JP3836242B2 - Method for producing vitamin Ks and functional food, and functional food - Google Patents
Method for producing vitamin Ks and functional food, and functional food Download PDFInfo
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Description
【０００１】
【発明の属する技術分野】
本発明は、ビタミンＫ類及びビタミンＫ類含有機能性食品の製造方法及び該方法により製造されるビタミンＫ類含有機能性食品に関する。
【０００２】
【従来の技術】
ビタミンＫ類は、血液凝固系に関与するビタミンであり、これが欠乏すると低プロトロンビン血症や出血傾向を招来することが知られている。
【０００３】
また、骨代謝に関与すると考えられている蛋白質であるオステオカルシンはビタミンＫ類依存性に形成されたγ−カルボキシグルタミン酸を含むことが明らかになり、ビタミンＫ類は、骨代謝においても重要な役割を担っていると考えられるようになっている。さらに、骨粗鬆症の発症を予防するためには、従来生体における必要量と考えられていた量よりもはるかに多量のビタミンＫ類を摂取することが必要であることが明らかになってきている。従って、栄養補助食品等により日常的にビタミンＫ類を摂取することにより、骨粗鬆症の予防や治療を行うことができると考えられる。
【０００４】
ビタミンＫ類を多く含む食品の一つとして、納豆が知られている。納豆は、原料の大豆に由来するものに加え、Bacillus subtilisに属する納豆菌により産生されるビタミンＫ類、特にメナキノン（ビタミンＫ2）の一種であるＭＫ−７を多く含み、糸引き納豆においてＭＫ−７の含有割合が８６３６ｎｇ／ｇである例が報告されている（平内三政、ビタミン、６２巻、３９３、１９８８）。しかしながら、ビタミンＫ類をさらに多く含み、且つ安価に提供される機能性食品が求められている。
【０００５】
【発明が解決しようとする課題】
本発明の目的は、簡便且つ低コストなビタミンＫ類の製造方法を提供することにある。
【０００６】
本発明の別の目的は、簡便且つ低コストにビタミンＫ類を著しく多く含む機能性食品を製造することができる製造方法を提供することにある。
【０００７】
本発明の別の目的は、安価に提供でき、安全性が高い、骨粗鬆症、低プロトロンビン血症、出血傾向等の予防や治療に有用な機能性食品を提供することにある。
【０００８】
【課題を解決するための手段】
本発明によれば、オカラをBacillus sp TT-52( 工業技術院生命工学工業技術研究所寄託番号 FERM P-16530) により発酵させる工程を含むことを特徴とするビタミンＫ類の製造方法が提供される。
【０００９】
また、本発明によれば、オカラをBacillus sp TT-52( 工業技術院生命工学工業技術研究所寄託番号 FERM P-16530) により発酵させて得られるビタミンＫ類含有機能性食品の製造方法が提供される。
【００１０】
さらに本発明によれは、前記方法で得られるビタミンＫ類含有機能性食品が提供される。
【００１１】
【発明の実施の形態】
本発明において、ビタミンＫ類とは、フィロキノン（ビタミンＫ1）及び／又はメナキノン類（ビタミンＫ2）を含む。前記メナキノン類は、ＭＫ−４〜ＭＫ−８等の各種のメナキノンを含む。
【００１２】
本発明のビタミンＫ類の製造方法は、オカラを納豆菌で発酵させる工程を含む。
【００１３】
前記オカラとしては、特に限定されず、豆腐、豆乳、凍豆腐、大豆油、大豆タンパク質等の製造の過程で発生するもの、又はこれらの混合物等のあらゆるオカラを挙げることができる。また、発酵に供する際のオカラの水分含量は約７０〜９０重量％であることが好ましい。従って、発酵に際してオカラの水分含量がこの範囲外である場合、ローラープレスによる脱水又は水分の添加等により、適宜水分含量を好ましい範囲に調節することが好ましい。
【００１４】
前記納豆菌は、 Bacillus sp TT-52(工業技術院生命工学工業技術研究所寄託番号FERM P-16530)を用いることが、高効率にビタミンＫ類を生産することができるため好ましい。
【００１５】
前記発酵の具体的操作としては、前記オカラを前記納豆菌で発酵させる限りにおいて特に限定されないが、例えば以下の方法に従うことが好ましい。
【００１６】
まず、発酵に供するオカラの水分含量を必要に応じて約７０〜９０重量％の範囲に調節した後、加熱処理する。加熱処理は、圧力釜等を用い、高圧下で行うことが好ましい。加熱条件としては、加熱温度が１０５〜１３０℃、圧力が１．２〜３ｋｇ／ｃｍ2、加熱時間が２０〜５０分間であることが好ましい。前記加熱処理により、発酵を妨げる雑菌の滅菌、殺菌等を行うことができる。
【００１７】
続いて、前記加熱処理を行ったオカラに前記納豆菌を接種する。前記納豆菌の接種量は、１０5〜１０6／ｇ（乾燥オカラ換算重量）が好ましい。
【００１８】
続いて、前記納豆菌を接種したオカラを発酵させる。発酵に際しては、酸素の供給を十分に行うことが好ましい。具体的にはステンレス金網等の網状の支持体の上に、前記オカラを１ｃｍ程度の層にして載置して発酵に供することが好ましい。
【００１９】
前記発酵の際の発酵温度が２０〜４０℃であることが好ましい。また発酵時間は、２４〜１４４時間であることが好ましく、３０〜７２時間であることがさらに好ましい。
【００２０】
さらに好ましくは、３５〜３８℃で４５〜５０時間発酵させた後、温度を２０〜２５℃としてさらに１５〜２０時間発酵することにより、粘性が低く、アンモニア臭の低い発酵物が得られる。
【００２１】
以上のような方法に従う場合、菌の培養を助けるブドウ糖等の培養助成剤を全く添加しなくても、発酵を行うことができる。
【００２２】
尚、前記Bacillus sp TT-52等の納豆菌を、液体培地で培養することによっても、ビタミンＫ類を含む発酵物を得ることができる。この場合、Bacillus subtilisが増殖可能な組成の培地を殺菌後、１０5〜１０6／ｇ（乾燥オカラ換算重量）の納豆菌を接種し、例えば約４０℃、４８〜１４４時間の条件で振とう培養することにより、ビタミンＫ類を含む発酵物を得ることができる。
【００２３】
得られた前記発酵物について、必要に応じてさらに乾燥、精製、添加物の添加、その他各種の製剤化のための処理を行って、ビタミンＫ類を得ることができる。
【００２４】
本発明の機能性食品の製造方法は、前記オカラを前記納豆菌で発酵させる工程を含む。前記納豆菌としては、特にBacillus sp TT-52(工業技術院生命工学工業技術研究所寄託番号FERM P-16530)を用いることが、高効率に機能性食品を産生することができるため好ましい。
【００２５】
ここで発酵の具体的操作は、前記ビタミンＫ類の製造方法と同様に行うことができる。前記発酵により得られた発酵物は、そのまま、又は必要に応じてさらに乾燥、精製、添加物の添加、その他各種の処理を行って、ビタミンＫ類を含む機能性食品とすることができる。
【００２６】
本発明のビタミンＫ類含有機能性食品は、前記製造方法により得られるものである。
【００２７】
本発明のビタミンＫ類含有機能性食品は、前記発酵物を、好ましくは５〜１００重量％、さらに好ましくは２０〜１００重量％の割合で含有する。前記発酵物の含有割合を５重量％以上とすることにより、好ましい本発明の効果を得ることができる。また、前記発酵物は、ビタミンＫ類を、全量中好ましくは１〜５０μｇ／ｇ、さらに好ましくは５〜３５μｇ／ｇの割合で含有する。このような発酵物は、例えば前述の方法により製造することができる。
【００２８】
本発明のビタミンＫ類含有機能性食品は、前記発酵物を含むが、さらにこの他にブドウ糖、乳糖、粉末状水あめ、ショ糖、デキストリン等の添加物、ビタミン類、有機酸等の他の添加物を含んでも良い。
【００２９】
本発明のビタミンＫ類含有機能性食品の形態としては、特に限定されないが、乾燥、精製等を行わない前記発酵物そのまま、凍結乾燥等により乾燥し微粉末としたもの、又は前記発酵物をさらに加工し必要に応じ他の添加物を加え、散剤、顆粒剤、錠剤等の剤形としたもの、若しくは腸溶カプセル等のカプセルに充填しカプセル剤としたもの、半生タイプのスティック、ビスケット状等の形状、他の食品中に混入したもの、精製した前記発酵物を溶解し甘味料、香料等を加えドリンク剤としたもの等を挙げることができる。
【００３０】
本発明のビタミンＫ類含有機能性食品の投与量は、例えばヒトの場合、ビタミンＫに換算して０．０１〜５μｇ／ｋｇ／日、好ましくは０．０４〜３μｇ／ｋｇ／日であることが望ましい。
【００３１】
本発明のビタミンＫ類含有機能性食品を摂取することにより、骨粗鬆症、低プロトロンビン血症、出血傾向等のビタミンＫ類の欠乏に起因すると考えられる各種の疾患を治療若しくは予防することができ、又はその症状を緩和することができる。特に、本発明の機能性食品は、摂取者に対する負担が少なく日常的に摂取することが可能なので、前記各種疾患の予防のために摂取するのに特に適している。
【００３２】
前記発酵物は、大豆粕であるオカラの納豆菌発酵物であるので、納豆とほぼ同様な発酵物であり、長期の食経験でその安全性が十分に保証されていることから、本発明のビタミンＫ類含有機能性食品の安全性は高いものと考えられる。
【００３３】
【発明の効果】
本発明のビタミンＫ類の製造方法は、通常は廃棄されているオカラを原材料としてビタミンＫ類を簡便に製造できるので、ビタミンＫ類の簡便且つ低コストな製造方法として有用である。
【００３４】
本発明のビタミンＫ類含有機能性食品の製造方法は、通常は廃棄されているオカラを原材料として、ビタミンＫ類を著しく多く含む機能性食品を簡便に製造できるので、前記ビタミンＫ類含有機能性食品の簡便且つ低コストな製造方法として有用である。
【００３５】
本発明のビタミンＫ類含有機能性食品は、通常は廃棄されているオカラを原材料として簡便に得られる機能性食品であるため安価に提供でき、天然の食品由来であるため安全性が高いので、骨粗鬆症、低プロトロンビン血症、出血傾向等の予防や治療のために継続的に投与しうるビタミンＫ類含有機能性食品として有用である。
【００３６】
【実施例】
以下実施例によりさらに詳細に説明するが、本発明はこれらに限定されるものではない。
【００３７】
【実施例１】
豆腐の製造過程で発生したオカラ（水分含有率８０重量％、種皮部混入割合６重量％）を、圧力釜中１２１℃、圧力１．３ｋｇ／ｃｍ2にて２０分間加熱処理した。
【００３８】
処理後のオカラに納豆菌Bacillus sp TT-52を１０5／ｇ（乾燥オカラ換算）の割合で接種し、ステンレス製金網上に厚さ約１ｃｍの層として載置し、３７℃で４８時間発酵させ、その後温度を２５℃とし、さらに１７時間発酵させ、発酵物を得た。
【００３９】
【実施例２】
実施例１で得られた発酵物５．０ｇを正確にとり、水２０．０ｍｌ、イソプロパノール６０．０ｍｌ、ｎ−ヘキサン３０．０ｍｌを加え、室温で３０分間振とうした後×８００Ｇで５分間遠心分離した。上層のｎ−ヘキサン層を取り、溶媒を減圧留去した。残留物にｎ−ヘキサン２．０ｍｌを加え、あらかじめエーテル：ｎ−ヘキサン（４：９６、容量比）１０．０ｍｌで洗浄したＳｅｐ−ｐａｋシリカカートリッジ（ウォーターズ社製）に負荷した。カートリッジをｎ−ヘキサン１０．０ｍｌで洗浄した後エーテル：ｎーヘキサン（４：９６、容量比）５．０ｍｌを加えて溶出した。得られた溶出液を集めたものを試料として以下の条件でＨＰＬＣで分析し、ビタミンＫ類（ビタミンＫ1及びメナキノンＭＫ−４〜８）の含有量を乾燥オカラ発酵物中の含有割合として求めた。結果を表１に示す。
【００４０】
カラム：ＨＩＳＩＬ Ｃ１８（１５０×４．５ｍｍ、ＨＩＧＧＩＮＳ ＡＮＡＬＹＴＩＣＡＬ社製）
移動相：メタノール：エタノール（７：３、容量比）
流速：１．０ｍｌ／分
検出波長：励起波長（Ｅｘ）３２０ｎｍ、蛍光波長（Ｅｍ）４３０ｎｍ
還元装置：白金カラム
【００４１】
【表１】
【参考例１】
ペプトン（Ｄｉｆｃｏ製）５．０重量％、ブドウ糖５．０重量％、ＫＨ2ＰＯ4０．１重量％、ＭｇＳＯ4 ０．０５重量％、ＮａＣｌ ０．０５重量％及びビオチン１００μｇ／ｌを水に溶解した培地を１２１℃で１５分間殺菌したのち、１０5／ｇの納豆菌Bacillus sp TT-52を接種し、４０℃で１４４時間振とう培養した。
【００４３】
得られた培養物５０．０ｇを約２０．０ｍｌまで減圧下で濃縮し、イソプロパノール６０．０ｍｌ、ｎ−ヘキサン３０．０ｍｌを加え、室温で３０分間振とうした後×８００Ｇで５分間遠心分離した。上層のｎ−ヘキサン層を取り、溶媒を減圧留去した。残留物にｎ−ヘキサン２．０ｍｌを加え、あらかじめエーテル：ｎ−ヘキサン（４：９６、容量比）１０．０ｍｌで洗浄したＳｅｐ−ｐａｋシリカカートリッジ（ウォーターズ社製）に負荷した。カートリッジをｎ−ヘキサン１０．０ｍｌで洗浄した後エーテル：ｎーヘキサン（４：９６、容量比）５．０ｍｌを加えて溶出した。得られた溶出液を集めたものを試料として、実施例２で行ったものと同様の条件でＨＰＬＣによる分析を行い、ビタミンＫ類（ビタミンＫ1及びメナキノンＭＫ−４〜８）の含有量を培養物中の含有割合として求めた。結果を表２に示す。
【００４４】
【表２】
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing vitamin Ks and a functional food containing vitamin Ks, and a functional food containing vitamin Ks produced by the method.
[0002]
[Prior art]
Vitamin Ks are vitamins involved in the blood clotting system, and it is known that deficiency causes hypoprothrombinemia and bleeding tendency.
[0003]
In addition, osteocalcin, a protein believed to be involved in bone metabolism, has been found to contain γ-carboxyglutamic acid formed in a vitamin K-dependent manner, and vitamin Ks also play an important role in bone metabolism. It has come to be considered to be responsible. Furthermore, in order to prevent the onset of osteoporosis, it has become clear that it is necessary to ingest much higher amounts of vitamin K than the amount conventionally considered to be a necessary amount in a living body. Therefore, it is considered that osteoporosis can be prevented or treated by ingesting vitamin Ks on a daily basis with dietary supplements.
[0004]
Natto is known as one of foods rich in vitamin K. Natto contains a lot of vitamin Ks produced by natto bacteria belonging to Bacillus subtilis, in particular MK-7, which is a kind of menaquinone (vitamin K 2 ), in addition to those derived from soybean as a raw material. An example in which the content ratio of -7 is 8636 ng / g has been reported (Minami Hirauchi, Vitamin, 62, 393, 1988). However, there is a demand for functional foods that contain more vitamin Ks and that are provided at low cost.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a simple and low-cost method for producing vitamin Ks.
[0006]
Another object of the present invention is to provide a production method capable of producing a functional food containing a significant amount of vitamin K at a simple and low cost.
[0007]
Another object of the present invention is to provide a functional food that can be provided at low cost and is highly safe and useful for the prevention and treatment of osteoporosis, hypoprothrombinemia, bleeding tendency, and the like.
[0008]
[Means for Solving the Problems]
According to the present invention, there is provided a method for producing vitamin Ks comprising the step of fermenting okara with Bacillus sp TT-52 ( Accessory No. FERM P-16530, Biotechnology Institute of Industrial Science and Technology ). The
[0009]
Further, according to the present invention, there is provided a method for producing a functional food containing vitamin Ks obtained by fermenting okara with Bacillus sp TT-52 ( Institute of Biotechnology, Industrial Technology Research Institute deposit number FERM P-16530). Is done.
[0010]
Furthermore, according to this invention, the vitamin K containing functional food obtained by the said method is provided.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, vitamin Ks include phylloquinone (vitamin K 1 ) and / or menaquinones (vitamin K 2 ). The menaquinones include various menaquinones such as MK-4 to MK-8.
[0012]
The method for producing vitamin K of the present invention includes a step of fermenting okara with natto bacteria.
[0013]
The okara is not particularly limited, and examples thereof include any okara that is generated in the process of producing tofu, soy milk, frozen tofu, soybean oil, soybean protein, or a mixture thereof. Moreover, it is preferable that the water | moisture content of the okara at the time of using for fermentation is about 70 to 90 weight%. Therefore, when the moisture content of okara is outside this range during fermentation, it is preferable to appropriately adjust the moisture content to a preferred range by dehydration using a roller press or addition of moisture.
[0014]
The Bacillus natto, the use of B acillus sp TT-52 (Agency of Industrial Science Technology Institute Accession No. FERM P-16530) is preferable because it is possible to produce vitamin K to high efficiency.
[0015]
The specific operation of the fermentation is not particularly limited as long as the Okara is fermented with the Bacillus natto, but it is preferable to follow, for example, the following method.
[0016]
First, the moisture content of okara to be subjected to fermentation is adjusted to a range of about 70 to 90% by weight as necessary, followed by heat treatment. The heat treatment is preferably performed under high pressure using a pressure cooker or the like. As heating conditions, it is preferable that the heating temperature is 105 to 130 ° C., the pressure is 1.2 to 3 kg / cm 2 , and the heating time is 20 to 50 minutes. By the heat treatment, sterilization, sterilization and the like of various bacteria that hinder fermentation can be performed.
[0017]
Subsequently, the heat-treated Okara is inoculated with the Bacillus natto. The inoculum of the Bacillus natto is preferably 10 5 to 10 6 / g (weight in terms of dry okara).
[0018]
Subsequently, the okara inoculated with the Bacillus natto is fermented. In fermentation, it is preferable to sufficiently supply oxygen. Specifically, it is preferable that the okara be placed in a layer of about 1 cm on a net-like support such as a stainless steel wire mesh for fermentation.
[0019]
It is preferable that the fermentation temperature in the case of the said fermentation is 20-40 degreeC. The fermentation time is preferably 24 to 144 hours, and more preferably 30 to 72 hours.
[0020]
More preferably, after fermenting at 35 to 38 ° C. for 45 to 50 hours, fermentation is further performed at a temperature of 20 to 25 ° C. for 15 to 20 hours to obtain a fermented product having low viscosity and low ammonia odor.
[0021]
In the case of following the above method, fermentation can be performed without adding any culture aid such as glucose to assist the cultivation of bacteria.
[0022]
A fermented product containing vitamin Ks can also be obtained by culturing natto bacteria such as Bacillus sp TT-52 in a liquid medium. In this case, after sterilizing a medium having a composition capable of growing Bacillus subtilis, 10 5 to 10 6 / g (weight in terms of dry okara) is inoculated and shaken, for example, at about 40 ° C. for 48 to 144 hours. By culturing, a fermented material containing vitamin Ks can be obtained.
[0023]
The obtained fermented product can be further subjected to drying, purification, addition of additives, and various other preparations as necessary to obtain vitamin Ks.
[0024]
The method for producing a functional food of the present invention includes a step of fermenting the Okara with the Bacillus natto. As Bacillus natto, it is particularly preferable to use Bacillus sp TT-52 (Deposit number FERM P-16530, Biotechnology Institute of Industrial Technology, National Institute of Industrial Science and Technology) because it can produce a functional food with high efficiency.
[0025]
Here, the specific operation of fermentation can be performed in the same manner as in the method for producing vitamin Ks. The fermented product obtained by the fermentation can be made into a functional food containing vitamin Ks as it is or after further drying, purification, addition of additives, and other various treatments as necessary.
[0026]
The vitamin K-containing functional food of the present invention is obtained by the above production method.
[0027]
The vitamin K-containing functional food of the present invention contains the fermented product in a proportion of preferably 5 to 100% by weight, more preferably 20 to 100% by weight. By making the content of the fermented product 5% by weight or more, a preferable effect of the present invention can be obtained. The fermented product contains vitamin Ks in a total amount of preferably 1 to 50 μg / g, more preferably 5 to 35 μg / g. Such a fermented material can be manufactured by the above-mentioned method, for example.
[0028]
The vitamin K-containing functional food of the present invention contains the fermented product, but in addition, other additives such as glucose, lactose, powdered syrup, sucrose, dextrin, vitamins, organic acids, etc. You may include things.
[0029]
The form of the vitamin K-containing functional food of the present invention is not particularly limited, but the fermented product that is not dried, purified or the like is dried as it is by lyophilization or the like, or the fermented product is further added. Processed and added with other additives as necessary to form powders, granules, tablets, etc., filled into capsules such as enteric capsules, semi-life type sticks, biscuits, etc. And the like, those mixed in other foods, those obtained by dissolving the purified fermented product and adding sweeteners, fragrances and the like to give a drink.
[0030]
For example, in the case of humans, the dose of the functional food containing vitamin Ks of the present invention is 0.01 to 5 μg / kg / day, preferably 0.04 to 3 μg / kg / day in terms of vitamin K. Is desirable.
[0031]
By ingesting the functional food containing vitamin Ks of the present invention, it is possible to treat or prevent various diseases thought to be caused by deficiencies of vitamin Ks such as osteoporosis, hypoprothrombinemia, bleeding tendency, etc., or The symptoms can be alleviated. In particular, the functional food of the present invention is particularly suitable for ingestion for the prevention of the above-mentioned various diseases because it can be taken on a daily basis with little burden on the ingestor.
[0032]
Since the fermented product is a fermented natto fungus of Okara, which is soybean meal, it is a fermented product almost similar to natto, and its safety is sufficiently guaranteed by long-term eating experience. The safety of functional foods containing vitamin Ks is considered high.
[0033]
【The invention's effect】
The method for producing vitamin Ks of the present invention is useful as a simple and low-cost production method for vitamin K, because vitamin Ks can be easily produced using okara which is usually discarded as a raw material.
[0034]
The method for producing a functional food containing vitamin Ks of the present invention can easily produce a functional food containing a significant amount of vitamin Ks, starting from discarded okara as a raw material. It is useful as a simple and low-cost production method for food.
[0035]
Since the functional food containing vitamin Ks of the present invention is a functional food that can be easily obtained by using discarded okara as a raw material, it can be provided at low cost, and since it is derived from natural food, it is highly safe. It is useful as a functional food containing vitamin Ks that can be continuously administered for prevention and treatment of osteoporosis, hypoprothrombinemia, bleeding tendency and the like.
[0036]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[0037]
[Example 1]
Okara (moisture content 80% by weight, seed coat mixing ratio 6% by weight) generated in the production process of tofu was heat-treated in a pressure cooker at 121 ° C. and a pressure of 1.3 kg / cm 2 for 20 minutes.
[0038]
The treated okara is inoculated with Bacillus sp TT-52 at a rate of 10 5 / g (converted to dry okara), placed as a 1 cm thick layer on a stainless steel wire mesh, and fermented at 37 ° C for 48 hours. After that, the temperature was set to 25 ° C., and the mixture was further fermented for 17 hours to obtain a fermented product.
[0039]
[Example 2]
Take exactly 5.0 g of the fermented product obtained in Example 1, add 20.0 ml of water, 60.0 ml of isopropanol, 30.0 ml of n-hexane, shake for 30 minutes at room temperature, and then centrifuge at 800 g for 5 minutes. did. The upper n-hexane layer was taken and the solvent was distilled off under reduced pressure. To the residue, 2.0 ml of n-hexane was added, and the mixture was loaded on a Sep-pak silica cartridge (manufactured by Waters) which had been washed with 10.0 ml of ether: n-hexane (4:96, volume ratio) in advance. The cartridge was washed with 10.0 ml of n-hexane and then eluted with 5.0 ml of ether: n-hexane (4:96, volume ratio). The collected eluate is collected as a sample and analyzed by HPLC under the following conditions, and the content of vitamin Ks (vitamin K 1 and menaquinone MK-4 to 8) is determined as the content ratio in the dried okara fermented product. It was. The results are shown in Table 1.
[0040]
Column: HISIL C18 (150 × 4.5 mm, manufactured by HIGGINS ANALYTICAL)
Mobile phase: methanol: ethanol (7: 3, volume ratio)
Flow rate: 1.0 ml / min Detection wavelength: excitation wavelength (Ex) 320 nm, fluorescence wavelength (Em) 430 nm
Reduction device: platinum column [0041]
[Table 1]
[Reference Example 1]
Peptone (Difco) 5.0% by weight, glucose 5.0% by weight, KH 2 PO 4 0.1% by weight, MgSO 4 0.05% by weight, NaCl 0.05% by weight and biotin 100 μg / l in water The dissolved medium was sterilized at 121 ° C. for 15 minutes, inoculated with 10 5 / g of Bacillus natto Bacillus sp TT-52, and cultured with shaking at 40 ° C. for 144 hours.
[0043]
The obtained culture (50.0 g) was concentrated under reduced pressure to about 20.0 ml, isopropanol (60.0 ml) and n-hexane (30.0 ml) were added, and the mixture was shaken at room temperature for 30 minutes and then centrifuged at 800 g for 5 minutes. . The upper n-hexane layer was taken and the solvent was distilled off under reduced pressure. To the residue, 2.0 ml of n-hexane was added, and the mixture was loaded on a Sep-pak silica cartridge (manufactured by Waters) which had been washed with 10.0 ml of ether: n-hexane (4:96, volume ratio) in advance. The cartridge was washed with 10.0 ml of n-hexane and then eluted with 5.0 ml of ether: n-hexane (4:96, volume ratio). The collected eluate was collected as a sample and analyzed by HPLC under the same conditions as those used in Example 2 to determine the content of vitamin Ks (vitamin K 1 and menaquinone MK-4 to 8). It calculated | required as a content rate in a culture. The results are shown in Table 2.
[0044]
[Table 2]
Claims (3)
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| Application Number | Priority Date | Filing Date | Title |
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| JP00631298A JP3836242B2 (en) | 1998-01-16 | 1998-01-16 | Method for producing vitamin Ks and functional food, and functional food |
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| Application Number | Priority Date | Filing Date | Title |
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| JP00631298A JP3836242B2 (en) | 1998-01-16 | 1998-01-16 | Method for producing vitamin Ks and functional food, and functional food |
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| Publication Number | Publication Date |
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| JPH11196820A JPH11196820A (en) | 1999-07-27 |
| JP3836242B2 true JP3836242B2 (en) | 2006-10-25 |
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