CN108047313B (en) * 2017-12-29 2020-09-15 中国农业大学 Chicken blood cell antioxidant peptide and fermentation preparation method thereof
CN112812986A (en) * 2020-12-21 2021-05-18 苏州微克生活科技有限公司 Bacillus subtilis XTK1001 capable of producing natto kinase and vitamin K2 at high yield and application thereof

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