JP6057203B2 - Method for producing folic acid-rich yeast, folic acid-rich yeast, folic acid-rich yeast crushed material, and food - Google Patents
Method for producing folic acid-rich yeast, folic acid-rich yeast, folic acid-rich yeast crushed material, and food Download PDFInfo
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- JP6057203B2 JP6057203B2 JP2012192145A JP2012192145A JP6057203B2 JP 6057203 B2 JP6057203 B2 JP 6057203B2 JP 2012192145 A JP2012192145 A JP 2012192145A JP 2012192145 A JP2012192145 A JP 2012192145A JP 6057203 B2 JP6057203 B2 JP 6057203B2
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- folic acid
- yeast
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- acid
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- 239000011724 folic acid Substances 0.000 title claims description 206
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Description
本発明は、葉酸高含有酵母の製造方法、葉酸高含有酵母、葉酸高含有酵母破砕物、並びに該葉酸高含有酵母及び該葉酸高含有酵母破砕物の少なくともいずれかを含有する食品に関する。 The present invention relates to a method for producing a folic acid-rich yeast, a folic acid-rich yeast, a folic acid-rich yeast crushed material, and a food containing at least one of the folic acid-rich yeast and the folic acid-rich yeast crushed material.
葉酸は、ビタミンＭ、ビタミンＢ９、プテロイルグルタミン酸とも呼ばれ、水溶性ビタミンに分類される生理活性物質である。 Folic acid is also called vitamin M, vitamin B9, and pteroylglutamic acid, and is a physiologically active substance classified as a water-soluble vitamin.
葉酸の生理的作用としては、二分脊椎症、心筋梗塞、脳卒中、認知症、うつ病等の予防などが報告されている（非特許文献１参照）。葉酸の欠乏による症状としては、口腔の炎症、肌荒れ、疲労感などがあり、葉酸欠乏症としては、巨赤芽球性貧血(悪性貧血)が知られている。また、特に妊娠期に葉酸が欠乏すると、神経管閉鎖障害が起こり、臨床的には無脳症、二分脊椎症、髄膜瘤などのリスクが高まることが知られている（非特許文献２参照）。 As the physiological action of folic acid, prevention of spina bifida, myocardial infarction, stroke, dementia, depression and the like has been reported (see Non-Patent Document 1). Symptoms caused by folic acid deficiency include inflammation of the oral cavity, rough skin, and fatigue. Giant erythroblastic anemia (malignant anemia) is known as folic acid deficiency. Further, it is known that when folic acid is deficient particularly during pregnancy, neural tube closure disorder occurs, and clinical risks such as anencephalopathy, spina bifida, meningocele increase (see Non-Patent Document 2). .
このように、葉酸は、生体において種々の重要な役割を果たしていることから、米国、オーストラリアなど世界５２ヶ国では必須栄養素とされ、１日の必要量が４００μｇと定められている。ヒトをはじめとする哺乳類は、葉酸を産生することができないため、葉酸の主要な供給源は、葉酸を含有する食品である。前記食品としては、レバー、うなぎ、緑黄色野菜、海苔、茶葉などが知られている。これらの中でもレバー、海苔、茶葉に特に多く含まれている。ただし、調理や長期間保存による酸化によって葉酸は壊れるため、日常食において前記摂取基準値を達成することは容易ではない。 Thus, since folic acid plays various important roles in the living body, it is considered as an essential nutrient in 52 countries around the world such as the United States and Australia, and the daily required amount is set to 400 μg. Since mammals including humans cannot produce folic acid, the main source of folic acid is food containing folic acid. Known foods include liver, eel, green-yellow vegetables, laver, and tea leaves. Among these, it is particularly abundant in liver, nori and tea leaves. However, since folic acid is broken by oxidation due to cooking or long-term storage, it is not easy to achieve the intake standard value in daily food.
一方、酵母は、古くから人類が食品素材として利用しており、例えば、ビール酵母が食物繊維、ビタミンあるいはミネラル分の供給源としても用いられてきた。特に菌体内に葉酸を取り込ませた酵母又はその破砕物は、葉酸を補強した食品素材として、かつ葉酸の吸収効率に優れた安全な食品素材として利用可能であると考えられる。 On the other hand, yeast has long been used as a food material by humans. For example, brewer's yeast has been used as a source of dietary fiber, vitamins or minerals. In particular, it is considered that a yeast or a crushed product thereof in which folic acid is incorporated into the microbial cells can be used as a food material reinforced with folic acid and a safe food material excellent in folic acid absorption efficiency.
しかしながら、従来の酵母における葉酸含有量は、一般的に乾燥菌体重量（ｇ）当たり３０μｇ〜６０μｇ程度と低かった。そのため、サプリメント、飲料等の食品素材として用いるためには、より多く葉酸を含有させた酵母の提供が望まれているのが現状である。 However, the folic acid content in conventional yeasts was generally as low as about 30 μg to 60 μg per dry cell weight (g). Therefore, in order to use it as a food material for supplements, beverages, etc., it is currently desired to provide yeast containing more folic acid.
これまでに、生物に利用可能な葉酸を生産する方法として、モノグルタミル葉酸の量を増大させるように遺伝的に改変された食品グレードの微生物、及びこれを培養して葉酸を産生する方法が提案されている（特許文献１参照）。
しかしながら、前記提案には、遺伝子改変のためのコスト、及び遺伝子改変による生体への悪影響のリスクの点で問題があった。
また、葉酸活性を有する物質を効率よく製造する方法として、パラアミノ安息香酸を培地中に添加し、酵母を培養して葉酸を産生させる方法が提案されている（特許文献２参照）。
しかしながら、前記提案には、パラアミノ安息香酸を通常の培地に使用する量よりも過剰量使用するため、製造コストが高く、また、この方法によって産生された葉酸は、代謝により菌体外へ排出されるため、葉酸高含有酵母の製造には不向きである点で問題があった。
To date, food-grade microorganisms genetically modified to increase the amount of monoglutamyl folic acid and methods for producing folic acid by culturing it have been proposed as methods for producing folic acid that can be used by organisms. (See Patent Document 1).
However, the proposal has problems in terms of the cost for genetic modification and the risk of adverse effects on the living body due to the genetic modification.
In addition, as a method for efficiently producing a substance having folic acid activity, a method in which paraaminobenzoic acid is added to a medium and yeast is cultured to produce folic acid has been proposed (see Patent Document 2).
However, in the above proposal, paraaminobenzoic acid is used in an excessive amount as compared with the amount used in a normal medium, so that the production cost is high, and folic acid produced by this method is excreted outside the cells by metabolism. Therefore, there is a problem in that it is not suitable for the production of yeast with a high content of folic acid.
このため、低コストで、葉酸を高濃度に含有する酵母を製造する方法、及び葉酸を効率良く生体内に摂取及び吸収することができ、安全性が高く、二分脊椎症、動脈硬化症、認知症などの予防乃至改善に用いられるサプリメント、飲料などの食品素材として好適な葉酸高含有酵母及び葉酸高含有酵母破砕物、並びに該葉酸高含有酵母及び葉酸高含有酵母破砕物の少なくともいずれかを含有するサプリメント、飲料等の食品の提供が望まれているのが現状である。 Therefore, a method for producing yeast containing folic acid at a high concentration at a low cost, and folic acid can be efficiently ingested and absorbed in the living body, and it is highly safe, with spina bifida, arteriosclerosis, cognition Containing at least one of folic acid-rich yeast and folic acid-rich yeast crushed material, and folic acid-rich yeast and folic acid-rich yeast crushed material suitable for food materials such as supplements and beverages used to prevent or improve diseases Currently, it is desired to provide foods such as supplements and beverages.
本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、葉酸高含有酵母を効率良く簡単に低コストで製造可能な葉酸高含有酵母の製造方法を提供すること、葉酸を高含有する葉酸高含有酵母、及び葉酸高含有酵母破砕物、並びに、葉酸を効率良く生体内に摂取及び吸収することができ、安全性の高い各種の食品を提供することを目的とする。 An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention provides a method for producing a folic acid-rich yeast that can efficiently and easily produce a folic acid-rich yeast, a folic acid-rich yeast, and a folic acid-rich yeast crushed material In addition, an object is to provide various foods that can efficiently ingest and absorb folic acid into a living body and have high safety.
前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ メチオニンを０．２質量％〜０．６質量％含み、かつ、パラアミノ安息香酸（ｐＡＢＡ）を０．０００２質量％〜０．０００４質量％含む培養液中で酵母を培養することを特徴とする葉酸高含有酵母の製造方法である。
＜２＞ 酵母が食用酵母である前記＜１＞に記載の葉酸高含有酵母の製造方法である。
＜３＞ 酵母が清酒酵母である前記＜１＞に記載の葉酸高含有酵母の製造方法である。
＜４＞ 清酒酵母がきょうかい酵母６号、６０１号、７号、７０１号、８号、９号、９０１号、１１号、１２号、１３号、１４号、１５号、及び１７０１号の少なくともいずれかである前記＜３＞に記載の葉酸高含有酵母の製造方法である。
＜５＞ 培養が通気培養である前記＜１＞から＜４＞のいずれかに記載の葉酸高含有酵母の製造方法である。
＜６＞ 培養が流加培養である前記＜１＞から＜５＞のいずれかに記載の葉酸高含有酵母の製造方法である。
Means for solving the problems are as follows. That is,
<1> Yeast is cultured in a culture solution containing 0.2% by mass to 0.6% by mass of methionine and 0.0002% by mass to 0.0004% by mass of paraaminobenzoic acid (pABA). And a method for producing a yeast with a high content of folic acid.
<2> The method for producing a yeast with a high folic acid content according to <1>, wherein the yeast is an edible yeast.
<3> The method for producing a yeast with a high folic acid content according to <1>, wherein the yeast is sake yeast.
<4> Sake yeast is Kyoto yeast No. 6, 601, No. 7, No. 701, No. 8, No. 9, No. 901, No. 11, No. 12, No. 13, No. 14, No. 15, and No. 1701 The method for producing a yeast with a high content of folic acid according to <3>, which is any of the above.
<5> The method for producing a folic acid-rich yeast according to any one of <1> to <4>, wherein the culture is aeration culture.
<6> The method for producing a folic acid-rich yeast according to any one of <1> to <5>, wherein the culture is fed-batch culture.
＜７＞ 前記＜１＞から＜６＞のいずれかに記載の製造方法によって製造されたことを特徴とする葉酸高含有酵母である。
＜８＞ 菌体における葉酸含有量が乾燥菌体１ｇ当たり少なくとも１６０μｇであることを特徴とする葉酸高含有酵母である。
＜９＞ 菌体における葉酸含有量が乾燥菌体１ｇ当たり少なくとも１８０μｇであることを特徴とする葉酸高含有酵母である。
＜１０＞ 酵母が食用酵母である前記＜７＞から＜９＞のいずれかに記載の葉酸高含有酵母である。
＜１１＞ 酵母が清酒酵母である前記＜７＞から＜９＞のいずれかに記載の葉酸高含有酵母である。
＜１２＞ 清酒酵母がきょうかい酵母６号、６０１号、７号、７０１号、８号、９号、９０１号、１１号、１２号、１３号、１４号、１５号、及び１７０１号の少なくともいずれかである前記＜１１＞に記載の葉酸高含有酵母である。
＜１３＞ 食品に添加されて用いられる前記＜７＞から＜１２＞のいずれかに記載の葉酸高含有酵母である。
<7> A folic acid-rich yeast produced by the production method according to any one of <1> to <6>.
<8> The folic acid-rich yeast, wherein the folic acid content in the microbial cells is at least 160 μg per gram of the dry microbial cells.
<9> The folic acid-rich yeast, wherein the folic acid content in the microbial cells is at least 180 μg per gram of the dry microbial cells.
<10> The folic acid-rich yeast according to any one of <7> to <9>, wherein the yeast is an edible yeast.
<11> The folic acid-rich yeast according to any one of <7> to <9>, wherein the yeast is sake yeast.
<12> Sake yeast is Kyoto yeast No. 6, 601, No. 7, No. 701, No. 8, No. 9, No. 901, No. 11, No. 12, No. 13, No. 14, No. 15, and No. 1701 The folic acid-rich yeast according to <11>, which is any one of the above.
<13> The folic acid-rich yeast according to any one of <7> to <12>, which is used by being added to food.
＜１４＞ 前記＜７＞から＜１３＞のいずれかに記載の葉酸高含有酵母の破砕物を少なくとも含むことを特徴とする葉酸高含有酵母破砕物である。
＜１５＞ 葉酸高含有酵母の破砕物が乾燥物である前記＜１４＞に記載の葉酸高含有酵母破砕物である。
＜１６＞ 葉酸高含有酵母の破砕物が液状物である前記＜１４＞に記載の葉酸高含有酵母破砕物である。
＜１７＞ 食品に添加されて用いられる前記＜１４＞から＜１６＞のいずれかに記載の葉酸高含有酵母破砕物である。
<14> A folic acid-rich yeast crushed product comprising at least the folic acid-rich yeast crushed product according to any one of <7> to <13>.
<15> The folic acid-rich yeast crushed product according to <14>, wherein the folic acid-rich yeast crushed product is a dried product.
<16> The folic acid-rich yeast crushed product according to <14>, wherein the folic acid-rich yeast crushed product is a liquid product.
<17> The folic acid-rich yeast crushed product according to any one of <14> to <16>, which is used by being added to food.
＜１８＞ 前記＜７＞から＜１３＞のいずれかに記載の葉酸高含有酵母を少なくとも含むことを特徴とする食品である。
＜１９＞ 前記＜１４＞から＜１７＞のいずれかに記載の葉酸高含有酵母破砕物を少なくとも含むことを特徴とする食品である。
<18> A food comprising at least the yeast with a high content of folic acid according to any one of <7> to <13>.
<19> A food comprising at least the folic acid-rich yeast crushed material according to any one of <14> to <17>.
本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、葉酸高含有酵母を効率良く簡単に低コストで製造可能な葉酸高含有酵母の製造方法を提供すること、葉酸を高含有する葉酸高含有酵母、及び葉酸高含有酵母破砕物、並びに、葉酸を効率良く生体内に摂取及び吸収することができ、安全性の高い各種の食品を提供することができる。 According to the present invention, there is provided a method for producing a folic acid-rich yeast that can solve the above-mentioned problems and can achieve the above-mentioned object, and that can produce a folic acid-rich yeast efficiently and easily at low cost. Further, folic acid-rich yeast, folic acid-rich yeast crushed material, and folic acid can be efficiently ingested and absorbed in the living body, and various foods with high safety can be provided.
（葉酸高含有酵母の製造方法）
本発明の葉酸高含有酵母の製造方法は、メチオニンを０．２質量％〜０．６質量％含み、かつ、パラアミノ安息香酸（ｐＡＢＡ）を０．０００２質量％〜０．０００４質量％含む培養液中で酵母を培養する工程を含み、更に必要に応じてその他の工程を含む。
なお、本発明において「葉酸」とは、プテロイルモノグルタミン酸のみではなく、補酵素型、つまり還元型、一炭素単位置換型及びこれらのポリグルタミン酸型をも含む。
(Method for producing yeast with high folic acid content)
The method for producing a folic acid-rich yeast of the present invention comprises 0.2% to 0.6% by weight of methionine and 0.0002% to 0.0004% by weight of paraaminobenzoic acid (pABA). It includes a step of culturing yeast in the inside, and further includes other steps as necessary.
In the present invention, “folic acid” includes not only pteroyl monoglutamic acid but also a coenzyme type, that is, a reduced type, a one-carbon unit substitution type, and a polyglutamic acid type thereof.
＜培養液＞
本発明の葉酸高含有酵母の製造方法に用いる培養液としては、メチオニンを０．２質量％〜０．６質量％含み、かつ、パラアミノ安息香酸（ｐＡＢＡ）を０．０００２質量％〜０．０００４質量％含む限り特に制限はなく、目的に応じて公知のものの中から適宜選択することができ、アミノ酸、ビタミン等の他、糖、ｐＨ調整試薬、水などを含んでいてもよい。
前記培養液中におけるメチオニンの含有量としては、０．２質量％〜０．６質量％であれば、特に制限はなく、目的に応じて適宜選択することができるが、０．２質量％超０．６質量％未満が好ましい。
前記培養液中におけるパラアミノ安息香酸の含有量としては、０．０００２質量％〜０．０００４質量％であれば、特に制限はなく、目的に応じて適宜選択することができるが、０．０００２質量％超０．０００４質量％未満が好ましい。
<Culture solution>
The culture solution used in the method for producing a yeast with a high content of folic acid of the present invention contains 0.2% to 0.6% by mass of methionine and 0.0002% to 0.0004% of paraaminobenzoic acid (pABA). There is no particular limitation as long as it is contained by mass%, and it can be appropriately selected from known ones according to the purpose, and may contain amino acids, vitamins, etc., sugars, pH adjusting reagents, water and the like.
The content of methionine in the culture solution is not particularly limited as long as it is 0.2% by mass to 0.6% by mass, and can be appropriately selected according to the purpose. Less than 0.6 mass% is preferable.
The content of paraaminobenzoic acid in the culture solution is not particularly limited as long as it is 0.0002% by mass to 0.0004% by mass, and can be appropriately selected according to the purpose. More than% and less than 0.0004 mass% are preferable.
−アミノ酸、ビタミン等−
本発明における前記アミノ酸、ビタミン等としては、メチオニン及びパラアミノ安息香酸が必須であり、これら以外のアミノ酸、ビタミン等を含んでいてもよい。メチオニン以外のアミノ酸としては、例えば、ヒスチジン、グルタミン酸塩酸塩、グルタミン酸、グリシン、セリン、プロリン、トリプトファンなどが挙げられる。また、パラアミノ安息香酸以外のビタミンとしては、ビタミンＢ２、ビタミンＢ６、ビタミンＢ１２などが挙げられる。
これらは、１種単独で使用してもよいし、２種以上を併用してもよい。
-Amino acids, vitamins, etc.-
As the amino acid, vitamin and the like in the present invention, methionine and paraaminobenzoic acid are essential, and amino acids and vitamins other than these may be contained. Examples of amino acids other than methionine include histidine, glutamic acid hydrochloride, glutamic acid, glycine, serine, proline, and tryptophan. Examples of vitamins other than paraaminobenzoic acid include vitamin B2, vitamin B6, vitamin B12, and the like.
These may be used individually by 1 type and may use 2 or more types together.
前記培養液における前記メチオニン以外のアミノ酸の含有量としては、培養液の総液量に対し、０．０１質量％〜１．２０質量％とすることができるが、０．０８質量％〜０．９０質量％が好ましく、０．２０質量％〜０．６０質量％がより好ましい。前記含有量が０．０１質量％未満であると、葉酸高含有酵母を製造することができないことがある。前記アミノ酸の含有量が１．２０質量％を超えると、葉酸含有量が減少することがある。
前記パラアミノ安息香酸以外のビタミンの含有量としては、培養液の総液量に対し、０．０００１質量％〜０．００１５質量％とすることができるが、０．０００１５質量％〜０．０００８０質量％が好ましく、０．０００２０質量％〜０．０００４０質量％がより好ましい。前記含有量が０．０００１質量％未満、又は０．００１５質量％を超えると、菌体内の葉酸含有量が減少することがある。
前記アミノ酸、ビタミン等は、培地に初期添加させておいてもよく、流加液中に添加させておき、これを前記培養液中に流加することにより、培養液中の葉酸化合物濃度を制御してもよいが、初期添加することが乾燥菌体当たりの葉酸含有量を増加させることができる点で好ましい。
なお、前記培養液に添加するアミノ酸、ビタミン等の量は、公知の方法で測定することができ、例えば、全自動アミノ酸分析機（ＪＬＣ−５００／Ｖ、日本電子株式会社製）、ＨＰＬＣによりそれぞれ測定することができる。
The content of amino acids other than the methionine in the culture solution may be 0.01% by mass to 1.20% by mass with respect to the total amount of the culture solution, but 0.08% by mass to 0.00%. 90 mass% is preferable and 0.20 mass%-0.60 mass% is more preferable. When the content is less than 0.01% by mass, a folic acid-rich yeast may not be produced. If the amino acid content exceeds 1.20% by mass, the folic acid content may decrease.
The content of vitamins other than paraaminobenzoic acid can be 0.0001% by mass to 0.0015% by mass with respect to the total amount of the culture broth, but 0.00015% by mass to 0.00080% by mass. % Is preferable, and 0.00020 mass% to 0.00040 mass% is more preferable. When the content is less than 0.0001% by mass or exceeds 0.0015% by mass, the folic acid content in the cells may be decreased.
The amino acids, vitamins, etc. may be added to the culture medium in the initial stage, and added to the feeding solution and fed into the culture solution to control the folate compound concentration in the culture solution. However, initial addition is preferable in that the folic acid content per dry cell can be increased.
The amount of amino acids, vitamins, etc. added to the culture solution can be measured by a known method, for example, using a fully automatic amino acid analyzer (JLC-500 / V, manufactured by JEOL Ltd.) and HPLC, respectively. Can be measured.
上述した培養液に添加するアミノ酸、ビタミン等の量は、該培養液の総液量に対する量であり、前記アミノ酸、ビタミン等を初期添加する場合は、初期の培地中の濃度を意味する。流加する場合は、培地（初期培養液）と、流加液（流加した量）との合計量に対する添加量（質量％）を意味する。
なお、流加液中のアミノ酸、ビタミン等の添加量としては、培養液の総液量に対するアミノ酸、ビタミン等の添加量が上述の範囲内となる限り特に制限はなく、目的に応じて適宜選択することができる。
The above-mentioned amounts of amino acids, vitamins, etc. added to the culture solution are amounts relative to the total amount of the culture solution. When the amino acids, vitamins, etc. are initially added, they mean the initial concentration in the medium. In the case of feeding, it means the added amount (% by mass) with respect to the total amount of the culture medium (initial culture solution) and the fed solution (amount fed).
The addition amount of amino acids, vitamins, etc. in the fed-batch solution is not particularly limited as long as the addition amount of amino acids, vitamins, etc. with respect to the total amount of the culture solution is within the above range, and is appropriately selected according to the purpose. can do.
前記流加培養の方法としては、例えば、流加連続培養、流加回分（バッチ）培養などが挙げられる。 Examples of the fed-batch culture method include fed-batch continuous culture and fed-batch (batch) culture.
前記流加培養に用いる流加液としては、特に制限はなく、目的に応じて公知のものの中から適宜選択することができるが、例えば、廃糖蜜などが好適に挙げられる。なお、流加液は、適宜、前記培養液の成分を含んでいてもよい。 There is no restriction | limiting in particular as a feeding solution used for the said fed-batch culture, Although it can select suitably from well-known things according to the objective, For example, waste molasses etc. are mentioned suitably. The fed-batch solution may appropriately contain the components of the culture solution.
＜酵母＞
前記培養を行う酵母としては、特に制限はなく、目的に応じて適宜選択することができるが、食品素材などとして用いる場合には、食用酵母であることが特に好ましい。前記酵母は、１種単独で培養してもよいし、２種以上を培養してもよい。
<Yeast>
There is no restriction | limiting in particular as yeast which performs the said culture | cultivation, Although it can select suitably according to the objective, When using as a foodstuff raw material etc., it is especially preferable that it is edible yeast. The yeast may be cultivated singly or in combination of two or more.
前記食用酵母としては、特に制限はなく、公知のものの中から適宜選択することができるが、パン酵母、ビール酵母、ワイン酵母、清酒酵母、及び味噌醤油酵母から選択されることが好ましく、葉酸を高含有させることができる点で、清酒酵母が特に好ましい。 The edible yeast is not particularly limited and may be appropriately selected from known ones, but is preferably selected from baker's yeast, beer yeast, wine yeast, sake yeast, and miso soy yeast, Sake yeast is particularly preferable because it can be contained in a high amount.
前記清酒酵母としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、きょうかい酵母１号〜１５号、６０１号、７０１号、９０１号、１００１号、１６０１号、１７０１号などが挙げられる。これらの中でも、他の清酒酵母に比べて培養後の葉酸含有量が多い点で、きょうかい酵母６号、６０１号、７号、７０１号、８号、９号、９０１号、１１号、１２号、１３号、１４号、１５号、１７０１号などが好ましい。 There is no restriction | limiting in particular as said sake yeast, According to the objective, it can select suitably, For example, Kyoto yeast Nos. 1-15, 601, 701, 901, 1001, 1601, 1701 Etc. Among these, Kyokai yeast Nos. 6, 601, 7, 701, 8, 9, 901, 11, 12 are more folic acid content after cultivation than other sake yeasts. No. 13, No. 13, No. 15, No. 15, No. 1701, etc. are preferable.
前記食用酵母の菌株としては、サッカロミセス（Ｓａｃｃｈａｒｏｍｙｃｅｓ）属、トルロプシス（Ｔｏｒｕｌｏｐｓｉｓ）属、ミコトルラ（Ｍｙｃｏｔｏｒｕｌａ）属、トルラスポラ（Ｔｏｒｕｌａｓｐｏｒａ）属、キャンディダ（Ｃａｎｄｉｄａ）属、ロードトルラ（Ｒｈｏｄｏｔｏｒｕｌａ）属、ピキア（Ｐｉｃｈｉａ）属などが挙げられる。 The strain of the edible yeast, Saccharomyces (Saccharomyces) genus Torulopsis (Torulopsis) genus Mikotorura (Mycotorula) genus Torulaspora (Torulaspora) genus Candida (Candida) genus Rhodotorula (Rhodotorula) genus Pichia (Pichia) sp Etc.
前記食用酵母の菌株の具体例としては、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃａｒｌｓｂｅｒｇｅｎｓｉｓ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｕｖａｒｕｍ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｒｏｕｘｉｉ、Ｔｏｒｕｌｏｐｓｉｓ ｕｔｉｌｉｓ、Ｔｏｒｕｌｏｐｓｉｓ ｃａｎｄｉｄａ、Ｍｙｃｏｔｏｒｕｌａ ｊａｐｏｎｉｃａ、Ｍｙｃｏｔｏｒｕｌａ ｌｉｐｏｌｙｔｉｃａ、Ｔｏｒｕｌａｓｐｏｒａ ｄｅｌｂｒｕｅｃｋｉｉ、Ｔｏｒｕｌａｓｐｏｒａ ｆｅｒｍｅｎｔａｔｉ、Ｃａｎｄｉｄａ ｓａｋｅ、Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ、Ｃａｎｄｉｄａ ｕｔｉｌｉｓ、Ｈａｎｓｅｎｕｌａ ａｎｏｍａｌａ、Ｈａｎｓｅｎｕｌａ ｓｕａｖｅｏｌｅｎｓ、Ｓａｃｃｈａｒｏｍｙｃｏｐｓｉｓ ｆｉｂｌｉｇｅｒａ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｌｉｐｏｌｙｔｉｃａ、Ｒｈｏｄｏｔｏｒｕｌａ ｒｕｂｒａ、Ｐｉｃｈｉａ ｆａｒｉｎｏｓａなどが挙げられる。
これらの中でも、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃａｒｌｓｂｅｒｇｅｎｓｉｓが好ましく、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅが特に好ましい。
Specific examples of strains of the edible yeast, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces uvarum, Saccharomyces rouxii, Torulopsis utilis, Torulopsis candida, Mycotorula japonica, Mycotorula lipolytica, Torulaspora delbrueckii, Torulaspora fermentati, Candida sake, Candida tropicalis, Candida utilis, Hansenula anomala , Hansenula suaveolens , Sacchar omycopsis fibrigera , Saccharomyces lipolytica , Rhodotorula rubra , Pichia farinosa and the like.
Among these, Saccharomyces cerevisiae and Saccharomyces carlsbergensis are preferable, and Saccharomyces cerevisiae is particularly preferable.
前記酵母の前記培養液への接種量としては、特に制限はなく適宜決定することができるが、通常５質量％程度である。 There is no restriction | limiting in particular as an inoculation amount to the said culture solution of the said yeast, Although it can determine suitably, it is about 5 mass% normally.
＜培養方法＞
前記酵母の培養方法としては、葉酸を取り込むことができれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、回分培養法、半回分培養法、連続培養法などが挙げられる。これらの中でも、前記糖及び前記ｐＨ調整試薬を流加して培養することが、乾燥菌体当たりの葉酸含有量を増加させることができる点で好ましい。
なお、流加培養を行う場合は、培養０時間（培養開始時）から培養終了時までの間、連続的に流加することが好ましく、定値流加することがより好ましい。
前記培養は、ジャーファーメンターを用いて好適に行うことができる。
<Culture method>
The yeast culture method is not particularly limited as long as it can incorporate folic acid, and can be appropriately selected according to the purpose. Examples thereof include a batch culture method, a semi-batch culture method, and a continuous culture method. Among these, it is preferable that the sugar and the pH adjusting reagent are fed and cultured in terms of increasing the folic acid content per dry cell.
In addition, when performing fed-batch culture, it is preferable to feed continuously from culture | cultivation 0 time (at the time of culture | cultivation start) to the time of completion | finish of culture | cultivation, and it is more preferable to carry out constant value feeding.
The culture can be suitably performed using a jar fermenter.
−培養温度−
前記培養の温度としては、特に制限はなく、目的に応じて適宜選択することができるが、２８℃〜３３℃が好ましく、葉酸を高含有させる観点から３０℃がより好ましい。
-Culture temperature-
There is no restriction | limiting in particular as temperature of the said culture | cultivation, Although it can select suitably according to the objective, 28 to 33 degreeC is preferable and 30 degreeC is more preferable from a viewpoint of making folic acid highly contained.
−培養時間−
前記培養時間としては、特に制限はなく、目的に応じて適宜選択することができるが、６時間〜２４時間が好ましく、８時間〜１６時間がより好ましい。前記培養時間が、６時間未満であると、所望の乾燥菌体当たりの葉酸含有量を得ることができないことがあり、２４時間を超えると、製造上効率が悪い。
-Culture time-
There is no restriction | limiting in particular as said culture | cultivation time, Although it can select suitably according to the objective, 6 to 24 hours are preferable and 8 to 16 hours are more preferable. If the culture time is less than 6 hours, the desired folic acid content per dry cell may not be obtained, and if it exceeds 24 hours, the production efficiency is poor.
−通気−
前記培養時は、通気しても、通気しなくてもよいが、通気することが、葉酸を高含有させることができる点で好ましい。
前記通気量としては、特に制限はなく、目的に応じて適宜選択することができるが、０．５ｖｖｍ〜４ｖｖｍが好ましく、１ｖｖｍ〜２ｖｖｍがより好ましい。前記通気量が、０．５ｖｖｍ未満であると、葉酸の取り込みが不安定になることがあり、４ｖｖｍを超えると、培養時に激しい発泡を生じ、工業生産に支障をきたすことがある。
-Ventilation-
At the time of the culture, aeration may or may not be performed, but aeration is preferable from the viewpoint that folic acid can be highly contained.
There is no restriction | limiting in particular as said ventilation | gas_flowing amount, Although it can select suitably according to the objective, 0.5 vvm-4 vvm are preferable and 1 vvm-2 vvm are more preferable. If the aeration rate is less than 0.5 vvm, folic acid uptake may become unstable, and if it exceeds 4 vvm, severe foaming may occur during cultivation, which may hinder industrial production.
−攪拌速度−
前記培養時の攪拌速度としては、菌体が沈まない程度であれば、特に制限はなく、目的に応じて適宜選択することができるが、１００ｒｐｍ〜６００ｒｐｍが好ましく、４００ｒｐｍ〜５００ｒｐｍがより好ましい。
-Agitation speed-
The stirring speed at the time of culturing is not particularly limited as long as the cells do not sink, and can be appropriately selected according to the purpose, but is preferably 100 rpm to 600 rpm, more preferably 400 rpm to 500 rpm.
（葉酸高含有酵母、葉酸高含有酵母破砕物）
本発明の葉酸高含有酵母は、本発明の葉酸高含有酵母の製造方法により好適に製造することができる。
(High-folic acid yeast, folic acid-rich yeast crushed material)
The folic acid-rich yeast of the present invention can be suitably produced by the method for producing the folic acid-rich yeast of the present invention.
＜葉酸含有量＞
前記葉酸高含有酵母における葉酸の含有量としては、乾燥菌体１ｇ当たり少なくとも１６０μｇであるが、該葉酸高含有酵母を葉酸強化食品素材などとして用いる場合には多いほど好ましく、１７０μｇ以上がより好ましく、１８０μｇ以上が特に好ましい。前記葉酸含有量の上限としては、特に制限はなく、目的に応じて適宜選択することができる。
前記菌体における葉酸含有量は、前記菌体が２５℃のイオン交換水で洗浄した菌体であっても、乾燥菌体１ｇ当たり少なくとも１６０μｇであり、洗浄後においても葉酸含有量が高く維持される。
<Folic acid content>
The content of folic acid in the folic acid-rich yeast is at least 160 μg per gram of dry cells, but is more preferable when the folic acid-rich yeast is used as a folic acid-enriched food material, and more preferably 170 μg or more. 180 μg or more is particularly preferable. There is no restriction | limiting in particular as an upper limit of the said folic acid content, According to the objective, it can select suitably.
The folic acid content in the cells is at least 160 μg per gram of dry cells even if the cells are washed with ion-exchanged water at 25 ° C., and the folic acid content is maintained high even after washing. The
前記葉酸高含有酵母における葉酸含有量を測定する方法としては、特に制限はなく、公知の測定方法から適宜選択することができ、例えば、前記葉酸高含有酵母より葉酸を抽出後、吸光度を測定する方法、液体クロマトグラフィーで分析する方法、質量分析する方法などが挙げられる。
前記葉酸を抽出する方法としては、特に制限はなく、公知の測定方法から適宜選択することができ、例えば、熱抽出法などが挙げられる。また、測定機器としては、ＨＰＬＣ（株式会社島津製作所製）などが挙げられ、例えば、テトラヒドロ葉酸（ＴＨＦ）と５−メチル−ＴＨＦとの合計を葉酸含有量として測定する。
The method for measuring the folic acid content in the yeast with a high content of folic acid is not particularly limited and can be appropriately selected from known measuring methods. For example, after extracting folic acid from the yeast with a high content of folic acid, the absorbance is measured. The method, the method of analyzing by liquid chromatography, the method of mass spectrometry, etc. are mentioned.
The method for extracting folic acid is not particularly limited and may be appropriately selected from known measurement methods. Examples thereof include a heat extraction method. Moreover, HPLC (made by Shimadzu Corporation) etc. is mentioned as a measuring instrument, For example, the total of tetrahydrofolic acid (THF) and 5-methyl-THF is measured as folic acid content.
本発明の葉酸高含有酵母は、前記葉酸を菌体内部において保持しており、洗浄を行っても該葉酸は除去されず菌体内部に保持されたままであるので、食品素材などとして用いた場合、添加した食品の風味などを損なうことながなく、食品素材などとして好適である。 The yeast with high folic acid content of the present invention retains the folic acid inside the microbial cell, and even when washed, the folic acid is not removed and remains retained inside the microbial cell, so when used as a food material, etc. It is suitable as a food material and the like without impairing the flavor of the added food.
前記葉酸高含有酵母は、生菌乃至未乾燥の状態であってもよいし、乾燥された状態であってもよく、また、菌体が破砕された破砕物の状態であってもよい。
なお、前記葉酸高含有酵母破砕物とは、顕微鏡観察下で未破砕菌体がなくなった状態をいう。
The yeast with a high content of folic acid may be a live bacterium or an undried state, may be a dried state, or may be a crushed material in which the microbial cells are crushed.
The folic acid-rich yeast crushed material refers to a state in which unbroken microbial cells disappeared under microscopic observation.
前記葉酸高含有酵母を乾燥する方法としては、特に制限はなく、公知の方法から適宜選択することができ、例えば、噴霧乾燥、流動層乾燥、凍結乾燥などが挙げられる。 There is no restriction | limiting in particular as a method of drying the said yeast with high content of folic acid, It can select suitably from a well-known method, For example, spray drying, fluidized-bed drying, freeze-drying etc. are mentioned.
前記葉酸高含有酵母を破砕する方法としては、顕微鏡観察下で未破砕菌体がなくなる方法であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、物理的破砕処理法、化学的破砕処理法などが挙げられる。 The method for crushing the folic acid-rich yeast is not particularly limited as long as it is a method that eliminates unbroken cells under a microscope, and can be appropriately selected according to the purpose. For example, a physical crushing method And chemical crushing method.
前記物理的破砕処理法の具体例としては、０．５ｍｍ径ビーズをシリンダーに５０容量％充填したダイノミルを用いる方法などが好適に挙げられる。
前記ダイノミルとしては、特に制限はなく、目的に応じて適宜選択することができるが、例えば、ＷＡＢ社製のＤｙｎｏｍｉｌｌ Ｍｏｄｅｌ Ｔｙｐｅ ＫＤＬなどが挙げられる。
As a specific example of the physical crushing method, a method using a dynomill in which 0.5% diameter beads are filled in a cylinder by 50% by volume is preferably used.
There is no restriction | limiting in particular as said dynomill, Although it can select suitably according to the objective, For example, Dynamo Model Type KDL by WAB company etc. are mentioned.
前記シリンダーの容量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、実験的には、０．６Ｌ程度である。前記葉酸高含有酵母の懸濁液（３０質量％）の前記ダイノミルにおける前記シリンダー内への流速としては、特に制限はなく、目的に応じて適宜選択することができるが、２．１６Ｌ／時間程度が好ましい。
また、前記葉酸高含有酵母の前記ダイノミルにおける前記シリンダー内での滞在時間としては、１０分間程度が好ましい。
なお、前記破砕の前に、イオン交換水で前記ダイノミルにおける前記シリンダー内を予め洗浄しておくことが好ましい。また、前記顕微鏡観察下での未破砕菌体の有無は、適宜サンプリングをして顕微鏡観察を行うことにより確認することができる。
There is no restriction | limiting in particular as a capacity | capacitance of the said cylinder, According to the objective, it can select suitably, For example, it is about 0.6L experimentally. There is no restriction | limiting in particular as a flow rate in the said cylinder in the said dynomill of the suspension (30 mass%) of the said folic acid high content yeast, Although it can select suitably according to the objective, About 2.16L / hour Is preferred.
Moreover, as a residence time in the said cylinder in the said dynomill of the said folic acid high content yeast, about 10 minutes are preferable.
In addition, it is preferable to wash | clean the inside of the said cylinder in the said dynomill previously with ion-exchange water before the said crushing. In addition, the presence or absence of unbroken cells under the microscope observation can be confirmed by appropriately sampling and performing a microscope observation.
前記葉酸高含有酵母破砕物における乾燥菌体当たりの葉酸含有量としては、特に制限はなく、目的に応じて適宜選択することができるが、破砕菌体を食品素材などとして利用する観点からは、破砕を行う前の菌体における乾燥菌体当たりの葉酸含有量に対し、７０質量％以上が好ましく、８０質量％以上がより好ましく、９０質量％以上が特に好ましい。 The folic acid content per dry cell in the high folic acid-containing yeast crushed material is not particularly limited and can be appropriately selected according to the purpose, from the viewpoint of using the crushed cell as a food material, etc. 70 mass% or more is preferable with respect to the folic acid content per dry cell in the microbial cell before crushing, 80 mass% or more is more preferable, 90 mass% or more is especially preferable.
前記葉酸高含有酵母破砕物の態様としては、用途などに応じて適宜選択することができ、例えば、乾燥物のみの態様（破砕物から液状物を除去し、スプレードライ等により乾燥したものなど）であってもよいし、固形物のみの態様（破砕物から液状物を除去したものなど）であってもよいし、液状物のみの態様（破砕物から固形分を除去したものなど）であってもよく、あるいはこれらを含む態様（破砕しただけのものなど）であってもよい。なお、前記葉酸高含有破砕物の調製は、特に制限はなく、公知の装置などを用い、公知の方法に従って行うことができる。 The mode of the folic acid-rich yeast crushed material can be appropriately selected depending on the application and the like. For example, only the dried product (such as a product obtained by removing the liquid material from the crushed product and drying it by spray drying or the like) It may be a solid-only mode (such as a crushed product from which a liquid product has been removed) or a liquid-only mode (such as a crushed product from which a solid content has been removed). Alternatively, it may be an embodiment including these (such as a crushed one). In addition, there is no restriction | limiting in particular in the preparation of the said folic acid high content crushed material, It can carry out according to a well-known method using a well-known apparatus etc.
＜酵母の種類＞
本発明の葉酸高含有酵母の種類としては、前記食用酵母が好適に挙げられ、該食用酵母の菌株の属及び具体例としては、上述の通りのものが挙げられる。
<Type of yeast>
The above-mentioned edible yeast is preferably exemplified as the kind of folic acid-rich yeast of the present invention, and the genus and specific examples of the edible yeast strain include those described above.
＜用途＞
本発明の葉酸高含有酵母の用途としては、特に制限はなく、目的に応じて適宜選択することができるが、食品に添加されて用いられる食品素材、飼料、餌料などとしての用途が好ましく、これらの中でも、前記食品素材としての用途が特に好ましい。本発明の葉酸高含有酵母を用いることにより、葉酸高含有食品、葉酸高含有飼料、葉酸高含有餌料などが得られる。
<Application>
The use of the folic acid-rich yeast of the present invention is not particularly limited and can be appropriately selected according to the purpose, but is preferably used as a food material, feed, feed or the like used by being added to food. Among these, the use as the food material is particularly preferable. By using the yeast with a high content of folic acid according to the present invention, a food with a high content of folic acid, a feed with a high content of folic acid, a feed with a high content of folic acid, and the like can be obtained.
（食品）
本発明の食品は、前記葉酸高含有酵母及び葉酸高含有酵母破砕物の少なくともいずれかを含み、必要に応じて、更にその他の成分を含む。
ここで、前記食品とは、人の健康に危害を加えるおそれが少なく、通常の社会生活において、経口又は消化管投与により摂取されるものをいい、行政区分上の食品、医薬品、医薬部外品などの区分に制限されるものではなく、例えば、経口的に摂取される一般食品、健康食品、保健機能食品、医薬部外品、医薬品などを幅広く含むものを意味する。
(Food)
The food of the present invention contains at least one of the folic acid-rich yeast and the folic acid-rich yeast crushed material, and further contains other components as necessary.
Here, the food is one that is less likely to harm human health and is taken by oral or gastrointestinal administration in normal social life. It is not limited to such categories as, for example, it means a wide range of foods that are taken orally, such as general foods, health foods, health functional foods, quasi drugs, and pharmaceuticals.
前記食品中の前記葉酸高含有酵母及び葉酸高含有酵母破砕物の少なくともいずれかの含有量としては、特に制限はなく、本発明の効果を損なわない範囲内で、対象となる食品の種類に応じて適宜配合することができる。 The content of at least one of the yeast with a high content of folic acid and a yeast with a high content of folic acid in the food is not particularly limited, and is within a range that does not impair the effects of the present invention, depending on the type of the target food. Can be appropriately blended.
＜食品の種類＞
前記食品の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料等の飲料；アイスクリーム、アイスシャーベット、かき氷等の冷菓；そば、うどん、はるさめ、ぎょうざの皮、しゅうまいの皮、中華麺、即席麺等の麺類；飴、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、パン等の菓子類；カニ、サケ、アサリ、マグロ、イワシ、エビ、カツオ、サバ、クジラ、カキ、サンマ、イカ、アカガイ、ホタテ、アワビ、ウニ、イクラ、トコブシ等の水産物；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及び油脂加工食品；ソース、たれ等の調味料；カレー、シチュー、親子丼、お粥、雑炊、中華丼、かつ丼、天丼、うな丼、ハヤシライス、おでん、マーボドーフ、牛丼、ミートソース、玉子スープ、オムライス、餃子、シューマイ、ハンバーグ、ミートボール等のレトルトパウチ食品；流動食等の種々の形態の栄養補助食品、健康食品、医薬品、医薬部外品などが挙げられる。
<Type of food>
The type of the food is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include beverages such as soft drinks, carbonated drinks, nutrition drinks, fruit drinks, and lactic acid drinks; ice cream, ice sherbet, shaved ice Noodles such as buckwheat noodles, udon, harusame, gyoza peel, Chinese noodles, instant noodles; rice cake, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery , Confectionery such as bread; crab, salmon, clams, tuna, sardines, shrimp, skipjack, mackerel, whale, oyster, saury, squid, red sea bream, scallop, abalone, sea urchin, sea bream, tocobushi, etc .; Fishery and livestock processed foods such as sausages; Dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, show Oil and fat processed foods such as ning, whipped cream and dressing; seasonings such as sauces and sauces; curry, stew, oyakodon, rice bowl, miscellaneous cooking, Chinese rice bowl, and rice cake, tempura, eel rice, hayashi rice, oden, marvodorf, Retort pouch foods such as beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger, meatballs; various forms of nutritional supplements such as liquid foods, health foods, pharmaceuticals, and quasi drugs.
＜その他の成分＞
前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、食品を製造するにあたって通常用いられる、補助的原料又は添加物などが挙げられる。
前記補助的原料又は添加物としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ブドウ糖、果糖、ショ糖、マルトース、ソルビトール、ステビオサイド、ルブソサイド、コーンシロップ、乳糖、クエン酸、酒石酸、リンゴ酸、コハク酸、乳酸、Ｌ−アスコルビン酸、ｄｌ−α−トコフェロール、エリソルビン酸ナトリウム、グリセリン、プロピレングリコール、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、ショ糖脂肪酸エステル、ソルビタン脂肪酸エステル、アラビアガム、カラギーナン、カゼイン、ゼラチン、ペクチン、寒天、ビタミンＢ類、ニコチン酸アミド、パントテン酸カルシウム、アミノ酸類、カルシウム塩類、色素、香料、保存剤などが挙げられる。
前記その他の成分の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
<Other ingredients>
There is no restriction | limiting in particular as said other component, According to the objective, it can select suitably, For example, the auxiliary | assistant raw material or additive etc. which are normally used in manufacturing a foodstuff are mentioned.
The auxiliary raw material or additive is not particularly limited and may be appropriately selected depending on the intended purpose. For example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid , Tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, Arabia Examples include gum, carrageenan, casein, gelatin, pectin, agar, vitamin Bs, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives and the like.
There is no restriction | limiting in particular as content of the said other component, According to the objective, it can select suitably.
以下に本発明の実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples of the present invention, but the present invention is not limited to these examples.
（試験例１：菌株の検討）
以下に示す方法を用い、下記表１に示す菌株における酵母菌体内の葉酸含有量について測定を行った。
(Test Example 1: Examination of strains)
Using the method shown below, the folic acid content in yeast cells in the strains shown in Table 1 below was measured.
＜培養液の作製＞
水１Ｌに１０ｇの酵母エキス、２０ｇのペプトン、及び２０ｇのグルコース（Ｄｉｆｃｏ製）を加え、１２０℃で２０分間オートクレーブすることにより、ＹＰＤ培地を作製した。
<Production of culture solution>
10 g yeast extract, 20 g peptone, and 20 g glucose (manufactured by Difco) were added to 1 L of water, and autoclaved at 120 ° C. for 20 minutes to prepare a YPD medium.
＜酵母の培養＞
表１に示す清酒酵母の菌株それぞれについて、前記培養液５ｍＬ中に湿菌体５０ｍｇ（１質量％／総液量）を接種して３０℃、２４時間振とう培養を行い前培養とした。その後、前培養菌液を前記培養液１００ｍＬ中に２×１０５ｃeｌｌｓ／ｍＬとなるように５００ｍＬ容のバッフル付フラスコ内に収容させ、前記酵母を振とう培養し、本培養とした。
本培養の条件としては、培養温度を３０℃、培養時間を９６時間、振とう速度を１２０ｒｐｍ（中型振とう機 ダブルシェーカーＮＲ−３０、タイテック社製）とし、通気は行わなかった。
<Culture of yeast>
For each sake yeast strain shown in Table 1, 50 mg (1% by mass / total liquid amount) of wet cells were inoculated into 5 mL of the above culture solution, followed by shaking culture at 30 ° C. for 24 hours for preculture. Thereafter, the precultured bacterial solution was placed in a 500 mL baffled flask so as to be 2 × 10 5 cels / mL in 100 mL of the culture solution, and the yeast was cultured with shaking to obtain main culture.
As the conditions of the main culture, the culture temperature was 30 ° C., the culture time was 96 hours, the shaking speed was 120 rpm (medium shaker double shaker NR-30, manufactured by Taitec Corporation), and aeration was not performed.
その後、培養開始から１２時間後、２４時間後、４８時間後、７２時間後、及び９６時間後において前記培養液適量を４，０００ｒｐｍにて２分間遠心分離し、上清を廃棄した。菌体（沈殿）に２５℃の滅菌水１５ｍＬを添加し懸濁後、４，０００ｒｐｍにて２分間遠心分離し、上清を廃棄して洗浄した。この洗浄を２回行った後、菌体を凍結乾燥して乾燥させた。
各時間における乾燥菌体重量（ＤＣＷ）１ｇ当たりの葉酸含有量（質量％）を、下記方法にて測定した結果を表１に示す。また、図１Ａ及び図１Ｂに乾燥菌体１ｇ当たりの葉酸含有量（μｇ）の経時変化を示す。
Thereafter, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after the start of the culture, the appropriate amount of the culture solution was centrifuged at 4,000 rpm for 2 minutes, and the supernatant was discarded. To the cells (precipitate), 15 mL of 25 ° C. sterilized water was added and suspended, followed by centrifugation at 4,000 rpm for 2 minutes, and the supernatant was discarded and washed. After performing this washing twice, the cells were freeze-dried and dried.
Table 1 shows the results of measuring the folic acid content (% by mass) per gram of dry cell weight (DCW) at each time by the following method. In addition, FIG. 1A and FIG. 1B show changes with time in the folic acid content (μg) per gram of dry cells.
＜葉酸含有量の測定方法＞
−葉酸の抽出−
前記酵母約２０ｍｇ（乾燥重量）を、０．１Ｍリン酸ｂｕｆｆｅｒ(ｐＨ６．１)と２質量％アスコルビン酸ナトリウムと０．１質量％２-メルカプトエタノール溶液の混合溶液１０ｍＬに溶解して熱抽出し、遠心上清をラット血清を用いてｃｏｎｊｕｇａｓｅ処理を行うことにより、測定に供する試料溶液とした。
<Measurement method of folic acid content>
-Extraction of folic acid-
About 20 mg (dry weight) of the yeast was dissolved in 10 mL of a mixed solution of 0.1 M phosphate buffer (pH 6.1), 2% by mass sodium ascorbate, and 0.1% by mass 2-mercaptoethanol solution, followed by heat extraction. The centrifugal supernatant was subjected to conjugase treatment using rat serum to obtain a sample solution for measurement.
−ＨＰＬＣによる葉酸含有量の測定−
まず、テトラヒドロ葉酸（ＴＨＦ）と５−メチル−ＴＨＦの標品を用いて検量線を得た。前記葉酸試料溶液を用い、下記ＨＰＬＣ測定条件により葉酸量を測定し、前記検量線から、酵母に含まれる葉酸含有量を定量し、乾燥菌体重量（ＤＣＷ）１ｇ当たりの葉酸含有量（μｇ）を算出した。なお、テトラヒドロ葉酸（ＴＨＦ）と５−メチル−ＴＨＦの合計を葉酸量（μｇ）とした。
-Measurement of folic acid content by HPLC-
First, a calibration curve was obtained using a sample of tetrahydrofolic acid (THF) and 5-methyl-THF. Using the folic acid sample solution, the amount of folic acid was measured under the following HPLC measurement conditions, the folic acid content contained in the yeast was quantified from the calibration curve, and the folic acid content (μg) per gram of dry cell weight (DCW) Was calculated. The total of tetrahydrofolic acid (THF) and 5-methyl-THF was defined as the amount of folic acid (μg).
−−ＨＰＬＣ測定条件−−
カラム：Ａｑｕａｓｉｌ Ｃ１８カラム（１５０ｍｍＩＤ×４．６ｍｍ）
溶媒：アセトニトリルと３０ｍＭ（或いはｍｇ／Ｌ）リン酸ｂｕｆｆｅｒ(ｐＨ２．３)の混合液
勾配溶離メソッド：
検出器：蛍光検出器
カラム温度：３０℃
--HPLC measurement conditions--
Column: Aquasil C18 column (150 mm ID × 4.6 mm)
Solvent: Mixture of acetonitrile and 30 mM (or mg / L) phosphate buffer (pH 2.3) Gradient elution method:
図１Ａ及び図１Ｂの結果から、上記の様々な菌株の中でも、清酒酵母が葉酸高含有酵母の製造に好適に用いることができ、清酒酵母の中でも、きょうかい酵母６０１号（Ｋ６０１）、７号（Ｋ７）、７０１号（Ｋ７０１）、９号（Ｋ９）、及び１５号（Ｋ１５）が葉酸高含有酵母の製造に好適に用いることができることがわかった。 From the results of FIG. 1A and FIG. 1B, among the various strains described above, sake yeast can be suitably used for the production of folic acid-rich yeast, and among sake yeasts, Kyoto yeast 601 (K601), No. 7 It was found that (K7), No. 701 (K701), No. 9 (K9), and No. 15 (K15) can be suitably used for the production of folic acid-rich yeast.
（実施例１：メチオニン及びパラアミノ安息香酸添加培地で培養）
＜培養液の作製＞
まず、水１Ｌに１７ｇのアミノ酸を含まないＹＮＢ（ｙｅａｓｔ ｎｉｔｒｏｇｅｎ ｂａｓｅ ｗ／ｏ Ａｍｉｎｏ Ａｃｉｄｓ ａｎｄ Ａｍｍｏｎｉｕｍ Ｓｕｌｆａｔｅ、Ｄｉｆｃｏ社製）、５０ｇの硫酸アンモニウム及び１００ｇのグルコース（和光純薬工業株式会社製）を加え、１２０℃で２０分間オートクレーブすることにより、１０倍濃度のＹＮＢ培地を作成した。その後、滅菌水を用いて１０倍希釈を行い、下記に示す組成及び濃度の基本培地（アミノ酸を含まないＹＮＢ培地）を作製した。
−基本培地−
グルコース １０ｇ／Ｌ
硫酸アンモニウム ５ｇ／Ｌ
リン酸一カリウム １．０ｇ／Ｌ
硫酸マグネシウム ０．５ｇ／Ｌ
硫酸亜鉛 ４００μｇ／Ｌ
硫酸マンガン ４００μｇ／Ｌ
硫酸銅 ４０μｇ／Ｌ
塩化カルシウム ０．１ｇ／Ｌ
塩化鉄 ２００μｇ／Ｌ
モリブデン酸ナトリウム ２００μｇ／Ｌ
ホウ酸 ５００μｇ／Ｌ
ヨウ化カリウム １００μｇ／Ｌ
ビオチン ２μｇ／Ｌ
パントテン酸カルシウム ４００μｇ／Ｌ
イノシトール ２，０００μｇ／Ｌ
チアミン塩酸塩 ４００μｇ／Ｌ
ピリドキシン塩酸塩 ４００μｇ／Ｌ
ｐ−アミノ安息香酸 ２００μｇ／Ｌ
塩化ナトリウム ０．１ｇ／Ｌ
リボフラビン ２００μｇ／Ｌ
ナイアシン ４００μｇ／Ｌ
葉酸 ２μｇ／Ｌ
(Example 1: Culture in a medium supplemented with methionine and paraaminobenzoic acid)
<Production of culture solution>
First, YNB (yeast nitrogen base w / o Amino Acids and Ammonium Sulfate, manufactured by Difco) not containing 17 g of amino acid in 1 L of water, 50 g of ammonium sulfate and 100 g of glucose (manufactured by Wako Pure Chemical Industries, Ltd.) were added. A 10-fold concentration of YNB medium was prepared by autoclaving at 20 ° C. for 20 minutes. Then, 10-fold dilution was performed using sterilized water to prepare a basic medium (YNB medium containing no amino acid) having the following composition and concentration.
-Basic medium-
Glucose 10g / L
Ammonium sulfate 5g / L
Monopotassium phosphate 1.0 g / L
Magnesium sulfate 0.5g / L
Zinc sulfate 400μg / L
Manganese sulfate 400μg / L
Copper sulfate 40μg / L
Calcium chloride 0.1g / L
Iron chloride 200μg / L
Sodium molybdate 200μg / L
Boric acid 500μg / L
Potassium iodide 100 μg / L
Biotin 2 μg / L
Calcium pantothenate 400μg / L
Inositol 2,000μg / L
Thiamine hydrochloride 400μg / L
Pyridoxine hydrochloride 400 μg / L
p-aminobenzoic acid 200 μg / L
Sodium chloride 0.1g / L
Riboflavin 200μg / L
Niacin 400μg / L
Folic acid 2μg / L
−メチオニン及びパラアミノ安息香酸添加培地−
前記基本培地中にメチオニンを濃度が４ｍｇ／ｍＬ、パラアミノ安息香酸を濃度が３μｇ／ｍＬとなるように添加してメチオニン及びパラアミノ安息香酸添加培地を作製した。
-Medium supplemented with methionine and paraaminobenzoic acid-
A methionine and paraaminobenzoic acid-added medium was prepared by adding methionine to the basic medium at a concentration of 4 mg / mL and paraaminobenzoic acid at a concentration of 3 μg / mL.
＜酵母の培養＞
清酒酵母菌株きょうかい酵母９号（Ｋ９）を前記基本培地５ｍＬを用いて３０℃で２４時間前培養を行った。前記前培養後の酵母菌体をメチオニン及びパラアミノ安息香酸添加培地１００ｍＬ中に、細胞濃度が２×１０５ｃｅｌｌｓ／ｍＬとなるように接種して５００ｍＬ容のバッフル付フラスコ内に収容させ、前記酵母を振とう培養した。
培養条件は、培養温度を３０℃、培養時間を７２時間、振とう速度を１２０ｒｐｍ（中型振とう機 ダブルシェーカーＮＲ−３０、タイテック社製）とし、通気は行わなかった。
<Culture of yeast>
Sake yeast strain Kyoto yeast 9 (K9) was pre-cultured at 30 ° C. for 24 hours using 5 mL of the basic medium. The yeast cells after the pre-culture were inoculated into 100 mL of methionine and paraaminobenzoic acid-added medium so as to have a cell concentration of 2 × 10 5 cells / mL and accommodated in a 500 mL baffled flask. Was cultured with shaking.
The culture conditions were a culture temperature of 30 ° C., a culture time of 72 hours, a shaking speed of 120 rpm (medium shaker double shaker NR-30, manufactured by Taitec Corporation), and aeration was not performed.
前記条件で培養した酵母菌体を経時的（培養開始から０時間後、１２時間後、２４時間後、適宜３６時間後、４８時間後、適宜７２時間後）に回収した。
その後、前記培養液１００ｍＬを４，０００ｒｐｍにて２分間遠心分離し、上清を廃棄した。菌体（沈殿）に２５℃の滅菌水１５ｍＬを添加し懸濁後、４，０００ｒｐｍにて２分間遠心分離し、上清を廃棄して洗浄した。この洗浄を２回行った後、菌体を凍結乾燥して乾燥させた。
各時間における乾燥菌体（ＤＣＷ）１ｇ当たりの葉酸含有量（μｇ）を前記試験例１に記載の方法と同様の方法にて測定した結果を、図２中、棒グラフで示す。酵母菌体の培地中における細胞濃度の経時変化を図２中、黒丸で示す。
The yeast cells cultured under the above conditions were collected over time (0 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours as appropriate).
Thereafter, 100 mL of the culture solution was centrifuged at 4,000 rpm for 2 minutes, and the supernatant was discarded. To the cells (precipitate), 15 mL of 25 ° C. sterilized water was added and suspended, followed by centrifugation at 4,000 rpm for 2 minutes, and the supernatant was discarded and washed. After performing this washing twice, the cells were freeze-dried and dried.
The results of measuring the folic acid content (μg) per gram of dried bacterial cells (DCW) at each time by the same method as described in Test Example 1 are shown in a bar graph in FIG. The time course of the cell concentration in the yeast cell culture medium is indicated by black circles in FIG.
（実施例２）
実施例１において、基本培地中にメチオニンを濃度が４ｍｇ／ｍＬ、パラアミノ安息香酸を濃度が３μｇ／ｍＬとなるように添加してした点を、メチオニンの濃度が６ｍｇ／ｍＬ、パラアミノ安息香酸を濃度が４μｇ／ｍＬとなるように添加した以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図３に示す。
(Example 2)
In Example 1, methionine was added to the basic medium at a concentration of 4 mg / mL and paraaminobenzoic acid was added at a concentration of 3 μg / mL. The concentration of methionine was 6 mg / mL, and the concentration of paraaminobenzoic acid was A dry cell was prepared in the same manner as in Example 1 except that the amount was 4 μg / mL, and the folic acid content per 1 g of the obtained dry cell was measured. The results are shown in FIG.
（比較例１）
実施例１において、基本培地に添加したアミノ酸、ビタミン等（メチオニン及びパラアミノ安息香酸）を添加しなかった以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図４に示す。
(Comparative Example 1)
In Example 1, except that the amino acid and vitamins (methionine and paraaminobenzoic acid) added to the basic medium were not added, a dry cell was prepared in the same manner as in Example 1, and the obtained dry cell The folic acid content per gram was measured. The results are shown in FIG.
（比較例２）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からヒスチジンに変更し、その濃度を０．１ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図５に示す。
(Comparative Example 2)
In Example 1, dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to histidine, and the concentration was changed to 0.1 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例３）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からグルタミン酸に変更し、その濃度を２ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図６に示す。
(Comparative Example 3)
In Example 1, dry cells were prepared in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to glutamic acid, and the concentration was changed to 2 mg / mL. Then, the folic acid content per 1 g of the obtained dried cells was measured. The results are shown in FIG.
（比較例４）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からセリンに変更し、その濃度を０．６ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図７に示す。
(Comparative Example 4)
In Example 1, dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to serine and the concentration was 0.6 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例５）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からプロリンに変更し、その濃度を０．２ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図８に示す。
(Comparative Example 5)
In Example 1, the dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to proline and the concentration was changed to 0.2 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例６）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からトリプトファンに変更し、その濃度を０．２ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図９に示す。
(Comparative Example 6)
In Example 1, dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to tryptophan and the concentration was 0.2 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例７）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からパラアミノ安息香酸に変更し、その濃度を３μｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１０に示す。
(Comparative Example 7)
In Example 1, dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to paraaminobenzoic acid, and the concentration was changed to 3 μg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例８）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からビタミンＢ２に変更し、その濃度を２μｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１１に示す。
(Comparative Example 8)
In Example 1, the amino acid and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to vitamin B2, and the concentration was changed to 2 μg / mL. The folic acid content per gram of the prepared and obtained dried cells was measured. The results are shown in FIG.
（比較例９）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からビタミンＢ６に変更し、その濃度を８μｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１２に示す。
(Comparative Example 9)
In Example 1, the amino acid, vitamin and the like added to the basic medium were changed from methionine and paraaminobenzoic acid to vitamin B6, and the concentration was changed to 8 μg / mL. The folic acid content per gram of the prepared and obtained dried cells was measured. The results are shown in FIG.
（比較例１０）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からビタミンＢ１２に変更し、その濃度を８μｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１３に示す。
(Comparative Example 10)
In Example 1, the amino acid and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to vitamin B12, and the concentration was changed to 8 μg / mL. The folic acid content per gram of the prepared and obtained dried cells was measured. The results are shown in FIG.
（比較例１１）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からグリシンに変更し、その濃度を３．１ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１４に示す。
(Comparative Example 11)
In Example 1, dried cells were changed in the same manner as in Example 1 except that amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to glycine and the concentration was 3.1 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例１２）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からグルタミン酸塩酸塩に変更し、その濃度を１ｍｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１５に示す。
(Comparative Example 12)
In Example 1, amino acids, vitamins and the like added to the basic medium were changed from methionine and paraaminobenzoic acid to glutamic acid hydrochloride, and the concentration was changed to 1 mg / mL. And the folic acid content per gram of the obtained dried cells was measured. The results are shown in FIG.
（比較例１３）
実施例１において、基本培地に添加するアミノ酸、ビタミン等をメチオニン及びパラアミノ安息香酸からメチオニン及びビタミンＢ２に変更し、その濃度をメチオニンが４ｍｇ／ｍＬ、ビタミンＢ２が２μｇ／ｍＬとした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１６に示す。
(Comparative Example 13)
In Example 1, the amino acids and vitamins added to the basic medium were changed from methionine and paraaminobenzoic acid to methionine and vitamin B2, and the concentrations were changed to 4 mg / mL for methionine and 2 μg / mL for vitamin B2. In the same manner as in Example 1, dry cells were prepared, and the folic acid content per gram of the obtained dry cells was measured. The results are shown in FIG.
（実施例３）
実施例１において、培養する菌株をきょうかい酵母９号から下記表３に示すそれぞれの菌株に変更し、培養時間を１２時間とした以外は、実施例１と同様にして、乾燥菌体を調製し、得られた乾燥菌体１ｇ当たりの葉酸含有量を測定した。結果を図１７に示す。
(Example 3)
In Example 1, the dried bacterial cells were prepared in the same manner as in Example 1 except that the strains to be cultured were changed from the yeast 9 to the respective strains shown in Table 3 below and the culture time was 12 hours. Then, the folic acid content per 1 g of the obtained dried cells was measured. The results are shown in FIG.
比較例１〜６の結果から、アミノ酸を添加しない培地及びヒスチジン、グルタミン酸、セリン、プロリン又はトリプトファンなどのアミノ酸を添加した培地で酵母を培養した場合には、酵母菌体内における葉酸含有量には顕著な変化が見られなかった。
比較例７〜１０の結果から、葉酸の前駆体であるパラアミノ安息香酸、葉酸代謝経路に重要な役割を果たしているビタミン等であるビタミンＢ２、Ｂ６、Ｂ１２を添加した培地で酵母を培養しても、酵母菌体内における葉酸含有量には顕著な変化が見られなかった。
比較例１１〜１２の結果から、グリシン、及びグルタミン酸塩酸塩のいずれかを添加した培地で酵母を培養することにより、葉酸の含有量が増加したり、減少せず維持したりする酵母が得られることが分かったが、葉酸の含有量は、本発明の葉酸高含有酵母に比べて低かった。
比較例１３の結果から、メチオニン及びビタミンＢ２の両方を添加した培地で酵母を培養した場合、細胞増殖が顕著に抑制され、葉酸含有量の減少が見られた。
From the results of Comparative Examples 1 to 6, when yeast was cultured in a medium to which no amino acid was added and a medium to which an amino acid such as histidine, glutamic acid, serine, proline or tryptophan was added, the folic acid content in the yeast cells was remarkable. No significant change was seen.
From the results of Comparative Examples 7 to 10, even when cultivating yeast in a medium supplemented with paraaminobenzoic acid, which is a precursor of folic acid, and vitamins B2, B6, B12 which are vitamins that play an important role in the folic acid metabolic pathway There was no significant change in the folic acid content in the yeast cells.
From the results of Comparative Examples 11 to 12, by culturing the yeast in a medium to which any of glycine and glutamate hydrochloride is added, a yeast that increases or maintains the content of folic acid can be obtained. However, the content of folic acid was lower than that of the high folic acid-containing yeast of the present invention.
From the results of Comparative Example 13, when yeast was cultured in a medium to which both methionine and vitamin B2 were added, cell growth was significantly suppressed and a decrease in folic acid content was observed.
一方、実施例１の結果から、メチオニン及びパラアミノ安息香酸の両方を添加した培地で酵母を培養することにより、葉酸をより高含有する酵母が得られることが分かった。特に、酵母を１２時間培養した場合には、乾燥菌体１ｇあたり１８０μｇ以上の葉酸を含有した酵母が得られた。
また、実施例２の結果から、メチオニン及びパラアモノ安息香酸の添加量を変えた場合であっても、本発明の方法の範囲内では、乾燥菌体１ｇ当たり１６０μｇ以上という、葉酸を高含有する酵母が得られることが分かった。
また、実施例３の結果から、葉酸をより高含有させるためには、清酒酵母の中でも、きょうかい酵母６号、６０１号、７号、７０１号、８号、９号、９０１号、１１号、１２号、１３号、１４号、１５号、及び１７０１号がより好ましいことが分かった。更に、きょうかい酵母６号、６０１号、７号、７０１号、８号、９号、９０１号、１２号、１３号、１４号、１５号、及び１７０１号では、乾燥菌体１ｇあたり１８０μｇ以上の葉酸を含有させることができた。
On the other hand, from the results of Example 1, it was found that yeast containing higher folic acid can be obtained by culturing yeast in a medium to which both methionine and paraaminobenzoic acid are added. In particular, when yeast was cultured for 12 hours, yeast containing 180 μg or more of folic acid per 1 g of dried cells was obtained.
Moreover, even if it is a case where the addition amount of methionine and para-amonobenzoic acid is changed from the result of Example 2, within the range of the method of this invention, it is 160 micrograms or more per gram of dry microbial cells and yeast with high content of folic acid. Was found to be obtained.
Moreover, from the result of Example 3, in order to contain folic acid at a higher level, among sake yeasts, Kyokai yeast Nos. 6, 601, 7, 701, 8, 9, 901, 11 No. 12, No. 13, No. 14, No. 15, and No. 1701 were found to be more preferable. Furthermore, in Kyokai Yeast No. 6, 601, No. 7, No. 701, No. 8, No. 9, No. 901, No. 12, No. 13, No. 14, No. 15, and No. 1701, 180 μg or more per 1 g of dry cells. Folic acid could be included.
本発明の葉酸高含有酵母の製造方法によれば、培養液中に安価なアミノ酸、ビタミン等を添加するだけで、簡単に葉酸を高含有した酵母を製造することができる。
本発明の葉酸高含有酵母及び葉酸高含有酵母破砕物は、葉酸を高濃度に含有するため、葉酸を効率良く生体内に摂取及び吸収することができ、食品素材として好適に利用可能である。
また、本発明の前記葉酸高含有酵母及び前記葉酸高含有酵母破砕物を含有する食品は、酵母を使用しているため安全性が高く、添加した食品の風味などを損なうことがなく、日常食として好適に利用可能である。
According to the method for producing a yeast with a high content of folic acid of the present invention, a yeast with a high content of folic acid can be easily produced simply by adding inexpensive amino acids, vitamins and the like to the culture solution.
Since the folic acid-rich yeast and the folic acid-rich yeast crushed material of the present invention contain folic acid in a high concentration, folic acid can be efficiently ingested and absorbed in the living body and can be suitably used as a food material.
Further, the food containing the folic acid-rich yeast and the folic acid-rich yeast crushed product of the present invention is highly safe because it uses yeast, and does not impair the flavor of the added food. Can be suitably used.
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