JP5721162B2 - Method for producing high purity lactic acid - Google Patents
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims description 276
- 239000004310 lactic acid Substances 0.000 title claims description 123
- 235000014655 lactic acid Nutrition 0.000 title claims description 123
- 238000004519 manufacturing process Methods 0.000 title claims description 40
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims description 148
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 claims description 114
- 229940022769 d- lactic acid Drugs 0.000 claims description 114
- 229930182843 D-Lactic acid Natural products 0.000 claims description 110
- 241000894006 Bacteria Species 0.000 claims description 67
- 238000000855 fermentation Methods 0.000 claims description 40
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- 241000517333 Pichia manshurica Species 0.000 claims description 35
- 241000235648 Pichia Species 0.000 claims description 32
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- 238000000034 method Methods 0.000 claims description 18
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- 229930006000 Sucrose Natural products 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
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- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 2
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
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- KVZLHPXEUGJPAH-BXRBKJIMSA-N (2s)-2-oxidanylpropanoic acid Chemical compound C[C@H](O)C(O)=O.C[C@H](O)C(O)=O KVZLHPXEUGJPAH-BXRBKJIMSA-N 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
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Description
本発明は、微生物を用いた高純度Ｄ−乳酸の製造方法に関し、詳しくは、Ｄ−乳酸及びＬ−乳酸を含む溶液からＬ−乳酸資化性菌によってＬ−乳酸を除去する高純度Ｄ−乳酸の製造方法に関する。 The present invention relates to a method for producing high-purity D-lactic acid using a microorganism, and more specifically, high-purity D- from which L-lactic acid is removed by a L-lactic acid-assimilating bacterium from a solution containing D-lactic acid and L-lactic acid. The present invention relates to a method for producing lactic acid.
近年、化石資源の枯渇への危惧や地球環境保全の観点から、生分解性プラスチックに対する関心が高まっており、中でもバイオマスを原料として生産が可能なポリ乳酸系バイオプラスチックが注目されている。 In recent years, interest in biodegradable plastics is increasing from the perspective of fossil resource depletion and global environmental protection, and polylactic acid based bioplastics that can be produced using biomass as raw materials have attracted attention.
ポリ乳酸のモノマーである乳酸は、光学活性が異なるＬ‐乳酸とＤ‐乳酸の２つのエナンチオマーが存在する。どちらかのエナンチオマーのみ、すなわち、Ｌ‐乳酸あるいはＤ‐乳酸でポリ乳酸を合成した場合、ラセミ体であるＤＬ‐乳酸を原料とした場合と比べて、結晶性や延伸性、耐熱性、成型加工性等の優れたポリマーができることがよく知られている。ちなみに、Ｌ‐乳酸で合成したポリ乳酸は、ポリ‐Ｌ‐乳酸、Ｄ‐乳酸で合成した場合は、ポリ‐Ｄ‐乳酸という。 Lactic acid, which is a polylactic acid monomer, has two enantiomers, L-lactic acid and D-lactic acid, which have different optical activities. When polylactic acid is synthesized using only one of the enantiomers, that is, L-lactic acid or D-lactic acid, crystallinity, stretchability, heat resistance, and molding processing are compared to the case where racemic DL-lactic acid is used as a raw material. It is well known that polymers having excellent properties can be produced. By the way, polylactic acid synthesized with L-lactic acid is called poly-L-lactic acid, and when synthesized with D-lactic acid, it is called poly-D-lactic acid.
ポリ‐Ｌ‐乳酸あるいはポリ‐Ｄ‐乳酸を合成する場合、その原料モノマーであるＬ‐乳酸あるいはＤ‐乳酸の純度が重合度やガラス転移点に大きく影響する。さらに、原料モノマーへの他方のエナンチオマーの混入は、合成されたポリ乳酸の融点を下げることが知られている。そのため、工業用プラスチック素材としてポリ乳酸を使用するためには、ポリ乳酸製造のための原料乳酸の光学純度が高いことが必要となる。 When synthesizing poly-L-lactic acid or poly-D-lactic acid, the purity of the raw material monomer L-lactic acid or D-lactic acid greatly affects the degree of polymerization and the glass transition point. Furthermore, it is known that mixing of the other enantiomer in the raw material monomer lowers the melting point of the synthesized polylactic acid. Therefore, in order to use polylactic acid as an industrial plastic material, it is necessary that the raw material lactic acid for producing polylactic acid has a high optical purity.
ところで、これまでのポリ乳酸は、人体で生成される乳酸がＬ‐乳酸であることなどの理由からポリ‐Ｌ‐乳酸が主流であった。そのため、その原料となるＬ‐乳酸生産のための研究やその光学純度を高める研究がなされてきた。例えば、特開平９−１２１８４４号公報には、高光学純度のＬ−乳酸を生産する新規バチルス(Bacillus) sp. SHO-1（FERM P‐15234）を培養し、この培養物から光学純度９５％以上のＬ−乳酸を採取することが開示されている（特許文献１）。 By the way, poly-L-lactic acid has been the mainstream so far because the lactic acid produced in the human body is L-lactic acid. Therefore, research for producing L-lactic acid as a raw material and research for enhancing its optical purity have been made. For example, in Japanese Patent Application Laid-Open No. 9-121844, a novel Bacillus sp. SHO-1 (FERM P-15234) that produces L-lactic acid with high optical purity is cultured, and an optical purity of 95% is obtained from this culture. Collecting the above L-lactic acid is disclosed (Patent Document 1).
ところが最近になって、ポリ‐Ｌ-乳酸とポリ‐Ｄ‐乳酸とを混合して得られるステレオコンプレックス型ポリ乳酸が、ポリ‐Ｌ‐乳酸あるいはポリ‐Ｄ‐乳酸と比較して、その強度や耐熱性の点でさらに優れた特性を有することが見いだされた。例えば、特開２０００‐１７１６３号公報には、Ｌ−乳酸、Ｄ−乳酸及び／又は乳酸以外の共重合成分により構成された非晶性ポリマーを特定の混合重量比で溶融ブレンドした結晶性ポリ乳酸ステレオコンプレックスポリマー組成物が、成形加工性に優れ、低コストで得られる旨が開示されている（特許文献２）。そのため、効率的なＤ‐乳酸生産方法および光学純度の高いＤ‐乳酸生産方法の開発が必要となった。 Recently, however, the stereocomplex polylactic acid obtained by mixing poly-L-lactic acid and poly-D-lactic acid is stronger than poly-L-lactic acid or poly-D-lactic acid. It has been found that it has more excellent characteristics in terms of heat resistance. For example, Japanese Patent Laid-Open No. 2000-17163 discloses crystalline polylactic acid obtained by melt-blending an amorphous polymer composed of L-lactic acid, D-lactic acid and / or a copolymerization component other than lactic acid at a specific mixing weight ratio. It is disclosed that a stereocomplex polymer composition is excellent in molding processability and can be obtained at low cost (Patent Document 2). Therefore, it was necessary to develop an efficient D-lactic acid production method and a high optical purity D-lactic acid production method.
一般的な乳酸の生産方法は、合成法及び発酵法の２つが知られている。合成法は、アセトアルデヒドに青酸を作用させ、生成したシアンヒドリンを加水分解して乳酸を合成する場合と、アセトアルデヒドと一酸化炭素とを高圧下で反応させて合成する場合が知られている。一方、発酵法は、ショ糖、ブドウ糖、デンプン、ジャガイモ等のバイオマス原料を乳酸生産菌によって発酵させて得た乳酸を精製する。 Two general lactic acid production methods are known: a synthesis method and a fermentation method. As the synthesis method, there are known cases in which hydrocyanic acid is allowed to act on acetaldehyde to synthesize lactic acid by hydrolyzing the produced cyanohydrin, and in a case in which acetaldehyde and carbon monoxide are reacted under high pressure. On the other hand, the fermentation method purifies lactic acid obtained by fermenting biomass raw materials such as sucrose, glucose, starch, and potato with lactic acid-producing bacteria.
合成法によって合成された乳酸の光学純度は、Ｌ‐乳酸：Ｄ‐乳酸比が１：１のラセミ体となる。 The optical purity of lactic acid synthesized by the synthesis method is a racemate having an L-lactic acid: D-lactic acid ratio of 1: 1.
一方、発酵法においては、発酵に用いる微生物を選択することによってＬ‐乳酸あるいはＤ-乳酸の光学純度を高めることができる。例えば、Ｄ‐乳酸生産菌を用いることによって、比較的純度の高いＤ‐乳酸を生産することができる。 On the other hand, in the fermentation method, the optical purity of L-lactic acid or D-lactic acid can be increased by selecting a microorganism to be used for fermentation. For example, D-lactic acid having a relatively high purity can be produced by using a D-lactic acid-producing bacterium.
また、遺伝子工学的手法によって改変した微生物を用いた高純度Ｄ‐乳酸の生産についての研究も行われており、例えば、Ishida, N.らは、高光学純度Ｄ‐乳酸の効率的生産を目的として、ピルビン酸デカルボキシラーゼ遺伝子を欠損したサッカロミセス セルビシエ（Saccharomyces cerevisiae）にロイコノストック メセンテロイデス（Leuconostoc mesenteroides subsp. mesenteroides） NBRC 3426株由来のＤ‐乳酸デヒドロゲナーゼ遺伝子を導入してグルコースからのＤ‐乳酸生産を試みたところ、光学純度９９．９％のＤ‐乳酸を生産したことが開示されている（非特許文献１、特許文献３）。 In addition, research on the production of high-purity D-lactic acid using microorganisms modified by genetic engineering techniques has also been conducted. For example, Ishida, N. et al. Aimed at efficient production of high-optical purity D-lactic acid. D-lactic acid production from glucose by introducing D-lactate dehydrogenase gene derived from Leuconostoc mesenteroides subsp. Mesenteroides NBRC 3426 into Saccharomyces cerevisiae lacking the pyruvate decarboxylase gene An attempt has been made to produce D-lactic acid with an optical purity of 99.9% (Non-patent Document 1, Patent Document 3).
ところで、Ｄ-乳酸およびＬ-乳酸のように光学活性の異なる物質を分割する方法には、（１）ラセミ体にキラル化合物（光学分割剤）を作用させてジアステレオマーを形成させ、ジアステレオマー間の物理的な性質、例えば溶解度などの差異を利用して、それぞれのジアステレオマーを分別結晶化した後、得られた単一のジアステレオマーから光学分割剤を取り除くことで目的のエナンチオマーを得る結晶化による光学分割法、（２）不斉要素をもつ固定相を用いたカラムクロマトグラフィーによって、その保持時間の差異を利用して分割する光学分割法、（３）酵素の高い不斉識別能によって一方のエナンチオマーを選択的に反応させる酵素法の３つが知られている。さらに、結晶化による光学分割方法には、優先晶出法、ジアステレオマー法、包接錯体法、優先富化法がある。 By the way, in the method of resolving substances having different optical activities such as D-lactic acid and L-lactic acid, (1) a diastereomer is formed by reacting a racemate with a chiral compound (an optical resolving agent) to form a diastereomer. The diastereomers are fractionally crystallized using differences in physical properties such as solubility between the dimers, and then the desired enantiomer is removed by removing the optical resolving agent from the obtained single diastereomers. Resolution method by crystallization to obtain (2) optical resolution method that utilizes the difference in retention time by column chromatography using a stationary phase having an asymmetric element, (3) high chirality of the enzyme Three enzyme methods are known in which one of the enantiomers is selectively reacted according to discrimination ability. Furthermore, optical resolution methods by crystallization include a preferential crystallization method, a diastereomer method, an inclusion complex method, and a preferential enrichment method.
合成法によって合成された乳酸の光学純度は、上記のようにＬ‐乳酸：Ｄ‐乳酸比が１：１のラセミ体となるため、Ｄ‐乳酸を高純度で製造することができない。 Since the optical purity of lactic acid synthesized by the synthesis method is a racemate having an L-lactic acid: D-lactic acid ratio of 1: 1 as described above, D-lactic acid cannot be produced with high purity.
乳酸菌により発酵法で生産される乳酸の光学純度は、Ｌ‐乳酸およびＤ‐乳酸のいずれにおいても、高いもので９７〜９８％程度であり、数パーセント含まれる光学異性体の存在が、ポリ乳酸の結晶性や融点の低下とそれに伴うバイオプラスチック強度の低下や生産効率の悪化の一因となっている。 The optical purity of lactic acid produced by fermentation using lactic acid bacteria is about 97 to 98% at high in both L-lactic acid and D-lactic acid, and the presence of optical isomers contained in several percent is polylactic acid. This is one of the causes of the decrease in crystallinity and melting point, and the resulting decrease in bioplastic strength and production efficiency.
また、遺伝子工学的手法による高光学純度乳酸生産菌の育種などに関する検討も現在進められているが、組み換え菌においても、その培養を繰り返すことで遺伝情報の欠落やそれに伴う形質の変化が生じることや、遺伝子組換え体の取り扱いが非常に難しいことなどの問題がある。 In addition, studies on the breeding of highly optically pure lactic acid-producing bacteria using genetic engineering methods are currently underway. However, in recombinant bacteria, the loss of genetic information and associated changes in traits may occur by repeated cultivation. In addition, there are problems such as extremely difficult handling of genetically modified organisms.
さらに、物理化学的な光学分割法により、ＤＬ‐混合乳酸から多量の高光学純度乳酸を製造することは必ずしも容易ではない。 Furthermore, it is not always easy to produce a large amount of high optical purity lactic acid from DL-mixed lactic acid by physicochemical optical resolution.
従って、本発明の目的は、生物学的手法を用いた簡便な高純度Ｄ-乳酸の生産であって、乳酸溶液中のＬ‐乳酸を選択的あるいは優先的に資化する菌によって、簡便、効率的かつ低コストで高純度Ｄ‐乳酸の製造方法を提供することにある。 Accordingly, an object of the present invention is to produce simple high-purity D-lactic acid using a biological technique, and can be easily performed by a bacterium that selectively or preferentially assimilate L-lactic acid in a lactic acid solution. An object is to provide a method for producing high-purity D-lactic acid efficiently and at low cost.
上記課題を解決するため、本発明は、Ｄ‐乳酸及びＬ‐乳酸を含む乳酸溶液を調製する乳酸溶液調製工程と、該乳酸溶液中でＬ‐乳酸資化性菌を培養し、該乳酸溶液中のＬ‐乳酸を資化させることによってＤ‐乳酸の純度を高めるＬ‐乳酸除去工程と、を有する高純度Ｄ‐乳酸の製造方法を提供するものである。 In order to solve the above problems, the present invention provides a lactic acid solution preparation step for preparing a lactic acid solution containing D-lactic acid and L-lactic acid, culturing L-lactic acid-assimilating bacteria in the lactic acid solution, The present invention provides a method for producing high-purity D-lactic acid, which comprises a step of removing L-lactic acid to increase the purity of D-lactic acid by assimilating L-lactic acid therein.
本発明に係る高純度乳酸の生産方法によれば、Ｌ‐乳酸を優先的に資化する能力が高い新規Ｌ‐乳酸資化性菌を使用し、Ｄ‐乳酸及びＬ‐乳酸を含む乳酸溶液中のＬ‐乳酸をこのＬ‐乳酸資化性菌によって資化させることで、簡便、効率的かつ低コストでＬ-乳酸を除去し、高純度のＤ‐乳酸の生産を製造することが可能となる。 According to the method for producing high-purity lactic acid according to the present invention, a novel L-lactic acid-assimilating bacterium having high ability to preferentially assimilate L-lactic acid is used, and a lactic acid solution containing D-lactic acid and L-lactic acid L-lactic acid can be assimilated with this L-lactic acid-assimilating bacterium to remove L-lactic acid easily, efficiently and at low cost, and to produce highly pure D-lactic acid It becomes.
本発明の高純度乳酸の製造方法は、Ｄ‐乳酸及びＬ‐乳酸を含む乳酸溶液を含む乳酸溶液を調製する乳酸溶液調製工程と、該乳酸溶液中でＬ‐乳酸資化性菌を培養し、該乳酸溶液中のＬ‐乳酸を資化させることによってＤ‐乳酸の純度を高めるＬ‐乳酸除去工程を有する。 The high purity lactic acid production method of the present invention comprises a lactic acid solution preparation step of preparing a lactic acid solution containing a lactic acid solution containing D-lactic acid and L-lactic acid, and culturing L-lactic acid-assimilating bacteria in the lactic acid solution. And an L-lactic acid removing step for enhancing the purity of D-lactic acid by assimilating L-lactic acid in the lactic acid solution.
本実施形態で使用されるＤ‐乳酸及びＬ‐乳酸を含む乳酸溶液は、公知の方法によって合成された合成乳酸、あるいはショ糖、ブドウ糖、デンプン、ジャガイモなどを原料として乳酸生産菌による乳酸発酵によって生成された発酵乳酸を用いることができる。Ｄ‐乳酸生産収率の観点からは、乳酸溶液は、Ｄ−乳酸生産菌による乳酸発酵で生産された乳酸であることが好ましい。 The lactic acid solution containing D-lactic acid and L-lactic acid used in this embodiment is synthesized by a known method, or lactic acid fermentation by lactic acid producing bacteria using sucrose, glucose, starch, potato, etc. as raw materials. The produced fermented lactic acid can be used. From the viewpoint of D-lactic acid production yield, the lactic acid solution is preferably lactic acid produced by lactic acid fermentation by D-lactic acid producing bacteria.
本実施形態で使用されるＤ‐乳酸生産菌としては、例えば、ラクトバチルス属（Lactobacillus）、ラクトコッカス属（Lactococcus）、ロイコノストック属（Leuconostoc）及びスポロラクトバチラス属（Sporolactobacillus）に属する微生物などが好ましく使用できる。 Examples of D-lactic acid producing bacteria used in the present embodiment include microorganisms belonging to the genus Lactobacillus, Lactococcus, Leuconostoc and Sporolactobacillus. Can be preferably used.
これらのうち、Ｄ−乳酸の生産能の高いものを選択することが好ましく、例えば、ラクトバチルス デルブリュッキ（Lactobacillus delbrueckii）、ラクトバチルス コリニフォルミス（Lactobacillus coryniformis）、ラクトバチルス カゼイ（Lactobacillus casei）、ラクトバチルス アシドフィルス（Lactobacillus acidophilus）、ラクトバチルス ビフィダス（Lactobacillus bifidus）、ラクトバチルス ビフィダス ペンシルバニカス（Lactobacillus bifidus Pennsylvanicus）、ラクトバチルス ブレビス（Lactobacillus brevis）、ラクトバチルス ブフネリ（Lactobacillus buchneri）、ラクトバチルス ブルガリカス（Lactobacillus bulgaricus）、ラクトバチルス コーカシカス（Lactobacillus caucasicus）、ラクトバチルス ファーメンタム（Lactobacillus fermentum）、ラクトバチルス ヒルガルディ（Lactobacillus hilgardii）、ラクトバチルス ラクティア（Lactobacillus lactia）、ラクトバチルス ライヒマニ（Lactobacillus leichmannii）、ラクトバチルス サーモフィルス（Lactobacillus thermophilus）、及びラクトバチルス トリコデス（Lactobacillus trichodes）等の、ラクトバチルス属（Lactobacillus）に属する微生物が好ましく、その中でもＤ‐乳酸の生産比率が高いラクトバチルス デルブリュッキー（Lactobacillus delbrueckii）であることがより好ましい。 Among these, it is preferable to select those having a high ability to produce D-lactic acid. For example, Lactobacillus delbrueckii, Lactobacillus coryniformis, Lactobacillus casei, Lactobacillus casei, Lactobacillus casei Lactobacillus acidophilus, Lactobacillus bifidus, Lactobacillus bifidus Pennsylvanicus, Lactobacillus binsylvanicus, Lactobacillus brevis, Lactobacillus buchlus Lactobacillus buch , Lactobacillus caucasicus, Lactobacillus fermentum, Lactobacillus hirugardi (Lactobacillus hi lgardii), Lactobacillus lactia, Lactobacillus leichmannii, Lactobacillus thermophilus, Lactobacillus trichodes, and the like, preferably belonging to the genus Lactobacillus trichodes Of these, Lactobacillus delbrueckii having a high production ratio of D-lactic acid is more preferable.
前記Ｄ‐乳酸生産菌を培養するための条件として、使用培地は、乳酸菌が増殖し、Ｄ‐乳酸を生産するためのものであればよく、乳酸菌の培養に一般的に用いられる様々な液体培地を用いることができる。好適な培地としては、ＧＹＰ培地、ＭＲＳ培地などを挙げることができ、その中でも、ＧＹＰ培地を用いることがより好ましい。また、培養温度は、１５〜５０℃の範囲で適宜決定することできるが、乳酸菌の培養至適温度を考慮すると２５〜４５℃に設定することが好ましい。乳酸発酵は嫌気的条件下で行われるため、静置培養又は嫌気的条件下での撹拌培養又はこれらの組み合わせで培養することが好ましい。 As a condition for culturing the D-lactic acid-producing bacterium, any medium can be used as long as lactic acid bacteria can grow and produce D-lactic acid. Various liquid media generally used for culturing lactic acid bacteria Can be used. Suitable media include GYP media, MRS media, etc. Among them, it is more preferable to use GYP media. Moreover, although culture | cultivation temperature can be suitably determined in the range of 15-50 degreeC, it is preferable to set to 25-45 degreeC when the optimal culture | cultivation temperature of lactic acid bacteria is considered. Since lactic acid fermentation is performed under anaerobic conditions, it is preferable to culture by stationary culture, stirring culture under anaerobic conditions, or a combination thereof.
本実施形態におけるＬ‐乳酸除去工程は、Ｄ‐乳酸およびＬ‐乳酸を含む乳酸溶液からＬ‐乳酸資化性菌によってＬ‐乳酸を除去することにより、乳酸溶液中のＤ−乳酸の純度を高める工程である。 In this embodiment, the L-lactic acid removal step removes L-lactic acid from the lactic acid solution containing D-lactic acid and L-lactic acid by L-lactic acid-assimilating bacteria, thereby improving the purity of D-lactic acid in the lactic acid solution. It is a process of increasing.
上述のように、Ｄ‐乳酸生産菌で乳酸発酵を実施した場合であっても、実際のＤ-乳酸の純度は高いもので９７〜９８％程度であり、Ｌ-乳酸が数％混在している。そのため、１００％に近い純度が要求されるバイオプラスチック製造においては、数パーセント含まれるＬ-乳酸の存在が、ポリ乳酸の結晶性や融点の低下とそれに伴うバイオプラスチック強度の低下や生産効率の悪化の一因となっている。従って、本実施形態の高純度乳酸の製造方法においては、Ｌ-乳酸を特異的に資化し、Ｄ-乳酸を資化しない又はＤ-乳酸資化能の低いＬ‐乳酸資化性菌を選択することが重要となる。 As described above, even when lactic acid fermentation is carried out with a D-lactic acid producing bacterium, the actual purity of D-lactic acid is about 97 to 98%, and a few percent of L-lactic acid is mixed. Yes. Therefore, in the production of bioplastics that require purity close to 100%, the presence of several percent of L-lactic acid reduces the crystallinity and melting point of polylactic acid and the resulting decrease in bioplastic strength and production efficiency. It is one of the causes. Therefore, in the method for producing high-purity lactic acid of this embodiment, L-lactic acid-assimilating bacteria that specifically assimilate L-lactic acid and do not assimilate D-lactic acid or have a low ability to assimilate D-lactic acid are selected. It is important to do.
本実施形態に使用されるＬ‐乳酸資化性菌は、ピキア マンシュリカ（Pichia manshurica）であることが好ましく、その中でも特に、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）は、Ｌ‐乳酸の選択的資化能及び前記乳酸溶液からのＤ‐乳酸生産収量の観点から好ましい。 L- lactic acid utilizing bacteria used in the present embodiment is preferably a Pichia Manshurika (Pichia manshurica), Among them, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) Is preferable from the viewpoint of the selective assimilation ability of L-lactic acid and the yield of D-lactic acid production from the lactic acid solution.
ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）は本発明者らが土壌からスクリーニングして得られた酵母であり、形態観察、炭素源資化性試験、26S rDNA-D1/D2塩基配列の解析を行い本菌の同定を試みた結果、ピキア マンシュリカ（Pichia manshurica）と同定されたものである。 Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) is a yeast present inventors have obtained by screening from soil, morphological observation, carbon source assimilation test, 26S rDNA-D1 As a result of analyzing the / D2 nucleotide sequence and attempting to identify this strain, it was identified as Pichia manshurica.
本菌株の栄養細胞は広楕円形であり、多極出芽による栄養増殖を示した。また、子のうにおける１〜４個の帽子形の胞子形成、および偽菌糸の形成が確認された（図１）。また、炭素源資化性試験から、ヘキサデカンを資化せず、N-アセチル-D-グルコサミンを資化することが明らかとなり（表１）、これらの性質はピキア マンシュリカ（Pichia manshurica）の性質と一致していた。さらに26S rDNA-D1/D2塩基配列は、子のう菌系酵母の一種であるピキア マンシュリカ（Pichia manshurica）の基準株IFO 10726Tに対して１００％の相同率を示した。 The vegetative cells of this strain were broad oval and showed vegetative growth by multipolar budding. In addition, formation of 1 to 4 hat-shaped spores and formation of pseudohyphae in the baby pupae were confirmed (FIG. 1). In addition, the carbon source assimilation test revealed that N-acetyl-D-glucosamine was assimilated without using hexadecane (Table 1), and these properties are similar to those of Pichia manshurica. It was consistent. Furthermore, the 26S rDNA-D1 / D2 nucleotide sequence showed 100% homology with the reference strain IFO 10726 T of Pichia manshurica, which is a kind of Ascomycetous yeast.
前記Ｌ‐乳酸除去工程の条件は適宜設定し得るが、例えば、Ｌ‐乳酸資化性菌を添加し、２５〜４０℃、ｐＨ２．６〜７．０で好気培養（120oscillations/min）することによって実施することができる。 The conditions for the L-lactic acid removal step can be appropriately set. For example, an L-lactic acid-assimilating bacterium is added and aerobic culture (120 oscillations / min) is performed at 25 to 40 ° C. and pH 2.6 to 7.0. Can be implemented.
本実施形態において、前記乳酸溶液調製工程の終了後に前記Ｌ‐乳酸除去工程を実施することができる。かかる場合、前記乳酸溶液調製工程の終了後に、乳酸溶液を遠心分離等によりＤ-乳酸生産菌を除去し、上清（Ｄ‐乳酸純度９７〜９８％）をＬ‐乳酸除去工程に供することが好ましい。これにより、Ｄ‐乳酸純度が９９．８％以上の高光学純度Ｄ‐乳酸溶液を得ることができる。 In this embodiment, the L-lactic acid removal step can be performed after the lactic acid solution preparation step. In such a case, after completion of the lactic acid solution preparation step, the lactic acid solution may be subjected to centrifugation to remove D-lactic acid-producing bacteria, and the supernatant (D-lactic acid purity 97 to 98%) may be subjected to the L-lactic acid removal step. preferable. Thereby, a high optical purity D-lactic acid solution having a D-lactic acid purity of 99.8% or more can be obtained.
また、前記乳酸溶液調製工程と、前記Ｌ‐乳酸除去工程とを同時に実施することもできる。かかる場合、Ｄ‐乳酸生産菌を培養する培地にＤ‐乳酸生産菌とＬ‐乳酸資化性菌を添加し、所定条件で培養することにより、Ｄ‐乳酸純度が９９．６％以上の高光学純度Ｄ‐乳酸溶液を得ることができる。このように、乳酸溶液調製工程とＬ‐乳酸除去工程とを同時に実施するとことで、より効率的に高光学純度Ｄ‐乳酸を得ることができる。 In addition, the lactic acid solution preparation step and the L-lactic acid removal step can be performed simultaneously. In such a case, a D-lactic acid purity of 99.6% or higher can be obtained by adding D-lactic acid-producing bacteria and L-lactic acid-assimilating bacteria to a medium for culturing D-lactic acid-producing bacteria and culturing under predetermined conditions. An optical purity D-lactic acid solution can be obtained. Thus, by carrying out the lactic acid solution preparation step and the L-lactic acid removal step at the same time, high optical purity D-lactic acid can be obtained more efficiently.
培養条件は、培養開始から２４時間経過まではＤ‐乳酸生産菌の至適生育条件で培養を実施し、培養開始から２４時間経過後はＬ‐乳酸資化性菌の至適生育条件で培養を実施することが好ましい。培養開始から２４時間経過までＤ‐乳酸生産菌の至適生育条件で培養することで、培地中に乳酸が蓄積されていき、培養開始から２４時間経過後はＬ‐乳酸資化性菌の至適生育条件で培養することで、培養液中に混在しているＬ‐乳酸をＬ‐乳酸資化性菌により資化させ、これを除去していくことができる。 The culture is performed under the optimal growth conditions for the D-lactic acid-producing bacteria until 24 hours after the start of the culture, and after 24 hours from the start of the culture, the culture is performed under the optimal growth conditions for the L-lactic acid-assimilating bacteria. It is preferable to implement. By culturing under the optimal growth conditions of D-lactic acid producing bacteria from the start of culture for 24 hours, lactic acid is accumulated in the medium, and after 24 hours from the start of culture, L-lactic acid-utilizing bacteria are By culturing under suitable growth conditions, L-lactic acid mixed in the culture solution can be assimilated by L-lactic acid-assimilating bacteria and removed.
Ｄ‐乳酸生産菌の至適生育条件は、３５〜４０℃、ｐＨ４．５〜５．５の静置培養あるいは嫌気条件下での撹拌培養とし、Ｌ‐乳酸資化性菌の至適生育条件は、３０〜３５℃、ｐＨ３．０〜３．８の振とう培養あるいは通気撹拌培養とすることが好ましい。かかる培養条件で培養することで、前半はＤ‐乳酸生産菌の乳酸発酵に適した嫌気的条件となり、後半はＬ‐乳酸資化性菌によるＬ−乳酸の資化・除去に適した好気的条件となるため、より効率よく高光学純度Ｄ‐乳酸を得ることができる。 Optimum growth conditions for D-lactic acid producing bacteria are static culture at 35 to 40 ° C. and pH 4.5 to 5.5 or stirring culture under anaerobic conditions, and optimal growth conditions for L-lactic acid-assimilating bacteria Is preferably shake culture or aeration and agitation culture at 30 to 35 ° C. and pH 3.0 to 3.8. By culturing under such culture conditions, the first half is anaerobic conditions suitable for lactic acid fermentation of D-lactic acid producing bacteria, and the second half is aerobic suitable for L-lactic acid utilization and removal by L-lactic acid-assimilating bacteria. Therefore, high optical purity D-lactic acid can be obtained more efficiently.
１．各種ピキア マンシュリカ（Pichia manshurica）の乳酸資化特性
（１）Ｌ‐乳酸資化性菌
ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）と既知のピキア マンシュリカ（Pichia manshurica）７菌株（NBRC1004株、NBRC1284株、NBRC1789株、NBRC1790株、NBRC10062株、NBRC100562株、NBRC10562株）のＬ‐乳酸資化特性を比較検討した。
1. Various Pichia Manshurika (Pichia manshurica) lactate assimilation properties of (1) L-lactic acid utilizing bacteria Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) and known Pichia Manshurika (Pichia manshurica) 7 The L-lactic acid utilization characteristics of the strains (NBRC1004 strain, NBRC1284 strain, NBRC1789 strain, NBRC1790 strain, NBRC10062 strain, NBRC100562 strain, NBRC10562 strain) were compared and examined.
（２）乳酸生産菌
乳酸生産菌としては、ラクトバチルス デルブリュッキ サブスピーシーズ デルブリュッキ（Lactobacillus delbrueckii subsp. delbrueckii）141株（Ｄ‐乳酸生産菌）、ラクトバチルス パラカゼイ サブスピーシーズ パラカゼイ（Lactobacillus paracasei subsp. paracasei）1532株（Ｌ−乳酸生産菌）およびラクトバチルス プランタラム（Lactobacillus plantarum）NRIC 1067株（ＤＬ−乳酸生産菌）を使用した。
(2) Lactic acid-producing bacteria The lactic acid-producing bacteria include Lactobacillus delbrueckii subsp. Delbrueckii 141 strain (D-lactic acid producing strain), Lactobacillus paracasei subsp. Paracasei strain 1532 (L-lactic acid producing bacteria) and Lactobacillus plantarum NRIC 1067 strain (DL-lactic acid producing bacteria) were used.
（３）乳酸資化特性の評価
ＹＭ培地を用いて培養した各Ｌ‐乳酸資化性菌の培養液を使用し、これらをそれぞれＬＹＰ培地（DL-Lactic acid：Purity 85.0~90.0%(Wako Pure Chemical Industries, Ltd.)2.5％、Peptone0.25％、Yeast extract(BD.Bacto)0.25％、KH2PO40.04％、pH3.8）に添加して30℃で72時間培養を行ったのち、培養液中に残存するＤ−乳酸およびＬ−乳酸の濃度を定量し、乳酸資化特性を評価した。
(3) Evaluation of Lactic Acid Utilization Characteristics Using the culture solution of each L-lactic acid assimilating bacterium cultivated using YM medium, each of them was treated with LYP medium (DL-Lactic acid: Purity 85.0-90.0% (Wako Pure Chemical Industries, Ltd.) 2.5%, Peptone 0.25%, Yeast extract (BD. Bacto) 0.25%, KH 2 PO 4 0.04%, pH 3.8) and cultured at 30 ° C. for 72 hours, The concentrations of D-lactic acid and L-lactic acid remaining in the culture medium were quantified to evaluate lactic acid utilization characteristics.
Ｌ‐乳酸及びＤ‐乳酸の分別定量は、酵素法（ＤＬ‐乳酸測定用Ｆキット：ロシュ・ダイアグノスティックス社製）によって行った。なお、ＤＬ‐乳酸の分別定量に供する培養液試料の調製は、以下のとおりである。すなわち、マイクロチューブに、培養液の遠心分離（4℃、12,000rpm、10分）上清を入れてキャップをロックした後、８０〜９０℃の温浴中で１５分間加温することにより、培養液中に含まれる各種酵素を失活させた。これを水浴中で冷却した後、遠心分離（4℃、12,000rpm、10分）し、回収した上清を定量試験に用いた。また、対照（Control）には試料液の代わりに同量の蒸留水を用いた。 The fractional quantification of L-lactic acid and D-lactic acid was carried out by an enzymatic method (DL-lactic acid measurement F kit: manufactured by Roche Diagnostics). In addition, the preparation of the culture solution sample used for the differential quantification of DL-lactic acid is as follows. That is, the culture solution is centrifuged (4 ° C., 12,000 rpm, 10 minutes) in a microtube, the cap is locked, and then heated in a warm bath at 80 to 90 ° C. for 15 minutes to obtain the culture solution. Various enzymes contained therein were inactivated. After cooling this in a water bath, it was centrifuged (4 ° C., 12,000 rpm, 10 minutes), and the recovered supernatant was used for a quantitative test. For the control, the same amount of distilled water was used instead of the sample solution.
結果を表２に示す。表２に示すように、いずれの菌においても、培養液中の残存乳酸濃度は、初発乳酸濃度の約１／２〜１／４程度となっており、用いた全ての菌が乳酸資化能を有することが明らかとなった。 The results are shown in Table 2. As shown in Table 2, in any bacterium, the residual lactic acid concentration in the culture solution is about 1/2 to 1/4 of the initial lactic acid concentration, and all the bacteria used were capable of utilizing lactic acid. It became clear to have.
また、乳酸資化の様相から、いずれもＬ−乳酸の資化力が強く、その特性がピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の場合と類似していることが明らかとなったが、Ｄ−乳酸の割合が最も高くなったピキア マンシュリカ（Pichia manshurica）NBRC1790株においても、その比はＤ：Ｌ＝81.66：18.34に過ぎなかった。 Also, the appearance of lactic acid-utilizing both strong assimilation force of L- lactic acid, its characteristics Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) is similar to the case that However, even in the Pichia manshurica NBRC1790 strain in which the ratio of D-lactic acid was highest, the ratio was only D: L = 81.66: 18.34.
以上のことから、Ｌ−乳酸資化能はピキア マンシュリカ（P. manshurica）に比較的広範に認められる共通の性質であるものの、他の菌よりもＤ−乳酸を資化せず特異的にＬ−乳酸を資化する特性を有するという観点からは、分離菌ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）は、極めて特異な菌であることが明らかとなった。 From the above, L-lactic acid assimilation ability is a common property that is relatively widely recognized in P. manshurica, but it does not assimilate D-lactic acid more specifically than other bacteria. - from the viewpoint of having the property of assimilates lactic acid, isolates Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) was found to be a very specific bacteria.
２．ピキア マンシュリカ（Pichia manshurica）LAAM001株の培養特性
（１）培養温度の検討
ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の培養温度条件を検討した。なお、該試験はＹＭ培地を用いて調製した菌体を種菌として用い、これをＬＹＰ培地に添加して、２５℃、３０℃、３５℃あるいは４０℃で７２時間振とう培養（120 oscillations/min）した。
2. Pichia Manshurika (Pichia manshurica) LAAM001 strain culture characteristics of (1) Examination of culture temperature Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) were studied culture temperature conditions. In this test, cells prepared using YM medium were used as seeds, added to LYP medium, and shake cultured at 25 ° C., 30 ° C., 35 ° C. or 40 ° C. for 72 hours (120 oscillations / min )did.
試験したすべての温度で菌体の増殖が認められた。特に、増殖速度は、培養温度３５℃までは温度の上昇と共に増加し、最大菌体濃度はＯＤ660値で約２５となった。一方、培養温度４０℃の場合では、菌体増殖速度及び菌体濃度ともに低下した。以上のことから、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の生育および乳酸資化に関わる最適培養温度は３０〜３５℃であると考えられた。 Cell growth was observed at all temperatures tested. In particular, the growth rate increased with increasing temperature up to a culture temperature of 35 ° C., and the maximum cell concentration was about 25 at the OD 660 value. On the other hand, when the culture temperature was 40 ° C., both the cell growth rate and the cell concentration decreased. From the above, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) optimum culture temperature involved in growth and lactic acid-utilizing the was considered 30 to 35 ° C..
（２）初発ｐＨの検討
ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の培養時における培地の初発ｐＨについて検討した。なお、該試験はＹＭ培地を用いて調製した菌体を種菌として用い、これを１Ｎ‐ＨＣｌあるいは６Ｎ‐ＮａＯＨによって初発ｐＨを２．６〜７．０に調整したＬＹＰ培地に添加し、３０℃で７２時間振とう培養（120 oscillations/min）した。
(2) Examination of initial pH Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) was examined initial pH of the medium during culturing. In this test, cells prepared using a YM medium were used as seeds, which were added to a LYP medium whose initial pH was adjusted to 2.6 to 7.0 with 1N-HCl or 6N-NaOH. For 72 hours with shaking (120 oscillations / min).
培地の初発ｐＨが３．０〜４．６の範囲で菌体の増殖が良好であり、培養７２時間後にＯＤ660値が約２０〜２５であった。また、初発ｐＨが３．０〜３．８の範囲で特に良好な乳酸資化が認められ、初発乳酸量の約６０％に相当する乳酸が資化された。一方、培地の初発ｐＨを２．６以下あるいは７．０以上にした場合は、菌体増殖及び乳酸資化力が低下した。特に、該菌株のＬ‐乳酸資化能を利用した高純度Ｄ‐乳酸生産において、培地の初発ｐＨは、菌体の増殖が最も良好であり、高い乳酸資化性が認められたｐＨ３．８が最も良いことが分かった。 The growth of the cells was good when the initial pH of the medium was in the range of 3.0 to 4.6, and the OD 660 value was about 20 to 25 after 72 hours of culture. Also, particularly good lactic acid utilization was observed in the initial pH range of 3.0 to 3.8, and lactic acid corresponding to about 60% of the initial lactic acid amount was utilized. On the other hand, when the initial pH of the medium was 2.6 or lower or 7.0 or higher, the bacterial cell growth and lactic acid assimilation ability decreased. In particular, in the production of high purity D-lactic acid utilizing the L-lactic acid assimilation ability of the strain, the initial pH of the medium is pH 3.8 where the cell growth is the best and the high lactic acid assimilation property is recognized. Was found to be the best.
（３）窒素源の検討
該菌株の培養時における培地の窒素源として添加する素材の種類について検討を行った。なお、該試験はＹＭ培地を用いて調製した菌体を種菌として用い、前記ＬＹＰ培地の窒素源をYeast extract ０．１％及び各種窒素源素材０．４％に置換した培地を用いて、３０℃で７２時間振とう培養（120 oscillations/min）を行った。
(3) Examination of nitrogen source The kind of the raw material added as a nitrogen source of the culture medium at the time of culture | cultivation of this strain was examined. In this test, the cells prepared using the YM medium were used as seeds, and the nitrogen source of the LYP medium was replaced with Yeast extract 0.1% and various nitrogen source materials 0.4%. The shaking culture (120 oscillations / min) was performed at ° C for 72 hours.
窒素源素材としてPeptoneあるいはExtract ehlrichを添加した場合、菌体の増殖が良好であり、培養７２時間後の菌体濃度がＯＤ660値で２０以上になり、初発乳酸量の約５５〜６０％が資化された。また、硫酸アンモニウムを添加した場合では、先の２つの条件の場合と比較して十分な菌体の増殖は認められなかった（ＯＤ660値は１２．８８）ものの、乳酸資化量は同程度であった。これらに対して、硝酸ナトリウムあるいは尿素を添加した場合では、菌体濃度（ＯＤ660値は１２以下）及び乳酸資化量（初発乳酸量の２３〜５０％程度）は低い値であった。このように、該菌株の培養時における培地の窒素源としては、有機態窒素及びアンモニア態窒素のいずれも使用可能であった。 When Peptone or Extract ehlrich is added as a nitrogen source material, the growth of the bacterial cells is good, the bacterial cell concentration after 72 hours of culture is 20 or more in OD 660 value, and about 55-60% of the initial lactic acid amount is Was assimilated. In addition, when ammonium sulfate was added, sufficient cell growth was not observed compared to the previous two conditions (OD 660 value was 12.88), but the amount of lactic acid utilization was similar. there were. On the other hand, when sodium nitrate or urea was added, the bacterial cell concentration (OD 660 value was 12 or less) and the amount of lactic acid utilization (about 23 to 50% of the initial lactic acid amount) were low. Thus, both organic nitrogen and ammonia nitrogen could be used as the nitrogen source of the medium during the cultivation of the strain.
（４）窒素源濃度の検討
さらに培地の窒素源の添加濃度についても検討を行った。なお、該試験はＹＭ培地を用いて調製した菌体を種菌として用い、本培養で使用するＬＹＰ培地の窒素源素材をYeast extractおよびPeptoneとした。
(4) Examination of nitrogen source concentration Furthermore, the addition concentration of the nitrogen source in the culture medium was also examined. In this test, cells prepared using a YM medium were used as inoculum, and the nitrogen source material of the LYP medium used in the main culture was Yeast extract and Peptone.
窒素源濃度０．３％以上の場合、菌体の増殖及び乳酸の資化が良好であり、乳酸資化速度も高い値を示した。中でも、窒素源濃度を０．７％とした場合、培養７２時間後に菌体濃度が最大（ＯＤ660値は２６．９）となり、初発乳酸量の約６３％が資化された。これに対し、窒素源濃度０．１％の場合は、培養７２時間後の菌体濃度（ＯＤ660値は９．７５）は小さく、資化された乳酸量も初発量の５０％以下であった。以上を総合的に考慮すれば、培地の窒素源及びその濃度は、Yeast extract ０．２５％とPeptone ０．２５％とを含む場合が最適であると判断された。ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の増殖範囲及び最適培養条件を表３に示す。 When the nitrogen source concentration was 0.3% or more, the cell growth and lactic acid utilization were good, and the lactic acid utilization rate was also high. In particular, when the nitrogen source concentration was 0.7%, the bacterial cell concentration reached its maximum after 72 hours of culture (OD 660 value was 26.9), and about 63% of the initial lactic acid amount was assimilated. In contrast, when the nitrogen source concentration was 0.1%, the cell concentration after 72 hours of culture (OD 660 value was 9.75) was small, and the amount of lactic acid utilized was 50% or less of the initial amount. It was. Considering the above comprehensively, it was determined that the nitrogen source of the medium and its concentration contained 0.25% Yeast extract and 0.25% Peptone when optimal. Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) growth ranges and optimal culture conditions shown in Table 3.
３．乳酸試薬からの高光学純度Ｄ-乳酸の生産
（１）実験方法
実験には、市販の乳酸（DL-乳酸比50：50）を用いて調製したLYP培地を使用し、ここにあらかじめYM培地を用いて培養したピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）を添加して振とう培養を行い、液中の乳酸光学純度を経時的に測定した。
3. Production of high optical purity D-lactic acid from lactic acid reagent (1) Experimental method For the experiment, LYP medium prepared using commercially available lactic acid (DL-lactic acid ratio 50:50) was used. using Pichia Manshurika cultured with (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) performs addition to shaking culture was measured over time lactate optical purity in the liquid.
（２）結果
図２はＤＬ-乳酸を含む培地（LYP培地）を乳酸溶液として高光学純度Ｄ‐乳酸の生産を実施した結果を示す図である。図２に示すように、培養開始直後より菌体の良好な増殖が認められ、培養開始４８時間で菌体量は定常となった。また、菌体増殖と連動するような型式でのＬ−乳酸濃度の急激な低下が観察され、これに起因すると思われる培養液ｐＨの上昇も認められた。しかしながら、培養液中のＤ−乳酸濃度にはほとんど変化が見られなかった。培養開始７２時間後における培養液中のＤＬ比は88.1：11.9であり、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の添加によるＤ−乳酸の光学純度向上が認められた。
(2) Results FIG. 2 is a diagram showing the results of production of high optical purity D-lactic acid using DL-lactic acid-containing medium (LYP medium) as a lactic acid solution. As shown in FIG. 2, good growth of the bacterial cells was observed immediately after the start of the culture, and the amount of the bacterial cells became steady 48 hours after the start of the culture. In addition, a rapid decrease in the L-lactic acid concentration was observed in a type linked to cell growth, and an increase in the pH of the culture solution, which was thought to be caused by this, was also observed. However, there was almost no change in the D-lactic acid concentration in the culture solution. DL ratio in the culture medium after the culture after 72 hours 88.1: 11.9, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) optical purity improvement of D- lactic acid by the addition of recognized It was.
以上のことから、Ｌ−乳酸を優先的あるいは選択的に資化するというピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の特性に着目し、本菌の培養系を利用することで、高光学純度Ｄ−乳酸の調製が十分に可能であることが示唆された。 From the above, L- lactic acid preferentially or selectively Pichia that assimilates Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) focused on the characteristics of, utilizing a culture system of this bacterium It was suggested that high optical purity D-lactic acid can be sufficiently prepared.
４．乳酸発酵液からの高光学純度Ｄ‐乳酸の生産
（１）乳酸発酵液の調製
乳酸溶液として、上記１．（２）に記載の乳酸生産菌を液体培養して得られた乳酸発酵液を使用した。その調製手順は以下の通りである。
4). Production of high optical purity D-lactic acid from lactic acid fermentation broth (1) Preparation of lactic acid fermentation broth A lactic acid fermentation broth obtained by liquid culture of the lactic acid-producing bacterium described in (2) was used. The preparation procedure is as follows.
すなわち、100ml容三角フラスコにMRS broth培地（Oxoid社製）50mlを入れ、121℃、10分の条件でオートクレーブ滅菌を行った。ここに前記１．（２）に記載の乳酸生産菌を１白金線接種し、各乳酸生産菌の適温（Lb. delbrueckii 141株：37℃、Lb. plantarum NRIC1067株：30℃、Lb. paracasei 1532株：30℃）で24時間静置培養した（前培養）。 That is, 50 ml of MRS broth medium (Oxoid) was placed in a 100 ml Erlenmeyer flask and autoclaved at 121 ° C. for 10 minutes. Here, the above 1. Inoculate one lactic acid-producing bacterium described in (2) with a platinum wire, and select the appropriate temperature for each lactic acid-producing bacterium (Lb. delbrueckii 141 strain: 37 ° C, Lb. plantarum NRIC1067 strain: 30 ° C, Lb. paracasei 1532 strain: 30 ° C) For 24 hours (pre-culture).
500ml容三角フラスコにＧＹＰ培地（Glucose 1%、Yeast extract 1%、Peptone 0.5%、CH3COONa・3H2O 0.1%、MgSO4・7H2O 0.02%、FeSO4・7H2O 0.001%、MnSO4・4H2O 0.001%、NaCl 0.001%、Tween80 0.05%）300mlを入れ、121℃、10分の条件でオートクレーブ滅菌をした後、ここに上述の前培養液3mlおよび、あらかじめ乾熱滅菌（180℃、120分）した炭酸カルシウム6.0gを添加し、マグネティックスターラーにより液を穏やかに撹拌しながら、各乳酸生産菌の生育最適温度で48時間培養（本培養）した。 GYP medium (Glucose 1%, Yeast extract 1%, Peptone 0.5%, CH 3 COONa · 3H 2 O 0.1%, MgSO 4 · 7H 2 O 0.02%, FeSO 4 · 7H 2 O 0.001%, MnSO 4 · 4H 2 O 0.001%, NaCl 0.001%, Tween80 0.05%) 300ml was added, autoclaved at 121 ° C for 10 minutes, then 3ml of the above pre-cultured solution and dry heat sterilization (180 Calcium carbonate (6.0 g, 120 minutes at 120 ° C.) was added, and the solution was cultured for 48 hours (main culture) at the optimum growth temperature for each lactic acid-producing bacterium while gently stirring the solution with a magnetic stirrer.
そして、培養液を遠心分離（4℃、10000G、30分）して得た上清を、121℃、10分の条件でオートクレーブ滅菌したあと流水で急冷し、これを乳酸発酵液とした。 Then, the supernatant obtained by centrifuging the culture solution (4 ° C., 10000 G, 30 minutes) was autoclaved at 121 ° C. for 10 minutes and then rapidly cooled with running water to obtain a lactic acid fermentation broth.
なお乳酸発酵液の名称は、その調製に使用した乳酸菌の種類によって、それぞれＤ‐乳酸発酵液（Lb. delbrueckii 141株により調製）、ＤＬ‐乳酸発酵液（Lb. plantarum NRIC 1067株により調製）およびＬ‐乳酸発酵液（Lb. paracasei 1532株により調製）とした。 The names of the lactic acid fermentation broth are D-lactic acid fermentation broth (prepared by Lb. delbrueckii 141 strain), DL-lactic acid fermentation broth (prepared by Lb. plantarum NRIC 1067 strain) and lactic acid bacteria used for the preparation, respectively. L-lactic acid fermentation broth (prepared by Lb. paracasei 1532 strain) was used.
各乳酸発酵液に含まれる乳酸のＤＬ比は乳酸生産菌の種類によって異なり、Ｄ‐乳酸発酵液はＤＬ比＝97.6：2.4、ＤＬ‐乳酸発酵液はＤＬ比＝51.4：48.6、Ｌ‐乳酸発酵液はＤＬ比＝6.1：93.9であった。 The DL ratio of lactic acid contained in each lactic acid fermentation broth varies depending on the type of lactic acid producing bacteria, DL-lactic acid fermentation broth has DL ratio = 97.6: 2.4, DL-lactic acid fermentation broth has DL ratio = 51.4: 48.6, L-lactic acid fermentation The liquid had a DL ratio = 6.1: 93.9.
（２）実験方法
Ｄ‐乳酸発酵液（ＤＬ比＝97.6：2.4）に、あらかじめＹＭ培地を用いて培養したピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）を添加してこれを３０℃で振とう培養し、培養液中の乳酸光学純度を経時的に測定した。また、比較対象として、ＤＬ‐乳酸発酵液（ＤＬ比＝51.4：48.6）、Ｌ‐乳酸発酵液（ＤＬ比＝6.1：93.9）を乳酸溶液として用いた場合についても同様に実施した。
(2) Experimental method D- lactic acid fermentation liquor (DL ratio = 97.6: 2.4) to advance Pichia cultured with YM medium Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) was added to This was cultured with shaking at 30 ° C., and the optical purity of lactic acid in the culture was measured over time. Moreover, it implemented similarly also when the DL-lactic acid fermentation liquid (DL ratio = 51.4: 48.6) and the L-lactic acid fermentation liquid (DL ratio = 6.1: 93.9) were used as a lactic acid solution as a comparison object.
（３）結果
図３はＤ‐乳酸発酵液を乳酸溶液として高光学純度Ｄ‐乳酸の生産を実施した結果を示す図である。図３に示すように、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）は培養開始直後より良好に生育し、培養開始４８時間でその菌体量はほぼ定常値となった。また、菌体増殖と連動して培養液中のＬ−乳酸もその大部分が資化されたが、Ｄ−乳酸は初発の半分以上が残存しており、培養開始７２時間のＤ−乳酸濃度は約8.8g/l（回収率57.33%）であった。また同時点における培養液中の乳酸のＤＬ比は約99.8：0.2であった。
(3) Results FIG. 3 is a diagram showing the results of producing high optical purity D-lactic acid using a D-lactic acid fermentation broth as a lactic acid solution. As shown in FIG. 3, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) is better grown immediately after the start of culture, the cell amount in the culture after 48 hours almost constant value It was. In addition, most of L-lactic acid in the culture solution was assimilated in association with cell growth, but more than half of the initial D-lactic acid remained, and the D-lactic acid concentration at 72 hours from the start of the culture Was about 8.8 g / l (recovery rate 57.33%). At the same time, the DL ratio of lactic acid in the culture broth was about 99.8: 0.2.
以上のことから、Ｄ−乳酸生産菌の発酵液を対象とした場合においても、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の乳酸資化特性を利用した高光学純度Ｄ−乳酸の調製が可能であることが示唆された。 From the above, when targeting the fermentation liquor of D- lactic acid-producing bacteria also Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) enantiopure utilizing lactic acid assimilation properties of It was suggested that preparation of D-lactic acid is possible.
図４及び図５はＤＬ‐乳酸発酵液及びＬ‐乳酸発酵液を乳酸溶液として高光学純度Ｄ‐乳酸の生産を実施した結果を示す図である。ＤＬ‐乳酸発酵液（ＤＬ比＝51：49）を使用してもＤ‐乳酸発酵液を使用した場合と比較してピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の生育にほとんど差は認められなかった。しかしながら、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）を添加した直後から、液中に多量に存在しているＬ−乳酸が直ちに資化され、これを伴って培養液ｐＨ値が急激に上昇した。 4 and 5 are diagrams showing the results of production of high optical purity D-lactic acid using DL-lactic acid fermentation broth and L-lactic acid fermentation broth as lactic acid solutions. DL- lactic acid fermentation liquor (DL ratio = 51: 49) be used compared to using the D- lactic acid fermentation liquor Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) of There was almost no difference in growth. However, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) immediately after adding, the L- lactic acid is present in large amounts in the liquid immediately assimilated culture with this The pH value increased rapidly.
分離菌ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）によるＬ−乳酸の資化は培養７２時間でほぼ停止し、その時点における液中の乳酸のＤＬ比は81：19であったことから、分離菌によるＤ−乳酸の光学純度向上が確認されたが、その光学純度や収量はＤ‐乳酸発酵液を用いた場合と比べると低い値であった。 Isolates Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) assimilation of by L- lactic acid almost stopped at 72 hours of culture, DL ratio of lactic acid in the liquid at that point 81:19 Therefore, although the optical purity improvement of D-lactic acid by isolate | separating bacteria was confirmed, the optical purity and yield were low values compared with the case where D-lactic acid fermentation liquor was used.
一方、液中のＬ−乳酸含量が極めて高いＬ‐乳酸発酵液（ＤＬ比＝6.1：93.9）を用いた場合には、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の添加によりＬ−乳酸が資化されたが、７２時間の培養を行っても系内にＬ−乳酸が多量に残存しており、乳酸のＤＬ比は11：89であったことからＤ−乳酸の光学純度が向上したと考えられるが、その程度は極めて低いものであった。 On the other hand, L- lactic acid content is very high L- lactic acid fermentation liquor in the liquid (DL ratio = 6.1: 93.9) in the case of using the Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) Although L-lactic acid was assimilated by the addition of L-lactic acid, a large amount of L-lactic acid remained in the system even after 72 hours of culture, and the DL ratio of lactic acid was 11:89. It is thought that the optical purity of lactic acid was improved, but the degree was extremely low.
以上のことから、ピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）のＬ−乳酸資化特性を利用すれば、乳酸発酵液の種類を問わずその液中Ｄ−乳酸の光学純度を向上させることが可能であることが示された。また、高光学純度の乳酸を高収量で得るためには、Ｄ−乳酸の初発光学純度や濃度が高い試料を用いることが望ましいことが推察された。 From the above, Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) By using the L- lactic acid assimilation properties of the liquid during the D- lactic acid regardless of the type of lactic acid fermentation liquor It was shown that the optical purity can be improved. Moreover, in order to obtain high optical purity lactic acid with high yield, it was guessed that it was desirable to use a sample with high initial optical purity and concentration of D-lactic acid.
４．混合培養法における高純度Ｄ‐乳酸生産
これまでの高光学純度Ｄ−乳酸の調製は、「Ｄ−乳酸溶液の調製」と「Ｄ−乳酸の高光学純度化」という２段階の工程からなっており、各工程で使用する種菌もそれぞれ別途用意する必要があることから、工程の運転・管理の簡素化や生産効率の向上に関して改善すべき問題点も少なくないと考えられた。そこで、これら問題の解決策の１つとして、ラクトバチルス デルブリュッキ（Lb. delbrueckii）141株とピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）との混合培養による高光学純度Ｄ−乳酸の生産を試みた。
4). Production of high-purity D-lactic acid in a mixed culture method The preparation of high-optical purity D-lactic acid so far consists of two steps: "preparation of D-lactic acid solution" and "high optical purity of D-lactic acid". In addition, since it is necessary to separately prepare the inoculum used in each process, it was thought that there were not a few problems that should be improved with regard to simplification of operation and management of the process and improvement of production efficiency. Therefore, as one of these solutions to problems, Lactobacillus Deruburyukki (Lb. delbrueckii) 141 strain and Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) and high optical purity D by mixed culture of -Attempted to produce lactic acid.
（１）実験方法
坂口フラスコを用いた振とう培養法によるラクトバチルス デルブリュッキ（Lb. delbrueckii）141株とピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）との混合培養を行い、高光学純度Ｄ−乳酸の生産を試みた。実験には、炭酸カルシウム０．５％を添加したＧＹＰ培地を使用し、培養開始から２４時間後まではＤ−乳酸生産菌であるラクトバチルス デルブリュッキ（Lb. delbrueckii）141株の最適生育温度である３７℃で静置培養を行い、培養２４時間目以降はＬ−乳酸資化性菌であるピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の最適生育温度である３０℃で振とう培養を行った。
(1) Lactobacillus Deruburyukki by shaking culture using experimental methods Sakaguchi flask (Lb. delbrueckii) 141 strain and Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) and mixed and incubated with An attempt was made to produce high optical purity D-lactic acid. In the experiment, a GYP medium supplemented with 0.5% calcium carbonate was used, and the optimal growth temperature of Lb. delbrueckii 141 strain, which is a D-lactic acid-producing bacterium, was maintained until 24 hours after the start of the culture. perform static culture at 37 ° C., Pichia Manshurika (Pichia manshurica) LAAM001 strain 24 hours after culturing is L- lactate-utilizing bacteria (consignment number: NITE P -902) 30 ℃ the optimum growth temperature of Incubated with shaking.
（２）結果
図６はラクトバチルス デルブリュッキー（Lb. delbrueckii）141株とピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）の混合培養法により高純度Ｄ‐乳酸生産を実施した結果を示す図である。図６に示すように、ＯＤ660で表される菌体の生育は典型的なシグモイド曲線となり、培地に添加したグルコースは培養開始後約４８時間で消費されたことから、いずれの菌も培養期間全体を通じて良好に生育していると考えられた。
(2) Results FIG. 6 Lactobacillus del Brussels key (Lb. delbrueckii) 141 strain and Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) of high purity D- lactic acid produced by mixed culture method It is a figure which shows the implemented result. As shown in FIG. 6, the growth of the microbial cell represented by OD 660 has a typical sigmoid curve, and glucose added to the medium was consumed about 48 hours after the start of the culture. It was thought that it grew well throughout.
また、Ｄ−乳酸の生産形態は菌体の増殖と連動しており、７２時間の混合培養により11.98g/l（対糖収率59.90%）のＤ−乳酸が得られた。一方、培養液中のＬ−乳酸濃度は培養開始２４時間目までは増加し、その濃度は最大で約0.2g/lに達したが、それ以後濃度は徐々に低下し、72時間の培養で0.05g/lとなった。72時間の培養で得られたＤ‐乳酸の光学純度は99.58%であり、ラクトバチルス デルブリュッキ（Lb. delbrueckii）141株とピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）との混合培養により、極めて高光学純度のＤ−乳酸を効率的に生産可能であることが明らかとなった。
The production form of D-lactic acid is linked to the growth of the bacterial cells, and 11.98 g / l (59.90% sugar yield) of D-lactic acid was obtained by the mixed culture for 72 hours. On the other hand, the L-lactic acid concentration in the culture solution increased until 24 hours after the start of the culture, and reached a maximum of about 0.2 g / l. Thereafter, the concentration gradually decreased, and the culture was continued for 72 hours. It became 0.05g / l. The optical purity of the obtained D- lactic acid in culture of 72 hours was 99.58%, Lactobacillus Deruburyukki (Lb. delbrueckii) 141 strain and Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) and It was revealed that extremely high optical purity D-lactic acid can be efficiently produced by the mixed culture.
Claims (8)
該乳酸溶液中でＬ−乳酸資化性菌であるピキア マンシュリカ（Pichia manshurica）LAAM001株（受託番号：ＮＩＴＥ Ｐ−９０２）を培養し、該乳酸溶液中のＬ−乳酸を資化させることによってＤ−乳酸の純度を高めるＬ−乳酸除去工程と、
を有する高純度乳酸の製造方法。 A lactic acid solution preparation step of preparing a lactic acid solution containing D-lactic acid and L-lactic acid;
In the lactic acid solution is L- lactate-utilizing bacteria Pichia Manshurika (Pichia manshurica) LAAM001 strain (consignment number: NITE P -902) culturing, by assimilating L- lactic acid the lactic acid solution An L-lactic acid removal step for increasing the purity of D-lactic acid;
A method for producing high-purity lactic acid.
前記Ｌ−乳酸資化性菌の最適生育条件が、３０〜３５℃、ｐＨ３．０〜３．８の好気培養である、
請求項７に記載の高純度乳酸の製造方法。 The optimal growth conditions for the D-lactic acid-producing bacterium are anaerobic culture at 35 to 40 ° C. and pH 4.5 to 5.5,
The optimal growth conditions for the L-lactic acid-assimilating bacterium are aerobic culture at 30 to 35 ° C. and pH 3.0 to 3.8.
The method for producing high-purity lactic acid according to claim 7.
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