JP5910978B2 - Non-protein amino acid-producing lactic acid bacteria and their uses - Google Patents
Non-protein amino acid-producing lactic acid bacteria and their uses Download PDFInfo
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- JP5910978B2 JP5910978B2 JP2012033583A JP2012033583A JP5910978B2 JP 5910978 B2 JP5910978 B2 JP 5910978B2 JP 2012033583 A JP2012033583 A JP 2012033583A JP 2012033583 A JP2012033583 A JP 2012033583A JP 5910978 B2 JP5910978 B2 JP 5910978B2
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- lactic acid
- acid bacteria
- bacteria
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- protein amino
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Description
本発明は非タンパク性アミノ酸を高生産する新規乳酸菌及びその用途などに関する。 The present invention relates to a novel lactic acid bacterium that highly produces non-protein amino acids and uses thereof.
アミノ酸にはL体とD体という二つの鏡像異性体が存在する。L-アミノ酸についてはタンパク質を構成するという重要な役割を果たすことに加え、生体内において様々な生理活性を発揮することが知られている。一方のD-アミノ酸は細胞壁の構成成分として細菌の生育に必須であることが知られている。最近になって、D-アミノ酸が真核生物においても重要な生理作用を有することが分かってきた。例えば、D-セリンについては、哺乳動物の脳に遊離状態で存在し、NMDA受容体のコアゴニストとして作用すること（非特許文献１）、統合失調症患者の脳脊髄液又は血清ではD-セリン含量が有意に低下しているとともに全セリン含量に占めるD-セリン含量の割合（D-セリン含量／全セリン含量）も有意に低下していること（非特許文献２、３）、さらにはアルツハイマー病患者の血清では全セリン含量に占めるD-セリン含量の割合（D-セリン含量／全セリン含量）が有意に低下していること（非特許文献４）等が報告されており、特に神経疾患との関係が注目されている。一方、D-セリンと同様に哺乳類体内で見出されるD-アスパラギン酸については、メラトニンの合成や分泌、テストステロンの産生などに関与することが知られている。D-アスパラギン酸には美肌効果があることも報告されている。また、D-アミノ酸と同様に非タンパク性アミノ酸であるγ−アミノ酪酸（GABA）は脳内神経物質として知られている。GABAは血圧降下作用、利尿作用、精神安定作用、脳機能改善作用などの生理活性も示し、食品ないし医薬素材として利用が図られている。 There are two enantiomers of amino acids, L and D. L-amino acids are known to exhibit various physiological activities in vivo in addition to the important role of constituting proteins. One D-amino acid is known to be essential for bacterial growth as a component of the cell wall. Recently, it has been found that D-amino acids have important physiological effects in eukaryotes. For example, D-serine exists in the mammalian brain in a free state and acts as a co-agonist of the NMDA receptor (Non-patent Document 1), and D-serine in cerebrospinal fluid or serum of schizophrenic patients. The ratio of D-serine content in the total serine content (D-serine content / total serine content) is also significantly decreased (Non-Patent Documents 2 and 3), and Alzheimer It has been reported that the ratio of D-serine content to total serine content (D-serine content / total serine content) in serum of patients with disease is significantly decreased (Non-patent Document 4), especially neurological diseases The relationship with is attracting attention. On the other hand, as with D-serine, D-aspartic acid found in mammals is known to be involved in the synthesis and secretion of melatonin and the production of testosterone. It has also been reported that D-aspartic acid has a skin beautifying effect. Further, γ-aminobutyric acid (GABA), which is a non-protein amino acid as well as D-amino acid, is known as an intracerebral nerve substance. GABA also exhibits physiological activities such as blood pressure lowering action, diuretic action, tranquilizing action, and brain function improving action, and is used as a food or pharmaceutical material.
最近になって特に注目されている非タンパク性アミノ酸の一つがオルニチンである。オルニチンはオルニチン回路を構成する重要な成分であり、アンモニアの解毒に関与する。オルニチンについては、疲労改善作用、基礎代謝改善作用、成長ホルモン分泌促進作用、免疫機能改善作用など、様々な生理機能を有することも知られており、機能性食品やサプリメントの有効成分として利用されている。 One of the non-protein amino acids that has recently attracted particular attention is ornithine. Ornithine is an important component of the ornithine cycle and is involved in ammonia detoxification. Ornithine is known to have various physiological functions such as fatigue improvement, basal metabolism improvement, growth hormone secretion promotion, immune function improvement, etc., and is used as an active ingredient in functional foods and supplements. Yes.
非タンパク性アミノ酸の製法としては、微生物由来の酵素を利用してD-アミノ酸を製造する方法（例えば特許文献１〜３）、乳酸発酵によりGABAを製造する方法（例えば特許文献４〜８）、微生物を用いてL-オルニチンを製造する方法（例えば特許文献９〜１２）等が知られている。 As a method for producing a non-protein amino acid, a method for producing a D-amino acid using an enzyme derived from a microorganism (for example, Patent Documents 1 to 3), a method for producing GABA by lactic acid fermentation (for example, Patent Documents 4 to 8), Methods for producing L-ornithine using microorganisms (for example, Patent Documents 9 to 12) are known.
非タンパク性アミノ酸の生理機能や有用性が次々に報告され、食品素材或いは医薬品素材等としての利用・応用が図られつつある中、非タンパク性アミノ酸を製造する手段に対するニーズは極めて高い。そこで本発明は、D-アミノ酸やGABA或いはL-オルニチンなど、その生理機能に注目が集まる非タンパク性アミノ酸を製造する手段及びその用途を提供することを課題とする。 Physiological functions and usefulness of non-protein amino acids are reported one after another, and use and application as food materials or pharmaceutical materials are being attempted. Therefore, needs for means for producing non-protein amino acids are extremely high. Accordingly, an object of the present invention is to provide a means for producing a non-protein amino acid such as D-amino acid, GABA or L-ornithine, which attracts attention for its physiological function, and its use.
上記課題に鑑み本発明者らは、京都の伝統的な漬け物の一つである「すぐき漬け」に注目し、非タンパク性アミノ酸の生産能に優れた新規乳酸菌を単離することを試みた。多種のすぐき漬けを用いた網羅的なスクリーニングの結果、非タンパク性アミノ酸を高生産する乳酸菌を単離することに成功した。単離した乳酸菌の菌種について16SリボソームRNA遺伝子（16S rDNA）の配列に基づき同定したところ、６種中の４種がラクトバチルス・ブレビスであり、残りの２種はラクトバチルス・プランタラムとラクトバチルス・ファーメンタムであった。尚、今後の利用・応用が大いに期待される非タンパク性アミノ酸を高生産する乳酸菌をすぐき漬けから単離できた事実は、すぐき漬けが有用乳酸菌の宝庫であること、即ち、すぐき漬けが有用乳酸菌をスクリーニングする際の優れたソースであることを意味する。 In view of the above-mentioned problems, the present inventors have focused on “Ikuki-zuke”, which is one of the traditional pickles in Kyoto, and have attempted to isolate a novel lactic acid bacterium excellent in the ability to produce non-protein amino acids. As a result of exhaustive screening using various types of pickles, we succeeded in isolating lactic acid bacteria that produce high amounts of non-protein amino acids. Species of isolated lactic acid bacteria were identified based on the sequence of the 16S ribosomal RNA gene (16S rDNA). Four of the six were Lactobacillus brevis, and the remaining two were Lactobacillus plantarum and lacto. It was Bacillus fermentum. The fact that lactic acid bacteria that produce highly non-protein amino acids that are highly expected to be used and applied in the future could be isolated from pickles is that pickles are a treasure trove of useful lactic acid bacteria. Means an excellent source for screening.
以下に列挙する本発明は主として上記成果に基づくものである。
［１］非タンパク性アミノ酸を高生産する乳酸菌であって、ラクトバチルス・ブレビス又はラクトバチルス・ファーメンタムである乳酸菌。
［２］受託番号がＮＩＴＥ Ｐ−１１６９又はＮＩＴＥ Ｐ−１１７１である、［１］に記載の乳酸菌。
［３］16SリボソームRNA遺伝子の配列が、配列番号１〜６のいずれかに示す配列を含む、［１］に記載の乳酸菌。
［４］すぐき漬け由来である、［１］〜［３］のいずれか一項に記載の乳酸菌。
［５］非タンパク性アミノ酸がD-アミノ酸、γ-アミノ酪酸又はL-オルニチンである、［１］〜［４］のいずれか一項に記載の乳酸菌。
［６］D-アミノ酸がD-アスパラギン酸である、［５］に記載の乳酸菌。
［７］［１］〜［６］のいずれか一項に記載の乳酸菌を培養するステップを含む、非タンパク性アミノ酸の製造方法。
［８］前記培養を培地中又は食品原料中で行う、［７］に記載の製造方法。
［９］前記ステップによって得られた培養物から、非タンパク性アミノ酸を分離するステップを更に含む、［７］に記載の製造方法。
［１０］［７］〜［９］のいずれか一項に記載の製造方法による製造物を含有した組成物。
［１１］医薬組成物、医薬部外組成物、化粧料組成物又は食品組成物である、［１０］に記載の組成物。
［１２］以下のステップ（１）〜（４）を含む、非タンパク性アミノ酸を高生産する乳酸菌のスクリーニング方法：
（１）すぐき漬けから生菌を分離するステップ；
（２）分離した生菌を培養するステップ；
（３）生じたコロニーから菌を採取するステップ；
（４）採取した菌の非タンパク性アミノ酸生産能を評価し、非タンパク性アミノ酸生産能が高い菌を選抜するステップ。
［１３］非タンパク性アミノ酸がD-アミノ酸、γ-アミノ酪酸又はL-オルニチンである、［１２］に記載のスクリーニング方法。
［１４］D-アミノ酸がD-アスパラギン酸である、［１３］に記載のスクリーニング方法。
［１５］ステップ（２）の培養を好気的条件下又は嫌気的条件下で行う、［１２］〜［１４］のいずれか一項に記載のスクリーニング方法。
The present invention listed below is mainly based on the above results.
[1] A lactic acid bacterium that highly produces non-protein amino acids, which is Lactobacillus brevis or Lactobacillus fermentum.
[2] The lactic acid bacterium according to [1], wherein the accession number is NITE P-1169 or NITE P-1171.
[3] The lactic acid bacterium according to [1], wherein the sequence of the 16S ribosomal RNA gene includes the sequence shown in any one of SEQ ID NOs: 1 to 6.
[4] The lactic acid bacterium according to any one of [1] to [3], which is derived from immediately pickled.
[5] The lactic acid bacterium according to any one of [1] to [4], wherein the non-protein amino acid is D-amino acid, γ-aminobutyric acid, or L-ornithine.
[6] The lactic acid bacterium according to [5], wherein the D-amino acid is D-aspartic acid.
[7] A method for producing a non-protein amino acid, comprising the step of culturing the lactic acid bacterium according to any one of [1] to [6].
[8] The production method according to [7], wherein the culture is performed in a medium or a food material.
[9] The production method according to [7], further comprising a step of separating non-protein amino acids from the culture obtained by the step.
[10] A composition containing a product produced by the production method according to any one of [7] to [9].
[11] The composition according to [10], which is a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, or a food composition.
[12] A screening method for lactic acid bacteria that highly produce non-protein amino acids, including the following steps (1) to (4):
(1) a step of separating live bacteria from pickles;
(2) culturing the separated viable bacteria;
(3) collecting bacteria from the resulting colonies;
(4) A step of evaluating the non-protein amino acid-producing ability of the collected bacteria and selecting bacteria having a high non-protein amino acid-producing ability.
[13] The screening method according to [12], wherein the non-protein amino acid is D-amino acid, γ-aminobutyric acid or L-ornithine.
[14] The screening method according to [13], wherein the D-amino acid is D-aspartic acid.
[15] The screening method according to any one of [12] to [14], wherein the culture in step (2) is performed under an aerobic condition or an anaerobic condition.
＜新規乳酸菌＞
本発明の第１の局面は非タンパク性アミノ酸の生産能に優れた新規乳酸菌を提供する。「非タンパク性アミノ酸」とは、タンパク質を構成するアミノ酸以外のアミノ酸のことである。本発明では特にD-アミノ酸（例えばD-アラニン、D-アスパラギン酸、D-グルタミン酸）、GABA及びL-オルニチンに注目し、これらのアミノ酸の中の少なくとも一つを高生産する乳酸菌を提供する。後述の実施例に示す通り、本発明者らは非タンパク性アミノ酸の生産能に優れた６種の乳酸菌を単離することに成功した。以下、各乳酸菌の主な特徴を示す。
<New lactic acid bacteria>
The first aspect of the present invention provides a novel lactic acid bacterium excellent in the ability to produce non-protein amino acids. A “non-protein amino acid” is an amino acid other than amino acids that constitute a protein. In particular, the present invention focuses on D-amino acids (for example, D-alanine, D-aspartic acid, D-glutamic acid), GABA and L-ornithine, and provides lactic acid bacteria that produce at least one of these amino acids at a high level. As shown in Examples described later, the present inventors have succeeded in isolating six types of lactic acid bacteria that are excellent in the ability to produce non-protein amino acids. The main features of each lactic acid bacterium are shown below.
（１）乳酸菌１ｂ
好気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・ブレビス（Lactobacillus brevis）であると同定された。乳酸菌１ｂの16S rDNAの一部の塩基配列を配列番号１に示す。乳酸菌１ｂは特にGABA、D-アラニン及びD-アスパラギン酸を高生産することができ、これらのアミノ酸を高濃度で培養液中に蓄積する。D-アラニンの生産能は極めて高い。乳酸菌１ｂは以下の通り所定の寄託機関に寄託されており、容易に入手可能である。尚、これまでにGABAを蓄積するラクトバチルス・ブレビスの報告はあるが、D-アスパラギン酸を蓄積するものは報告されていない。
寄託機関：独立行政法人製品評価技術基盤機構 特許微生物寄託センター（〒292-0818 日本国千葉県木更津市かずさ鎌足２−５−８）
寄託日：２０１１年１２月１日
受託番号：ＮＩＴＥ Ｐ−１１６９
(1) Lactic acid bacteria 1b
It was a lactic acid bacterium found by culture and screening under aerobic conditions and was identified as Lactobacillus brevis from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of lactic acid bacterium 1b is shown in SEQ ID NO: 1. Lactic acid bacteria 1b can particularly produce high GABA, D-alanine and D-aspartic acid, and accumulate these amino acids in the culture medium at a high concentration. The ability to produce D-alanine is extremely high. Lactic acid bacteria 1b are deposited with a predetermined depository as follows and are easily available. To date, there has been a report of Lactobacillus brevis that accumulates GABA, but none of D-aspartic acid accumulates.
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date: December 1, 2011 Deposit number: NITE P-1169
（２）乳酸菌４ｂ
好気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・ブレビス（Lactobacillus brevis）であると同定された。乳酸菌４ｂの16S rDNAの一部の塩基配列を配列番号２に示す。乳酸菌４ｂは特にD-アラニンを高生産することができ、当該アミノ酸を高濃度で培養液中に蓄積する。
(2)
It was a lactic acid bacterium found by culture and screening under aerobic conditions and was identified as Lactobacillus brevis from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of
（３）乳酸菌４ｃ
好気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・プランタラム（Lactobacillus plantarum）であると同定された。乳酸菌４ｃの16S rDNAの一部の塩基配列を配列番号３に示す。乳酸菌４ｃは特にL-オルニチンを高生産することができ、当該アミノ酸を高濃度で培養液中に蓄積する。
(3)
It was a lactic acid bacterium found by culture and screening under aerobic conditions, and was identified as Lactobacillus plantarum from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of
（４）乳酸菌２ｅ
嫌気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・ブレビス（Lactobacillus brevis）であると同定された。乳酸菌２ｅの16S rDNAの一部の塩基配列を配列番号４に示す。乳酸菌２ｅは特にGABAを高生産することができ、当該アミノ酸を高濃度で培養液中に蓄積する。
(4)
It was a lactic acid bacterium found by culture and screening under anaerobic conditions and was identified as Lactobacillus brevis from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of
（５）乳酸菌２ｆ
嫌気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・ブレビス（Lactobacillus brevis）であると同定された。乳酸菌２ｆの16S rDNAの一部の塩基配列を配列番号５に示す。乳酸菌２ｆは特にGABA、D-アラニン、D-アスパラギン酸、D-グルタミン酸及びL-オルニチンを高生産することができ、これらのアミノ酸を高濃度で培養液中に蓄積する。GABA、D-アラニン及びL-オルニチンの生産能は極めて高い。
(5)
It was a lactic acid bacterium found by culture and screening under anaerobic conditions and was identified as Lactobacillus brevis from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of
（６）乳酸菌４ｆ
嫌気的条件下での培養、スクリーニングによって見出された乳酸菌であり、16S rRNA遺伝子（16S rDNA）の解析結果より、ラクトバチルス・ファーメンタム（Lactobacillus fermentum）であると同定された。乳酸菌４ｆの16S rDNAの一部の塩基配列を配列番号６に示す。乳酸菌４ｆは特にD-アスパラギン酸、D-グルタミン酸及びL-オルニチンを高生産することができ、これらのアミノ酸を高濃度で培養液中に蓄積する。D-グルタミン酸とL-オルニチンの生産能は極めて高い。乳酸菌４ｆは以下の通り所定の寄託機関に寄託されており、容易に入手可能である。
寄託機関：独立行政法人製品評価技術基盤機構 特許微生物寄託センター（〒292-0818 日本国千葉県木更津市かずさ鎌足２−５−８）
寄託日：２０１１年１２月１日
受託番号：ＮＩＴＥ Ｐ−１１７１
(6)
It was a lactic acid bacterium found by culture and screening under anaerobic conditions and was identified as Lactobacillus fermentum from the analysis result of 16S rRNA gene (16S rDNA). A partial base sequence of 16S rDNA of
Depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan 292-0818)
Deposit date: December 1, 2011 Deposit number: NITE P-1171
D-アミノ酸の生産能の程度（高低）については、対応するL-アミノ酸の生産能を基準として表現することができる。例えば、最も高いD-アラニン生産能を示す乳酸菌２ｆは、L-アラニンの生産量の６０％以上に相当する生産量でD-アラニンを生産することができる（後述の実施例に示す培養条件及び測定条件の場合）。同様に、最も高いD-アスパラギン酸生産能を示す乳酸菌４ｆは、L-アスパラギン酸の生産量の５０％以上に相当する生産量でD-アスパラギン酸を生産することができる（後述の実施例に示す培養条件及び測定条件の場合）。また、最も高いD-グルタミン酸生産能を示す乳酸菌４ｆは、L-グルタミン酸の生産量の７５％以上に相当する生産量でD-グルタミン酸を生産することができる（後述の実施例に示す培養条件及び測定条件の場合）。
The degree of D-amino acid production ability (high or low) can be expressed based on the production ability of the corresponding L-amino acid. For example,
尚、上記６種の乳酸菌についてD-アスパラギン酸生産能、GABA生産能及びL-オルニチン生産能を比較すると次の通りである。
（１）D-アスパラギン酸の生産能
乳酸菌４ｆ＞乳酸菌１ｂ＞乳酸菌２ｆ＞＞乳酸菌４ｂ，乳酸菌４ｃ，乳酸菌２ｅ
（２）GABAの生産能
乳酸菌２ｆ＞乳酸菌１ｂ＞乳酸菌２ｅ＞＞乳酸菌４ｂ，乳酸菌４ｃ，乳酸菌４ｆ
（３）L-オルニチンの生産能
乳酸菌４ｆ＞乳酸菌２ｆ＞乳酸菌４ｃ＞＞乳酸菌１ｂ，乳酸菌４ｂ，乳酸菌２ｅ
In addition, it is as follows when D-aspartic acid production ability, GABA production ability, and L-ornithine production ability are compared about said 6 types of lactic acid bacteria.
(1) Production ability of D-aspartic acid Lactic
(2) GABA production ability
(3) L-ornithine production ability
後述の実施例に示す通り、本発明の乳酸菌は「すぐき漬け」から単離された。従って、「すぐき漬け由来である」との要件によって、本発明の乳酸菌を更に特徴付けることができる。すぐき漬けは、アブラナ科の二年草である「すぐき菜」を塩で漬け込み乳酸発酵させる、京都の伝統的な漬け物である。柴漬、千枚漬と並んで京都の三大漬物と呼ばれている。通常、室や樽の中に住み着いている乳酸菌による発酵により独特の風味が形成される。 As shown in the below-mentioned Examples, the lactic acid bacteria of the present invention were isolated from “Ikki-zuke”. Therefore, the lactic acid bacterium of the present invention can be further characterized by the requirement of “derived from immediately pickled”. Immediately pickled is a traditional Kyoto pickle that is made from a cruciferous biennial plant called “kikina”, soaked in salt and fermented with lactic acid. Along with Shiba-zuke and Sen-zuke, it is called the three major pickles in Kyoto. Usually, a unique flavor is formed by fermentation by lactic acid bacteria living in a room or barrel.
本発明の乳酸菌は、後述の実施例に示すスクリーニング方法ないし単離方法により、或いは諸条件（出発材料、生育状態など）に応じてこれらの方法に必要な修正を加えた方法により、単離した状態に調製することができる。また、寄託された乳酸菌については、所定の手続によって、寄託機関から分譲を受けることができる。 The lactic acid bacteria of the present invention were isolated by the screening method or isolation method shown in the examples described later, or by a method in which these methods were modified as necessary according to various conditions (starting material, growth state, etc.). Can be prepared in a state. In addition, the deposited lactic acid bacteria can be sold from the depository in accordance with a predetermined procedure.
＜新規乳酸菌の用途＞
本発明の第２の局面は本発明の乳酸菌の用途に関する。第１の用途として非タンパク性アミノ酸の製造方法が提供される。本発明の製造方法では本発明の乳酸菌を培養するステップを行う。本発明の乳酸菌が生育可能であり、且つ培養に伴い非タンパク性アミノ酸が生産（蓄積）される限りにおいて培養条件は特に限定されない。以下、培養に使用される培地及びその他の培養条件について説明する。
<Uses of new lactic acid bacteria>
The second aspect of the present invention relates to the use of the lactic acid bacteria of the present invention. As a first use, a method for producing a non-protein amino acid is provided. In the production method of the present invention, the step of culturing the lactic acid bacteria of the present invention is performed. The culture conditions are not particularly limited as long as the lactic acid bacteria of the present invention can grow and non-protein amino acids are produced (accumulated) with the culture. Hereinafter, the culture medium used for culture and other culture conditions will be described.
乳酸菌用の公知の培地を使用することができる。培地の例を挙げると、MRS培地（例えば関東化学株式会社が提供する）、ロゴサ培地（例えば関東化学株式会社が提供する）、オレンジセーラム培地（例えば関東化学株式会社が提供する）、LBS培地（例えば和光純薬工業株式会社が提供する）、GYP培地（例えば、1% Glucose, 1% Yeast extract, 0.5% Polypeptone, 0.2% Na-acetate・3H2O, 20ppm MgSO4・7H2O, 1ppm MnSO4・4H2O, 1ppm FeSO4・7H2O, 1ppm NaCl, 50ppm Tween 80の組成からなるもの）、FYP培地（例えば、1% fructose, 1% Yeast extract, 0.5% Polypeptone, 0.2% Na-acetate・3H2O, 20ppm MgSO4・7H2O, 1ppm MnSO4・4H2O, 1ppm FeSO4・7H2O, 1ppm NaCl, 50ppm Tween 80の組成からなるもの）、GAM培地（日水製薬株式会社製）である。 A known medium for lactic acid bacteria can be used. Examples of the medium include MRS medium (for example, provided by Kanto Chemical Co., Inc.), Rogosa medium (for example, provided by Kanto Chemical Co., Ltd.), Orange Salem medium (for example, provided by Kanto Chemical Co., Ltd.), LBS medium ( For example, provided by Wako Pure Chemical Industries, Ltd., GYP medium (eg 1% Glucose, 1% Yeast extract, 0.5% Polypeptone, 0.2% Na-acetate · 3H 2 O, 20ppm MgSO 4 · 7H 2 O, 1ppm MnSO 4 · 4H 2 O, 1ppm FeSO 4 · 7H 2 O, 1ppm NaCl, 50ppm Tween 80), FYP medium (eg 1% fructose, 1% Yeast extract, 0.5% Polypeptone, 0.2% Na-acetate・ 3H 2 O, 20ppm MgSO 4・ 7H 2 O, 1ppm MnSO 4・ 4H 2 O, 1ppm FeSO 4・ 7H 2 O, 1ppm NaCl, 50ppm Tween 80), GAM medium (Nissui Pharmaceutical Co., Ltd.) Made).
目的物（非タンパク性アミノ酸）の生産を促すため、培地中にアルギニン（オルニチンはアルギニンデイミナーゼ経路によりアルギニンからシトルリンを経て合成されていると予想される）、L-アスパラギン酸（アスパラギン酸ラセマーゼの基質）等を添加するとよい。目的物がGABAの場合には、グルタミン酸脱炭酸酵素の基質であるグルタミン酸又はその塩類を添加するとよい。 Arginine (ornithine is expected to be synthesized from arginine via citrulline via the arginine deiminase pathway), L-aspartic acid (aspartate racemase) Substrate) and the like may be added. When the target product is GABA, glutamic acid or a salt thereof, which is a substrate for glutamic acid decarboxylase, may be added.
培地に乳酸菌を接種して培養を開始するが、必要に応じて前培養を行っても良い。培養に供する乳酸菌として２種類以上の乳酸菌を併用することにしてもよい。培養は好気的条件下又は嫌気的条件下で行われる。例えば、乳酸菌１ｂ、乳酸菌４ｂ又は乳酸菌４ｃを培養するのであれば好気的条件を採用することが好ましく、他方、乳酸菌２ｅ、乳酸菌２ｆ又は乳酸菌４ｆを培養するのであれば嫌気的条件を採用することが好ましい。
The culture is inoculated with lactic acid bacteria in the medium, but pre-culture may be performed as necessary. Two or more types of lactic acid bacteria may be used in combination as lactic acid bacteria to be cultured. Culturing is performed under aerobic conditions or anaerobic conditions. For example, it is preferable to employ aerobic conditions when cultivating lactic acid bacteria 1b,
培養温度は、良好な生育を促すために、例えば15℃〜40℃であり、好ましくは25℃〜37℃である。培養液のpHは例えば4.8〜6.8であり、好ましくは6.0〜6.5である。培養時間については、目的とする収量、培養条件、培養スケールなどを考慮して決定すればよいが、例えば20時間〜3日である。 The culture temperature is, for example, 15 ° C. to 40 ° C., preferably 25 ° C. to 37 ° C., in order to promote good growth. The pH of the culture solution is, for example, 4.8 to 6.8, preferably 6.0 to 6.5. The culture time may be determined in consideration of the target yield, culture conditions, culture scale, etc., and is, for example, 20 hours to 3 days.
以上の培養によって培養液中又は菌体内に目的物が生産（蓄積）される。生産された目的物は必要に応じて回収、精製、濃縮、乾燥などの処理に供される。培養液から目的物を回収する場合には、例えば培養上清をろ過、遠心処理等することによって菌体等の不溶物を除去した後、限外ろ過膜による濃縮、硫安沈殿等の塩析、透析、各種クロマトグラフィーなどを適宜組み合わせて分離、精製を行うことにより目的物を得ることができる。他方、菌体内から回収する場合には、例えば菌体を加圧処理、超音波処理などによって破砕した後、上記と同様に分離、精製を行うことにより目的物を得ることができる。尚、ろ過、遠心処理などによって予め培養液から菌体を回収した後、上記一連の工程（菌体の破砕、分離、精製）を行ってもよい。 By the above culture, the target product is produced (accumulated) in the culture solution or in the fungus body. The produced object is subjected to treatments such as recovery, purification, concentration, and drying as necessary. When recovering the target product from the culture solution, for example, filtering the culture supernatant, removing insoluble matter such as cells by centrifugation, etc., followed by concentration by ultrafiltration membrane, salting out such as ammonium sulfate precipitation, The desired product can be obtained by performing separation and purification by appropriately combining dialysis, various chromatography and the like. On the other hand, when recovering from the bacterial cells, for example, the bacterial cells are crushed by pressure treatment, ultrasonic treatment, etc., and then separated and purified in the same manner as described above to obtain the target product. In addition, after collect | recovering a microbial cell from a culture solution previously by filtration, a centrifugation process, etc., you may perform said series of processes (crushing, isolation | separation, refinement | purification of a microbial cell).
以上のように回収等の処理を行えば、純度の高い目的物が得られる。一方、本発明の一態様では、目的物が蓄積された培養液をそのまま、或いはろ過、濃縮及び／又は乾燥させた後、各種利用に供する。 If the process such as recovery is performed as described above, a high-purity target product can be obtained. On the other hand, in one embodiment of the present invention, the culture solution in which the target product is accumulated is used as it is or after being filtered, concentrated and / or dried, for various uses.
上記製造方法では、培地に乳酸菌を接種して培養することにしたが、食品原料中で培養を行うことにしてもよい。当該方法によれば、非タンパク性アミノ酸を含有する又は非タンパク性アミノ酸の含量が増大した食品（例えば、ヨーグルト、チーズ、乳酸菌飲料などの発酵乳食品、ビール、発泡酒などの酒類、納豆、漬け物など）又は食品素材を得ることができる。 In the production method described above, the culture medium is inoculated with lactic acid bacteria, but the culture may be performed in a food material. According to the method, foods containing non-protein amino acids or having an increased content of non-protein amino acids (eg fermented milk foods such as yogurt, cheese, lactic acid bacteria beverages, alcoholic beverages such as beer and sparkling wine, natto, pickles Etc.) or a food material can be obtained.
食品原料は、使用する乳酸菌が生育可能な限り特に限定されない。食品原料の例を挙げると、乳類（生乳、加工乳、ヨーグルト等）、野菜類（トマト、ピーマン、人参、キャベツ、レタス、大根、ほうれん草など）、キノコ類（椎茸、シメジ、エノキダケなど）、果実類（リンゴ、オレンジ、グレープフルーツ、ブドウ、パイナップルなど）、穀類（米、小麦、大麦、ライ麦など）、芋類（ジャガイモ、サツマイモ、ナガイモなど）、豆類（大豆、エンドウ、小豆など）、肉類（牛肉、豚肉、鶏肉、羊肉等）、魚介類（アジ、鯖、サンマ、マグロ、アサリ、シジミなど）である。これらの食品原料は、必要に応じて、洗浄、粉砕、圧搾、加熱、混合等の前処理に供される。二種類以上の食品原料を組み合わせて用いることにしてもよい。乳酸菌の接種に際して或いは培養中において、乳酸菌の生育又は非タンパク性アミノ酸の生産を促す成分（糖、ビタミン、塩類、各種エキスなど）を添加することにしてもよい。乳酸菌の接種後の培養については、専用の培養工程を設けても良いし、通常の加工プロセスの中で培養が行われるようにしてもよい。 The food material is not particularly limited as long as the lactic acid bacteria to be used can grow. Examples of food ingredients include milk (raw milk, processed milk, yogurt, etc.), vegetables (tomatoes, peppers, carrots, cabbage, lettuce, radish, spinach, etc.), mushrooms (shiitake, shimeji, enoki mushrooms, etc.), Fruits (apples, oranges, grapefruits, grapes, pineapples, etc.), cereals (rice, wheat, barley, rye, etc.), potatoes (potatoes, sweet potatoes, yams, etc.), beans (soybeans, peas, red beans, etc.), meats ( Beef, pork, chicken, lamb, etc.) and seafood (aji, salmon, saury, tuna, clams, clams, swordfish, etc.). These food materials are subjected to pretreatment such as washing, crushing, pressing, heating, and mixing as necessary. Two or more kinds of food ingredients may be used in combination. At the time of inoculation with lactic acid bacteria or during culturing, ingredients that promote the growth of lactic acid bacteria or the production of non-protein amino acids (sugars, vitamins, salts, various extracts, etc.) may be added. About culture | cultivation after inoculation of lactic acid bacteria, you may provide a culture process for exclusive use, and you may make it culture | cultivate in a normal processing process.
本発明の製造方法によって食品素材が得られる場合のその最終形態は特に限定されない。例えば、粉末状、顆粒状、粒状、ゲル状、ペースト状、液状又は固形状に成形される。 The final form when the food material is obtained by the production method of the present invention is not particularly limited. For example, it is formed into powder, granule, granule, gel, paste, liquid or solid.
本発明の製造方法によって得られた非タンパク性アミノ酸は各種用途に利用可能である。そこで本願は、更なる発明として、本発明の製造方法によって得られた非タンパク性アミノ酸を含有する組成物を提供する。本発明の組成物の用途は特に限定されないが、好ましくは、医薬、医薬部外品、化粧料又は食品である。即ち、本発明は好ましい態様として、本発明の製造方法によって得られた非タンパク性アミノ酸を有効成分として含有する医薬組成物、医薬部外品組成物、化粧料組成物及び食品組成物を提供する。 The non-protein amino acid obtained by the production method of the present invention can be used for various applications. Therefore, the present application provides, as a further invention, a composition containing a non-protein amino acid obtained by the production method of the present invention. The use of the composition of the present invention is not particularly limited, but is preferably a medicine, a quasi-drug, a cosmetic, or a food. That is, the present invention provides, as a preferred embodiment, a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, and a food composition containing the non-protein amino acid obtained by the production method of the present invention as an active ingredient. .
本発明の医薬組成物及び医薬部外品組成物の製剤化は常法に従って行うことができる。製剤化する場合には、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を含有させることができる。賦形剤としては乳糖、デンプン、ソルビトール、D-マンニトール、白糖等を用いることができる。崩壊剤としてはデンプン、カルボキシメチルセルロース、炭酸カルシウム等を用いることができる。緩衝剤としてはリン酸塩、クエン酸塩、酢酸塩等を用いることができる。乳化剤としてはアラビアゴム、アルギン酸ナトリウム、トラガント等を用いることができる。懸濁剤としてはモノステアリン酸グリセリン、モノステアリン酸アルミニウム、メチルセルロース、カルボキシメチルセルロース、ヒドロキシメチルセルロース、ラウリル硫酸ナトリウム等を用いることができる。無痛化剤としてはベンジルアルコール、クロロブタノール、ソルビトール等を用いることができる。安定剤としてはプロピレングリコール、アスコルビン酸等を用いることができる。保存剤としてはフェノール、塩化ベンザルコニウム、ベンジルアルコール、クロロブタノール、メチルパラベン等を用いることができる。防腐剤としては塩化ベンザルコニウム、パラオキシ安息香酸、クロロブタノール等と用いることができる。 The pharmaceutical composition and quasi-drug composition of the present invention can be formulated according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
製剤化する場合の剤型も特に限定されず、例えば錠剤、散剤、細粒剤、顆粒剤、カプセル剤、シロップ剤、注射剤、外用剤、及び座剤などとして本発明の医薬組成物又は医薬部外品組成物を提供できる。 The dosage form in the case of formulating is also not particularly limited, and the pharmaceutical composition or pharmaceutical of the present invention can be used as tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, suppositories, etc. An quasi-drug composition can be provided.
本発明の医薬組成物には、期待される治療効果や予防効果を得るために必要な量（即ち治療上有効量）の有効成分が含有される。同様に本発明の医薬部外品組成物には、期待される改善効果や予防効果等を得るために必要な量の有効成分が含有される。本発明の医薬組成物又は医薬部外品組成物に含まれる有効成分量は一般に剤型や形態によって異なるが、所望の投与量を達成できるように有効成分量を例えば0.1重量％〜99重量％の範囲内で設定する。 The pharmaceutical composition of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect or preventive effect (that is, a therapeutically effective amount). Similarly, the quasi-drug composition of the present invention contains an active ingredient in an amount necessary for obtaining the expected improvement effect, prevention effect and the like. The amount of the active ingredient contained in the pharmaceutical composition or quasi-drug composition of the present invention generally differs depending on the dosage form and form, but the amount of the active ingredient is, for example, 0.1% by weight to 99% by weight so that a desired dosage can be achieved. Set within the range.
本発明の医薬組成物及び医薬部外品組成物はその剤型・形態に応じて経口又は非経口（静脈内、動脈内、皮下、筋肉、又は腹腔内注射、経皮、経鼻、経粘膜、塗布など）で対象に適用される。ここでの「対象」は特に限定されず、ヒト及びヒト以外の哺乳動物（ペット動物、家畜、実験動物を含む。具体的には例えばマウス、ラット、モルモット、ハムスター、サル、ウシ、ブタ、ヤギ、ヒツジ、イヌ、ネコ、ニワトリ、ウズラ等である）を含む。好ましい一態様では、適用対象はヒトである。 The pharmaceutical composition and quasi-drug composition of the present invention are oral or parenteral (intravenous, intraarterial, subcutaneous, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal depending on the dosage form and form. , Application, etc.). The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the application subject is a human.
本発明の医薬組成物及び医薬部外品組成物の投与量・使用量は、期待される効果が得られるように設定される。有効な投与量の設定においては一般に適用対象の症状、年齢、性別、体重などが考慮される。尚、当業者であればこれらの事項を考慮して適当な投与量を設定することが可能である。投与スケジュールとしては例えば一日一回〜数回、二日に一回、或いは三日に一回などを採用できる。投与スケジュールの作成においては、適用対象の症状や有効成分の効果持続時間などを考慮することができる。 The dosage and usage of the pharmaceutical composition and quasi-drug composition of the present invention are set so as to obtain the expected effect. In setting an effective dose, the symptom, age, sex, weight, etc. of the subject of application are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the symptom of the application target, the duration of effect of the active ingredient, and the like can be considered.
上記の通り本発明の一態様は、本発明の製造方法によって得られた非タンパク性アミノ酸を有効成分として含有する化粧料組成物である。例えば、本発明の化粧料組成物は、本発明の製造方法によって得られた非タンパク性アミノ酸と、化粧料に通常使用される成分・基材（例えば、各種油脂、ミネラルオイル、ワセリン、スクワラン、ラノリン、ミツロウ、変性アルコール、パルミチン酸デキストリン、グリセリン、グリセリン脂肪酸エステル、エチレングリコール、パラベン、カンフル、メントール、各種ビタミン、酸化亜鉛、酸化チタン、安息香酸、エデト酸、カミツレ油、カラギーナン、キチン末、キトサン、香料、着色料など）を配合することによって得ることができる。 As described above, one embodiment of the present invention is a cosmetic composition containing a non-protein amino acid obtained by the production method of the present invention as an active ingredient. For example, the cosmetic composition of the present invention comprises a non-protein amino acid obtained by the production method of the present invention, and ingredients / bases usually used in cosmetics (for example, various oils and fats, mineral oil, petrolatum, squalane, Lanolin, beeswax, denatured alcohol, dextrin palmitate, glycerin, glycerin fatty acid ester, ethylene glycol, paraben, camphor, menthol, various vitamins, zinc oxide, titanium oxide, benzoic acid, edetic acid, chamomile oil, carrageenan, chitin powder, chitosan , Fragrance, colorant, etc.).
化粧料組成物の形態として、フェイス又はボディー用の乳液、化粧水、クリーム、ローション、エッセンス、オイル、パック、シート、洗浄料などを例示できる。化粧料組成物における非タンパク性アミノ酸の添加量は特に限定されない。例えば0.1重量％〜10重量％となるように非タンパク性アミノ酸を添加するとよい。 Examples of the cosmetic composition include emulsions for face or body, lotions, creams, lotions, essences, oils, packs, sheets, and cleaning agents. The addition amount of the non-protein amino acid in the cosmetic composition is not particularly limited. For example, a non-protein amino acid may be added so as to be 0.1% by weight to 10% by weight.
上記の通り本発明の一態様は、本発明の製造方法によって得られた非タンパク性アミノ酸を含有する食品組成物である。本発明での「食品組成物」の例として一般食品（穀類、野菜、食肉、生乳、乳製品（例えばヨーグルト、加工乳、チーズ）などの各種加工食品、菓子類、清涼飲料水、アルコール飲料等）、栄養補助食品（サプリメント、栄養ドリンク等）、食品添加物、愛玩動物用食品、愛玩動物用栄養補助食品を挙げることができる。栄養補助食品又は食品添加物の場合、粉末、顆粒末、タブレット、ペースト、液体等の形状で提供することができる。食品組成物の形態で提供することによって、本発明の有効成分（本発明の製造方法によって得られた非タンパク性アミノ酸）を日常的に摂取したり、継続的に摂取したりすることが容易となる。 As described above, one embodiment of the present invention is a food composition containing a non-protein amino acid obtained by the production method of the present invention. Examples of “food composition” in the present invention include various processed foods such as general foods (cereals, vegetables, meat, raw milk, dairy products (eg yogurt, processed milk, cheese)), confectionery, soft drinks, alcoholic beverages, etc. ), Dietary supplements (supplements, energy drinks, etc.), food additives, pet foods, pet food supplements. In the case of a dietary supplement or food additive, it can be provided in the form of powder, granule powder, tablet, paste, liquid or the like. By providing it in the form of a food composition, it is easy to ingest the active ingredient of the present invention (non-protein amino acid obtained by the production method of the present invention) on a daily basis or continuously. Become.
本発明の食品組成物には、治療的又は予防的効果が期待できる量の有効成分が含有されることが好ましい。添加量は、それが使用される対象となる者の病状、健康状態、年齢、性別、体重などを考慮して定めることができる。 The food composition of the present invention preferably contains an active ingredient in an amount that can be expected to have a therapeutic or prophylactic effect. The amount added can be determined in consideration of the medical condition, health status, age, sex, weight, etc. of the person to whom it is used.
＜乳酸菌のスクリーニング方法＞
本発明の第３の局面は、すぐき漬けが有用乳酸菌の宝庫であるとの知見に基づくスクリーニング方法を提供する。本発明のスクリーニング方法では以下のステップ（１）〜（４）を行い、非タンパク性アミノ酸を高生産する乳酸菌をスクリーニングする。
（１）すぐき漬けから生菌を分離するステップ
（２）分離した生菌を培養するステップ
（３）生じたコロニーから菌を採取するステップ
（４）採取した菌の非タンパク性アミノ酸生産能を評価し、非タンパク性アミノ酸生産能が高い菌を選抜するステップ
<Screening method for lactic acid bacteria>
The third aspect of the present invention provides a screening method based on the finding that pickling is a treasure trove of useful lactic acid bacteria. In the screening method of the present invention, the following steps (1) to (4) are performed to screen for lactic acid bacteria that produce a high amount of non-protein amino acids.
(1) Step for separating live bacteria from pickles (2) Step for culturing the separated live bacteria (3) Step for collecting bacteria from the resulting colonies (4) Evaluation of the ability of the collected bacteria to produce non-protein amino acids And select bacteria with high ability to produce non-protein amino acids
ステップ（１）では、すぐき漬けを用意し、そこに含まれる生菌を分離する。現在、各種すぐき漬けが市販されている。本発明のスクリーニング方法では、このような市販のすぐき漬けを用いることができる。市販品ではなく、新たに製造したものを用いることにしてもよい。スクリーニング効率を高めるために、由来（典型的には製造元）の異なる２種類以上、好ましくは５種類以上、更に好ましくは１０種類以上のすぐき漬けを用意し、各々から生菌を分離するとよい。 In step (1), pickles are prepared immediately, and live bacteria contained therein are separated. Currently, various pickles are on the market. In the screening method of the present invention, such commercially available pickles can be used. You may decide to use what was newly manufactured instead of a commercial item. In order to increase the screening efficiency, two or more, preferably five or more, more preferably ten or more types of pickles that have different origins (typically manufacturers) are prepared, and live bacteria are separated from each.
すぐき漬けから生菌を分離するためには、例えば、用意したすぐき漬けと水（好ましくは滅菌水）を混合した後、滅菌水の一部を採取すればよい。すぐき漬けを水（好ましくは滅菌水）ですすぐことで付着している生菌を分離することにしてもよい。分離操作の後、顕微鏡観察などによって生菌の存在を確認することが好ましい。 In order to separate viable bacteria from the pickles, for example, the prepared pickles and water (preferably sterilized water) are mixed, and then a part of the sterilized water may be collected. Immediately pickling may be performed with water (preferably sterilized water) to separate viable bacteria attached thereto. It is preferable to confirm the presence of viable bacteria by microscopic observation after the separation operation.
続いて、分離した生菌を培養する（ステップ（２））。ここでの培養条件は、乳酸菌が生育可能な限り特に限定されず、本発明の第１の局面における乳酸菌の培養条件に準ずる。即ち、乳酸菌を選択的に培養するために、MRS培地、ロゴサ培地、オレンジセーラム培地、LBS培地等の乳酸菌用の培地を用い、乳酸菌の生育に適した温度条件及びpH条件で培養する。培養時間は例えば2時間〜48時間とする。好気的条件、嫌気的条件のいずれを採用してもよい。好気的条件を採用した培養と、嫌気的条件を採用した培養を並行して行うことにすれば、より多種多様な乳酸菌がスクリーニングされ得る。 Subsequently, the separated viable bacteria are cultured (step (2)). The culture conditions here are not particularly limited as long as lactic acid bacteria can grow, and conform to the culture conditions for lactic acid bacteria in the first aspect of the present invention. That is, in order to selectively cultivate lactic acid bacteria, a medium for lactic acid bacteria such as MRS medium, Rogosa medium, Orange Salem medium, LBS medium, etc. is used and cultured under temperature conditions and pH conditions suitable for the growth of lactic acid bacteria. The culture time is, for example, 2 hours to 48 hours. Either an aerobic condition or an anaerobic condition may be adopted. If culture using aerobic conditions and culture using anaerobic conditions are performed in parallel, a wider variety of lactic acid bacteria can be screened.
次に、培養によって生じたコロニーから菌を採取する（ステップ（３））。複数のコロニーが認められる場合には、コロニーの形状、色、サイズ等を考慮し、菌種が異なると予想される複数のコロニーについて、それぞれ菌の採取を行うとよい。 Next, a microbe is extract | collected from the colony produced by culture | cultivation (step (3)). In the case where a plurality of colonies are observed, taking into account the shape, color, size, etc. of the colonies, it is preferable to collect bacteria for each of the plurality of colonies expected to have different bacterial species.
続いて、採取した菌の非タンパク性アミノ酸生産能を評価し、非タンパク性アミノ酸生産能が高い菌を選抜する（ステップ（４））。ここでの非タンパク性アミノ酸はD-アミノ酸、γ-アミノ酪酸又はL-オルニチンである。D-アミノ酸の種類は特に限定されない。好ましいD-アミノ酸の一つとしてD-アスパラギン酸を例示することができる。 Subsequently, the ability of the collected bacteria to produce non-protein amino acids is evaluated, and bacteria having a high ability to produce non-protein amino acids are selected (step (4)). The non-protein amino acid here is D-amino acid, γ-aminobutyric acid or L-ornithine. The type of D-amino acid is not particularly limited. One preferred D-amino acid is D-aspartic acid.
D-アミノ酸の定量は公知の方法で行うことができる。D-アミノ酸の定量方法として、D-アミノ酸酸化酵素（DAO： EC 1.4.3.3）やD-アスパラギン酸酸化酵素（DDO： EC 1.4.3.1）等のD-アミノ酸特異的な酵素を利用した方法、クロマトグラフィー又は電気泳動によりD-アミノ酸を分離して定量する方法（Nimura, N., Fujiwara, T., Watanabe, A., Sekine, M., Furuchi, T., Yohda, M., Yamagishi, A., Oshima, T., & Homma, H. (2003) Anal. Biochem.,315,262-269.; Long, Z., Nimura, N., Adachi, M., Sekine, M., Hanai, T., Kubo, H., & Homma, H.(2001)J. Chromatogr. B ., 761, 99-106.; Song, Y., Feng, Y., LeBlanc, M.H., Zhao, S., & Liu, Y. M. (2006)Anal. Chem.,78,8121-8128.; Miao, H., Rubakhin, S.S., & Sweedler, J.V. (2005) Anal. Chem.,77,7190-7194.等を参照）などが知られている。クロマトグラフィーによりD-アミノ酸を分離して定量する方法としては、高速液体クロマトグラフィーを用いた方法が一般的である。当該方法の一つでは、アミノ酸を蛍光ジアステレオマーへと誘導体化した後、ODSカラムなどを用いてHPLCにて分離・定量する。D-セリンについては、本発明者らの研究グループによって開発された方法（WO/2008/047802）を利用することもできる。GABA及びL-オルニチンの定量についても常法で行えばよく、例えば高速液体クロマトグラフィーを用いた方法を採用すればよい。 D-amino acid can be quantified by a known method. As a method for quantifying D-amino acid, a method utilizing a D-amino acid-specific enzyme such as D-amino acid oxidase (DAO: EC 1.4.3.3) or D-aspartate oxidase (DDO: EC 1.4.3.1), Method for separating and quantifying D-amino acids by chromatography or electrophoresis (Nimura, N., Fujiwara, T., Watanabe, A., Sekine, M., Furuchi, T., Yohda, M., Yamagishi, A ., Oshima, T., & Homma, H. (2003) Anal. Biochem., 315,262-269 .; Long, Z., Nimura, N., Adachi, M., Sekine, M., Hanai, T., Kubo, H., & Homma, H. (2001) J. Chromatogr. B., 761, 99-106 .; Song, Y., Feng, Y., LeBlanc, MH, Zhao, S., & Liu, YM (2006) Anal. Chem., 78, 8121-8128 .; see Miao, H., Rubakhin, SS, & Sweedler, JV (2005) Anal. Chem., 77, 7190-7194. ing. As a method for separating and quantifying D-amino acids by chromatography, a method using high performance liquid chromatography is generally used. In one of the methods, amino acids are derivatized into fluorescent diastereomers and then separated and quantified by HPLC using an ODS column or the like. For D-serine, a method (WO / 2008/047802) developed by our research group can also be used. GABA and L-ornithine may be quantified by conventional methods, for example, a method using high performance liquid chromatography may be employed.
非タンパク性アミノ酸生産能の評価によって選抜された菌の中から、生育性（増殖性）、栄養要求性、培養条件（温度等で極端な条件を要しないこと）等の基準に基づき優良な菌を更に選抜することにしてもよい。 Excellent bacteria based on criteria such as growth (proliferation), auxotrophy, and culture conditions (not requiring extreme conditions such as temperature) among bacteria selected by evaluation of non-protein amino acid productivity May be further selected.
選抜された菌については、その菌種を同定するとよい。菌種の同定は常法で行えばよい。即ち、形成するコロニーの形状・形態、菌体の形状・形態、生育性、栄養要求性、グラム染色性、糖の資化性、酵素活性、16S rRNA遺伝子（16S rDNA）の配列等に基づき、菌種を同定することができる。 About the selected microbe, it is good to identify the bacterial species. Identification of the bacterial species may be performed by a conventional method. That is, based on the shape and form of colonies to be formed, the shape and form of bacterial cells, growth, auxotrophy, Gram staining, sugar assimilation, enzyme activity, 16S rRNA gene (16S rDNA) sequence, etc. Species can be identified.
スクリーニングによって選抜された菌は非タンパク性アミノ酸の生産能に優れるものであり、本発明の乳酸菌と同様の用途に適用可能である。 Bacteria selected by screening have excellent ability to produce non-protein amino acids, and can be applied to the same uses as the lactic acid bacteria of the present invention.
京都の伝統的な漬け物の一つであるすぐき漬けに注目し、非タンパク性アミノ酸の生産能に優れた新規乳酸菌を単離することを試みた。具体的には、京都市内で販売されている約10種のすぐき漬けを収集し、D-アミノ酸の生産能を指標として、乳酸菌を単離した。以下に単離方法（１．）及びD-アミノ酸の定量方法（２．）を示す。 Focusing on pickles, one of the traditional pickles in Kyoto, we tried to isolate a new lactic acid bacterium with excellent non-protein amino acid-producing ability. Specifically, about 10 kinds of pickles sold in Kyoto were collected, and lactic acid bacteria were isolated using D-amino acid production ability as an index. The isolation method (1.) and the D-amino acid quantification method (2.) are shown below.
１．漬物（京漬物、すぐき漬け）からの乳酸菌の単離方法
以下の操作はクリーンベンチ内において無菌的に行った。
(1)漬物0.5g程度および滅菌水500μLをマイクロチューブに入れ混合した。
(2)混合した滅菌水を一部採取し、顕微鏡観察により生菌の存在を確認した。
(3)混合した滅菌水をMRS寒天培地（下記のMRS培地に1.5% agarを添加した寒天培地）に塗布した。1プレートあたりに50〜200個程度コロニーが生える量を目安に（およそ30〜200μL）。
(4)植菌した寒天培地を30℃で好気条件下又は嫌気条件下で24〜48時間培養した。嫌気培養は密封容器を窒素で充填し、嫌気培養パウチ（三菱ガス化学社製のアネロパウチ（登録商標）ケンキ、脱酸素および炭酸ガス放出）を同封して培養した。
(5)寒天培地に生えたシングルコロニーを再度MRS寒天培地に塗布し、シングル化を行った（30℃で好気及び嫌気条件下で24〜48時間培養）。この際、コロニーの形状、色、サイズ等を観察し、菌種が異なると予想されるコロニーを採取した（1プレートから3〜5個程度）。
(6)シングル化したコロニーをMRS液体培地に植え継ぎ、30℃で24時間培養し、グリセロールストックを作成し-80℃で保存した。この際、好気的に単離した菌は振盪培養し、嫌気的に単離した菌は静置培養した。
MRS培地（peptone 10g, meat extract 10g, yeast extract 5g, glucose 20g, Tween80 1g, K2HPO4 2g, sodium acetate 5g, diammonium hydrogencitrate 2g, MgSO4・7H20 0.2g, MnSO4・nH2O 0.05g, distilled water 1 L, pH 6.2)
1. Isolation Method of Lactic Acid Bacteria from Pickles (Kyoto Pickles, Immediate Pickles) The following operations were performed aseptically in a clean bench.
(1) About 0.5 g of pickles and 500 μL of sterilized water were placed in a microtube and mixed.
(2) A part of the mixed sterilized water was collected, and the presence of viable bacteria was confirmed by microscopic observation.
(3) The mixed sterilized water was applied to an MRS agar medium (an agar medium obtained by adding 1.5% agar to the following MRS medium). Using approximately 50 to 200 colonies per plate as a guide (approximately 30 to 200 μL).
(4) The inoculated agar medium was cultured at 30 ° C. under aerobic or anaerobic conditions for 24-48 hours. Anaerobic culture was performed by filling a sealed container with nitrogen and enclosing an anaerobic culture pouch (Anero Pouch (registered trademark), deoxygenated and carbon dioxide released by Mitsubishi Gas Chemical Company).
(5) A single colony grown on an agar medium was again applied to an MRS agar medium to be singled (cultured at 30 ° C. under aerobic and anaerobic conditions for 24-48 hours). At this time, the shape, color, size, and the like of the colony were observed, and colonies expected to have different bacterial species were collected (about 3 to 5 from one plate).
(6) A single colony was inoculated into an MRS liquid medium and cultured at 30 ° C. for 24 hours to prepare a glycerol stock and stored at −80 ° C. At this time, the aerobically isolated bacteria were cultured with shaking, and the anaerobically isolated bacteria were statically cultured.
MRS medium (peptone 10g, meat extract 10g, yeast extract 5g, glucose 20g, Tween80 1g, K 2 HPO 4 2g, sodium acetate 5g, diammonium hydrogencitrate 2g, MgSO 4・ 7H 2 0 0.2g, MnSO 4・ nH 2 O 0.05 g, distilled water 1 L, pH 6.2)
２．単離した乳酸菌のD-アミノ酸生成能の検討
(1)MRS液体培地にて30℃で24時間培養（好気的に単離した菌は振盪培養し、嫌気的に単離した菌は静置培養）した。
(2)培養液を遠心分離（20,000 g, 10 min, 4℃）し、培地と菌体を分離した。
(3)除タンパク質のため、培地に等量の10% TCAを加え、氷上で15分間インキュベートした。その後、遠心分離（20,000 g, 15 min, 4℃）し、上清を得た。
(4)次いで、TCAを除去するためジエチルエーテル処理を3回繰り返した。ジエチルエーテル処理方法は次の通り：サンプルと等量の水飽和ジエチルエーテルを混合した後、5分間攪拌し、遠心分離（10,000 g, 5 min, 4℃）した後上層のジエチルエーテルを除去する。
(5)その後、アミノ酸を蛍光ジアステレオマーへと誘導体化（試薬：BOC-Cys-OHとOPA）した後に、HPLCにて分離・定量する（使用カラムはODSカラム）。
2. Examination of D-amino acid-producing ability of isolated lactic acid bacteria
(1) Cultured at 30 ° C. for 24 hours in an MRS liquid medium (aerobically isolated bacteria were cultured by shaking, and anaerobically isolated bacteria were statically cultured).
(2) The culture solution was centrifuged (20,000 g, 10 min, 4 ° C.) to separate the medium and the cells.
(3) For protein removal, an equal volume of 10% TCA was added to the medium and incubated on ice for 15 minutes. Thereafter, centrifugation (20,000 g, 15 min, 4 ° C.) was performed to obtain a supernatant.
(4) Next, diethyl ether treatment was repeated three times to remove TCA. The diethyl ether treatment method is as follows: After mixing the sample with an equal amount of water-saturated diethyl ether, the mixture is stirred for 5 minutes, centrifuged (10,000 g, 5 min, 4 ° C.), and then the upper layer diethyl ether is removed.
(5) After that, amino acids are derivatized into fluorescent diastereomers (reagents: BOC-Cys-OH and OPA), and then separated and quantified by HPLC (the column used is an ODS column).
以上の方法で新規乳酸菌の単離を試みた結果、特に有用な乳酸菌として、好気培養で３株（１ｂ、４ｂ、４ｃ）、嫌気培養でも３株（２ｅ、２ｆ、４ｆ）を取得できた。各乳酸菌のアミノ酸生産能を比較した結果を図１（好気的に単離した乳酸菌についての比較）及び図２（嫌気的に単離した乳酸菌についての比較）に示す。尚、有用アミノ酸として注目されているGABA及びL-オルニチンの生産能についても比較した。GABA及びL-オルニチンの定量は上記方法（２．）に準じた。 As a result of trying to isolate a new lactic acid bacterium by the above method, 3 strains (1b, 4b, 4c) were obtained by aerobic culture and 3 strains (2e, 2f, 4f) were obtained by anaerobic culture as particularly useful lactic acid bacteria. . FIG. 1 (comparison for an aerobically isolated lactic acid bacterium) and FIG. 2 (comparison for an anaerobically isolated lactic acid bacterium) show the results of comparing the amino acid producing ability of each lactic acid bacterium. In addition, the production ability of GABA and L-ornithine which are attracting attention as useful amino acids was also compared. GABA and L-ornithine were quantified according to the above method (2.).
図１に示すように、乳酸菌１ｂは特にGABA、D-アラニン及びD-アスパラギン酸を高生産する。D-アラニンの生産能は極めて高い。乳酸菌４ｂは特にD-アラニンを高生産する。また、乳酸菌４ｃは特にL-オルニチンを高生産する。尚、乳酸菌１ｂは寒天培地上にて白色円形のコロニーを形成する。乳酸菌４ｃも同様に、寒天培地上にて白色円形のコロニーを形成する。
As shown in FIG. 1, lactic acid bacteria 1b particularly produces high amounts of GABA, D-alanine and D-aspartic acid. The ability to produce D-alanine is extremely high.
一方、図２に示すように、乳酸菌２ｅは特にGABAを高生産する。乳酸菌２ｆは特にGABA、D-アラニン、D-アスパラギン酸、D-グルタミン酸及びL-オルニチンを高生産する。乳酸菌４ｆは特にD-アスパラギン酸、D-グルタミン酸及びL-オルニチンを高生産する。尚、乳酸菌４ｆは寒天培地上にて白色円形のコロニーを形成する。
On the other hand, as shown in FIG. 2, the
単離に成功した乳酸菌株の菌種を以下の方法（３．）で同定することにした。尚、乳酸菌株１ｂ、４ｆについては、２０１１年１２月１日付けで独立行政法人製品評価技術基盤機構 特許微生物寄託センターに寄託されている。 It was decided to identify the bacterial species of the lactic acid strain that was successfully isolated by the following method (3.). The lactic acid strains 1b and 4f have been deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation on December 1, 2011.
３．漬物（京漬物、すぐき漬け）から単離した乳酸菌種の同定方法
(1)MRS液体培地にて30℃で24時間培養した後、遠心分離（20,000 g, 2 min, 4℃）により菌体を回収した。
(2)ISOPLANT II（ニッポンジーン）を用いて回収した菌体からゲノムを抽出した。
(3)抽出したゲノムを鋳型として、各菌の16S rDNAの一部をPCRにより増幅した。その際、次のプライマーを用いた。520F: 5'-gtgccagcmgccgcgg-3'（配列番号７）および 1400R: 5'-acgggcggtgtgtrc-3'（配列番号８）
(4)増幅したDNA断片をアガロースゲル電気泳動した後、NucleoSpin（登録商標） Extract II（MACHEREY-NAGEL）を用いてゲルから抽出した。
(5)抽出したDNA断片を鋳型として塩基配列を解析し、ゲノムデータベースと比較することで菌種の同定をした。
3. Identification of lactic acid bacteria species isolated from pickles (Kyo pickles, pickles)
(1) After culturing in an MRS liquid medium at 30 ° C. for 24 hours, the cells were collected by centrifugation (20,000 g, 2 min, 4 ° C.).
(2) The genome was extracted from the cells collected using ISOPLANT II (Nippon Gene).
(3) Using the extracted genome as a template, a part of 16S rDNA of each bacterium was amplified by PCR. At that time, the following primers were used. 520F: 5'-gtgccagcmgccgcgg-3 '(SEQ ID NO: 7) and 1400R: 5'-acgggcggtgtgtrc-3' (SEQ ID NO: 8)
(4) The amplified DNA fragment was subjected to agarose gel electrophoresis and then extracted from the gel using NucleoSpin (registered trademark) Extract II (MACHEREY-NAGEL).
(5) The base sequence was analyzed using the extracted DNA fragment as a template, and the bacterial species was identified by comparing with the genome database.
結果、各乳酸菌は以下の通り同定された。尚、各乳酸菌の16S rDNA（一部）の配列を図３〜５に示す。
乳酸菌１ｂ：ラクトバチルス・ブレビス（Lactobacillus brevis）
乳酸菌４ｂ：ラクトバチルス・ブレビス（Lactobacillus brevis）
乳酸菌４ｃ：ラクトバチルス・プランタラム（Lactobacillus plantarum）
乳酸菌２ｅ：ラクトバチルス・ブレビス（Lactobacillus brevis）
乳酸菌２ｆ：ラクトバチルス・ブレビス（Lactobacillus brevis）
乳酸菌４ｆ：ラクトバチルス・ファーメンタム（Lactobacillus fermentum）
As a result, each lactic acid bacterium was identified as follows. In addition, the arrangement | sequence of 16S rDNA (part) of each lactic acid bacteria is shown to FIGS.
Lactic acid bacteria 1b: Lactobacillus brevis
＜まとめ＞
すぐき漬けから、非タンパク性アミノ酸（D-アミノ酸、GABA、L-オルニチン）を高生産する乳酸菌を単離及び同定することに成功した。これらの乳酸菌は非タンパク性アミノ酸の生産菌として有用である。非タンパク性アミノ酸を高生産する乳酸菌をすぐき漬けから単離できた事実は、すぐき漬けが有用乳酸菌をスクリーニングする際の優れたソースであることを意味する。
<Summary>
We succeeded in isolating and identifying lactic acid bacteria that produce high amounts of non-protein amino acids (D-amino acids, GABA, L-ornithine). These lactic acid bacteria are useful as non-protein amino acid producing bacteria. The fact that lactic acid bacteria producing high amounts of non-protein amino acids can be isolated from pickles means that pickles are an excellent source for screening useful lactic acid bacteria.
本発明の乳酸菌は非タンパク性アミノ酸の生産に優れる。D-アミノ酸、GABA、L-オルニチンの製造、又はこれらの非タンパク性アミノ酸を含む食品、化粧料、医薬の製造等への利用が期待される。 The lactic acid bacteria of the present invention are excellent in producing non-protein amino acids. It is expected to be used for the production of D-amino acids, GABA, L-ornithine, or for the production of foods, cosmetics and pharmaceuticals containing these non-protein amino acids.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
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| JP7190406B2 (en) * | 2019-08-02 | 2022-12-15 | サントリーホールディングス株式会社 | Beer-taste alcoholic beverage and method for producing the same |
| JP7428715B2 (en) * | 2019-08-02 | 2024-02-06 | サントリーホールディングス株式会社 | Composition for food and drink |
| JP7403096B2 (en) | 2020-02-06 | 2023-12-22 | 宮崎県 | Method for producing feed containing high GABA and ornithine content |
| CN111676166B (en) * | 2020-06-28 | 2022-03-01 | 千禾味业食品股份有限公司 | Lactic acid bacteria and application thereof in brewing of liquid vinegar |
| CN114480213A (en) * | 2022-03-07 | 2022-05-13 | 广东南芯医疗科技有限公司 | Lactobacillus fermentum E15 producing gamma-aminobutyric acid and application thereof in preparation of sleep improvement medicines |
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