JP6390476B2 - Method for producing D-lactic acid - Google Patents
Method for producing D-lactic acid Download PDFInfo
- Publication number
- JP6390476B2 JP6390476B2 JP2015051558A JP2015051558A JP6390476B2 JP 6390476 B2 JP6390476 B2 JP 6390476B2 JP 2015051558 A JP2015051558 A JP 2015051558A JP 2015051558 A JP2015051558 A JP 2015051558A JP 6390476 B2 JP6390476 B2 JP 6390476B2
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- Prior art keywords
- lactic acid
- producing
- medium
- production example
- fermentation
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- 238000004519 manufacturing process Methods 0.000 title claims description 115
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- 229940022769 d- lactic acid Drugs 0.000 title claims description 96
- 229930182843 D-Lactic acid Natural products 0.000 title claims description 93
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 72
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 claims description 66
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- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229940057867 methyl lactate Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000002791 naphthoquinones Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 239000010893 paper waste Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 150000004968 peroxymonosulfuric acids Chemical class 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- XTUSEBKMEQERQV-UHFFFAOYSA-N propan-2-ol;hydrate Chemical compound O.CC(C)O XTUSEBKMEQERQV-UHFFFAOYSA-N 0.000 description 1
- 239000013055 pulp slurry Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 238000007348 radical reaction Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 239000011122 softwood Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 150000008163 sugars Chemical group 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000001238 wet grinding Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、木質バイオマスを原料として使用し、ビタミン類を添加した培地でＤ−乳酸生産菌を培養することにより、Ｄ−乳酸生産菌を用いてＤ−乳酸を製造する方法に関する。 The present invention relates to a method for producing D-lactic acid using a D-lactic acid-producing bacterium by culturing the D-lactic acid-producing bacterium in a medium to which vitamins are added, using woody biomass as a raw material.
セルロースを含む木質系バイオマスを原料とし、微生物の働きにより、エタノール、ブ
タノール等の燃料として利用可能な物質や、有用物質を発酵生産する方法が実施されてい
る。発酵生産される成分のうち、乳酸は、生物の解糖系によりグルコースなどの糖分が分
解されて生産される有機酸であり、ＴＣＡ回路に誘導されて生体エネルギー産生の起点に
なる重要な化合物である。乳酸をエステル化した乳酸メチルや乳酸エチルはバイオ燃料と
して利用できる可能性がある。また乳酸は溶剤、食品の原料になりうる。さらに光学純度
の高い乳酸は、ポリ−Ｌ−乳酸又はポリ−Ｄ−乳酸を合成するための原料モノマーとなる
。原料モノマーの光学純度は、重合度やガラス転移点に影響するため、ポリ乳酸製造のた
めには原料である乳酸の光学純度が高いこと（例えば、９８％ee以上）が必要とされる。
A method of fermenting and producing a substance that can be used as a fuel such as ethanol and butanol by the action of microorganisms using woody biomass containing cellulose as a raw material has been implemented. Among the components produced by fermentation, lactic acid is an organic acid produced by the decomposition of sugars such as glucose by the biological glycolysis system, and is an important compound that is induced by the TCA circuit and becomes the starting point for bioenergy production. is there. There is a possibility that methyl lactate or ethyl lactate esterified with lactic acid can be used as a biofuel. Lactic acid can be a solvent and a raw material for food. Furthermore, lactic acid with high optical purity becomes a raw material monomer for synthesizing poly-L-lactic acid or poly-D-lactic acid. Since the optical purity of the raw material monomer affects the degree of polymerization and the glass transition point, the optical purity of lactic acid, which is the raw material, is required to be high for production of polylactic acid (for example, 98% ee or more).
乳酸の発酵生産を効率的に行うための方法が種々検討されている。例えば、特許文献１は、高い収率で、かつ安価に乳酸を製造できる方法として、乳酸菌の培養における培地が窒素源として乾燥酵母を含むことを特徴とする乳酸菌による乳酸の製造方法を提案している。また特許文献２は、リグノセルロース系バイオマスから、効率よく糖化液を製造する方法として、リグノセルロース系バイオマスを粉砕する工程と、得られた粉砕物を加水分解酵素を用いて加水分解する工程とを含む、糖化液の製造方法を提案する。さらに該糖化液を含む培地で微生物を培養することによる乳酸などの微生物代謝産物の製造方法を提案する。 Various methods for efficiently performing fermentation production of lactic acid have been studied. For example, Patent Document 1 proposes a method for producing lactic acid by lactic acid bacteria, characterized in that a medium in culturing lactic acid bacteria contains dry yeast as a nitrogen source as a method for producing lactic acid at a high yield and at low cost. Yes. Patent Document 2 discloses a method for efficiently producing a saccharified solution from lignocellulosic biomass, a step of pulverizing lignocellulosic biomass, and a step of hydrolyzing the obtained pulverized product using a hydrolase. A method for producing a saccharified solution is proposed. Furthermore, a method for producing a microbial metabolite such as lactic acid by culturing a microorganism in a medium containing the saccharified solution is proposed.
さらに、Ｄ−乳酸製造では発酵培地に窒素源を多量に添加することによる副生産物の産生が問題となっていた。例えば、特許文献３は、Ｄ−乳酸生産菌の培養における培地のシステイン含量を高めると共に、システイン以外の窒素源を含む培地成分を低減させることにより、副生産物が少なく、高光学純度のＤ−乳酸の製造方法を提案している。 Furthermore, in the production of D-lactic acid, the production of by-products by adding a large amount of nitrogen source to the fermentation medium has been a problem. For example, Patent Document 3 discloses that D-lactic acid with high optical purity is obtained by increasing the cysteine content of a medium in culturing D-lactic acid-producing bacteria and reducing medium components containing a nitrogen source other than cysteine, thereby reducing by-products. A method for producing lactic acid is proposed.
本発明は、木質バイオマスを原料として使用し、Ｄ−乳酸生産菌を用いて、高い光学純
度のＤ−乳酸を高い生産量で製造する方法を提供することを解決すべき課題とした。
This invention made it the subject which should be solved to provide the method of manufacturing D-lactic acid of high optical purity by a high production amount using woody biomass as a raw material and using D-lactic acid producing microbe.
乳酸菌の栄養要求性の研究は古くからなされており、乳酸菌の生育には複雑な栄養要求性があることが良く知られている。その結果、乳酸菌の培養においては多くのアミノ酸、ビタミン類、核酸、金属塩を含有する酵母抽出物等の天然培地が使用されている。本発明者らはバイオマス原料からのＤ−乳酸の製造における培地について検討する中でビタミン類に着目した。従来、乳酸菌の培養に使用される天然培地には微量のビタミン類が含まれており、目的とする乳酸菌の増殖に適した天然培地を選択すること、あるいは栄養要求性の少ない乳酸菌を選択することが一般的であった。一方、本発明者らは、ホモ乳酸発酵を行い、Ｄ−乳酸の生産性に優れた乳酸菌に対して、培地へのビタミン類の添加を検討して、Ｄ−乳酸の生産量と光学純度に影響を与えるビタミン類を見出した。関連するビタミン類のうち、シアノコバラミン（ビタミンＢ１２）とリボフラビン（ビタミンＢ２）の添加濃度を適切な濃度にすることにより、高い光学純度のＤ−乳酸を高い生産量で製造できることを見出し、本発明を完成するに至った。 Research on auxotrophy of lactic acid bacteria has been made for a long time, and it is well known that growth of lactic acid bacteria has complex auxotrophy. As a result, natural media such as yeast extract containing many amino acids, vitamins, nucleic acids, and metal salts are used for culturing lactic acid bacteria. The present inventors paid attention to vitamins in examining the culture medium in the production of D-lactic acid from biomass raw materials. Traditionally, natural media used for culturing lactic acid bacteria contain trace amounts of vitamins. Select natural media suitable for the growth of the desired lactic acid bacteria, or select lactic acid bacteria with low auxotrophy. Was common. On the other hand, the present inventors conducted homolactic fermentation and examined the addition of vitamins to the medium for lactic acid bacteria excellent in D-lactic acid productivity, and thus improved the production amount and optical purity of D-lactic acid. I found vitamins that have an effect. Among related vitamins, it has been found that high optical purity D-lactic acid can be produced in a high production amount by adjusting the addition concentrations of cyanocobalamin (vitamin B12) and riboflavin (vitamin B2) to an appropriate concentration. It came to be completed.
即ち、本発明によれば、以下の発明が提供される。
［１］Ｄ−乳酸生産菌を用いて木質バイオマスからＤ−乳酸を発酵生産するＤ−乳酸の製造方法であって、Ｄ−乳酸生産菌による発酵が、シアノコバラミンを添加した培地中で行われることを特徴とするＤ−乳酸の製造方法。
［２］Ｄ−乳酸生産菌を用いて木質バイオマスからＤ−乳酸を発酵生産するＤ−乳酸の製造方法であって、Ｄ−乳酸生産菌による発酵が、シアノコバラミンとリボフラビンを添加した培地中で行われることを特徴とする［１］に記載のＤ−乳酸の製造方法。
［３］Ｄ−乳酸生産菌を用いて木質バイオマスからＤ−乳酸を発酵生産するＤ−乳酸の製造方法であって、Ｄ−乳酸生産菌による発酵が、シアノコバラミン濃度が０．１ｍｇ／Ｌ以上を含む培地中で行われることを特徴とする［１］または［２］に記載のＤ−乳酸の製造方法。
［４］Ｄ−乳酸生産菌を用いて木質バイオマスからＤ−乳酸を発酵生産するＤ−乳酸の製造方法であって、Ｄ−乳酸生産菌による発酵が、シアノコバラミン濃度が０．１ｍｇ／Ｌ以上、かつリボフラビン濃度が０．２５ｍｇ／Ｌ以下を含む培地中で行われることを特徴とする［１］〜［３］のいずれか１項に記載のＤ−乳酸の製造方法。
［５］Ｄ―乳酸生産菌による発酵が、酵素及びＤ−乳酸生産菌を同時に作用させて糖化及び発酵を併行して行われることを特徴とする［１］〜［４］のいずれか１項に記載のＤ−乳酸の製造方法。
［６］糖化の前、又は糖化及び発酵を併行して行う前に、木質バイオマスに前処理を施すことを特徴とする［１］〜［５］のいずれか１項に記載のＤ−乳酸の製造方法。
［７］Ｄ−乳酸生産菌が、ラクトバシラス属に属する乳酸菌であることを特徴とする［１］〜［６］のいずれか１項に記載のＤ−乳酸の製造方法。
［８］Ｄ−乳酸生産菌が、ラクトバシラス・デルブルキに属する乳酸菌であることを特徴とする［１］〜［７］のいずれか１項に記載のＤ−乳酸の製造方法。
That is, according to the present invention, the following inventions are provided.
[1] A method for producing D-lactic acid by fermentative production of D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein the fermentation by the D-lactic acid-producing bacterium is performed in a medium to which cyanocobalamin has been added. A process for producing D-lactic acid, characterized in that
[2] A method for producing D-lactic acid by fermentatively producing D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein the fermentation by the D-lactic acid-producing bacterium is performed in a medium to which cyanocobalamin and riboflavin are added. The method for producing D-lactic acid according to [1], wherein
[3] A method for producing D-lactic acid by fermentatively producing D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein the fermentation by the D-lactic acid-producing bacterium has a cyanocobalamin concentration of 0.1 mg / L or more. The method for producing D-lactic acid according to [1] or [2], which is performed in a medium containing the same.
[4] A method for producing D-lactic acid by fermentatively producing D-lactic acid from woody biomass using a D-lactic acid-producing bacterium, wherein fermentation by the D-lactic acid-producing bacterium has a cyanocobalamin concentration of 0.1 mg / L or more, The method for producing D-lactic acid according to any one of [1] to [3], wherein the method is performed in a medium containing a riboflavin concentration of 0.25 mg / L or less.
[5] Any one of [1] to [4], wherein the fermentation with the D-lactic acid-producing bacterium is carried out by simultaneously causing the enzyme and the D-lactic acid-producing bacterium to act simultaneously with saccharification and fermentation. A method for producing D-lactic acid according to 1.
[6] Before the saccharification or before the saccharification and fermentation are performed in parallel, the woody biomass is pretreated, wherein the D-lactic acid according to any one of [1] to [5] Production method.
[7] The method for producing D-lactic acid according to any one of [1] to [6], wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to the genus Lactobacillus.
[8] The method for producing D-lactic acid according to any one of [1] to [7], wherein the D-lactic acid-producing bacterium is a lactic acid bacterium belonging to Lactobacillus delbruki.
本発明によれば、木質バイオマスを原料として、Ｄ−乳酸生産菌を用いて、高い光学純
度のＤ−乳酸を高い生産量で製造することができる。
ADVANTAGE OF THE INVENTION According to this invention, D-lactic acid with high optical purity can be manufactured with a high production amount using D-lactic acid-producing bacteria using woody biomass as a raw material.
以下、本発明について更に詳細に説明する。
＜木質バイオマス＞
本発明の方法で原料として使用する木質バイオマスとしては、製紙用樹木、林地残材、
間伐材等のチップ又は樹皮、木本性植物の切株から発生した萌芽、製材工場等から発生す
る鋸屑又はおがくず、街路樹の剪定枝葉、建築廃材等が挙げられる。さらに、木材由来の
紙、古紙、パルプ等を原料として利用することができる。これらの木質バイオマスは、単
独、あるいは複数を組み合わせて使用することができる。また、木質バイオマスは、乾燥
固形物であっても、水分を含んだ固形物であっても、スラリーであっても用いることがで
きる。
Hereinafter, the present invention will be described in more detail.
<Wood biomass>
As woody biomass used as a raw material in the method of the present invention, a tree for papermaking, a forest land residual material,
Chips or bark of thinned wood, germination generated from stumps of woody plants, sawdust or sawdust generated from sawmills, pruned branches of street trees, construction waste, etc. Furthermore, wood-derived paper, waste paper, pulp and the like can be used as raw materials. These woody biomass can be used alone or in combination. Further, the woody biomass can be used as a dry solid, a solid containing moisture, or a slurry.
木質バイオマスの原料としては、ユーカリ（Ｅｕｃａｌｙｐｔｕｓ）属植物、ヤナギ（
Ｓａｌｉｘ）属植物、ポプラ（Ｐｏｐｕｌｕｓ）属植物、アカシア（Ａｃａｃｉａ）属植物、スギ（Ｃｒｙｐｔｏｍｅｒｉａ）属植物、マツ（Ｐｉｎｕｓ）属植物等が利用できる。特に、ユーカリ属植物、アカシア属、ヤナギ属植物が原料として大量に採取し易いため好ましい。例えば、製紙原料用として一般に用いられるユーカリ（Ｅｕｃａｌｙｐｔｕｓ）属又はアカシア（Ａｃａｃｉａ）属等の樹種の樹皮は、製紙原料用の製材工場やチップ工場等から安定して大量に入手可能であるため、特に好適に用いられる。
The raw materials for woody biomass include Eucalyptus genus plants, willow (
Salix genus plants, Populus genus plants, Acacia genus plants, Cryptomeria genus plants, Pinus genus plants and the like can be used. In particular, Eucalyptus plants, Acacia plants, and Willow plants are preferable because they can be easily collected in large quantities as raw materials. For example, bark of tree species such as Eucalyptus genus or Acacia genus commonly used for papermaking raw materials can be obtained in large quantities stably from lumber mills and chip factories for papermaking raw materials. Preferably used.
（前処理）
本発明においては、木質バイオマスに、糖化処理及び発酵処理に適した前処理を施すこ
とができる。木質バイオマスに前処理を行ったものを、「前処理原料」と称することがあ
る。前処理としては、以下に何れかの処理を挙げることができる。このような前処理を行
うことにより、木質バイオマス中のリグノセルロースは、糖化発酵可能な状態となる。
機械的処理、化学的処理、水熱処理、加圧熱水処理、二酸化炭素添加水熱処理、蒸煮処理
、湿式粉砕処理、希硫酸処理、水蒸気爆砕処理、アンモニア爆砕処理、二酸化炭素爆砕処
理、超音波照射処理、マイクロ波照射処理、電子線照射処理、γ線照射処理、超臨界処理
、亜臨界処理、有機溶媒処理、相分離処理、木材腐朽菌処理、グリーン溶媒活性化処理、
各種触媒処理、ラジカル反応処理、オゾン酸化処理。
これらの処理は、各単独処理もしくは複数を組み合わせた処理のいずれであってもよい。
中でも、木質バイオマスに対し、アルカリ処理、加圧熱水処理、機械的処理から選択され
る1つ以上の前処理を行うことが好ましい。
(Preprocessing)
In the present invention, the woody biomass can be subjected to pretreatment suitable for saccharification and fermentation. A wood biomass that has been pretreated may be referred to as a “pretreated raw material”. As pre-processing, any of the following processes can be mentioned. By performing such pretreatment, the lignocellulose in the woody biomass is in a state capable of saccharification and fermentation.
Mechanical treatment, chemical treatment, hydrothermal treatment, pressurized hot water treatment, carbon dioxide added hydrothermal treatment, steaming treatment, wet grinding treatment, dilute sulfuric acid treatment, steam explosion treatment, ammonia explosion treatment, carbon dioxide explosion treatment, ultrasonic irradiation Treatment, microwave irradiation treatment, electron beam irradiation treatment, γ ray irradiation treatment, supercritical treatment, subcritical treatment, organic solvent treatment, phase separation treatment, wood decay fungus treatment, green solvent activation treatment,
Various catalyst treatment, radical reaction treatment, ozone oxidation treatment.
These processes may be either single processes or a combination of a plurality of processes.
Among these, it is preferable to perform one or more pretreatments selected from alkali treatment, pressurized hot water treatment, and mechanical treatment on woody biomass.
機械的処理としては、破砕、裁断、磨砕等の任意の機械的手段が挙げられ、木質バイオ
マス中のリグノセルロースを糖化発酵処理工程で糖化発酵され易い状態にすることである
。使用する機械装置については特に限定されないが、例えば、一軸破砕機、二軸破砕機、
ハンマークラッシャー、レファイナー、ニーダー等を用いることができる。
Examples of the mechanical treatment include arbitrary mechanical means such as crushing, cutting, and grinding, and making lignocellulose in the woody biomass easy to be saccharified and fermented in the saccharification and fermentation treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a uniaxial crusher, a biaxial crusher,
A hammer crusher, refiner, kneader, or the like can be used.
化学的処理は、酸やアルカリ等の薬品の水溶液に木質バイオマスを浸漬して、酵素糖化
処理に適した状態にする処理である。化学的処理に使用する薬品等については特に限定さ
れないが、例えば、アルカリ金属又はアルカリ土類金属の水酸化物、硫酸、希硫酸などの
硫化物、炭酸塩又は亜硫酸塩から１種以上選択されたものである。水酸化ナトリウム、水
酸化カルシウム、硫化ナトリウム、炭酸ナトリウム、炭酸カルシウム、亜硫酸ナトリウム
等から選択された１種以上の薬品の水溶液に浸漬してなるアルカリ処理等が化学処理とし
て好適である。また、オゾン、二酸化塩素などの酸化剤による化学的処理も可能である。
化学的処理は、前記機械的処理と組み合わせてそれらの前処理の後処理として行うことが
好適である。化学的処理に使用する薬品の添加量は、状況に応じて任意に調整可能である
が、薬品コスト低下の面から、またセルロースの溶出・過分解による収率低下防止の面か
ら、木質バイオマスの絶乾１００質量部に対して５０質量部以下であることが望ましい。
化学的処理における薬品の水溶液への浸漬時間及び処理温度は、使用する原料や薬品によ
って任意に設定可能であるが、処理時間３０分〜１時間、処理温度８０〜１３０℃が好ま
しい。処理条件を厳しくすることで、原料中のセルロースの液側への溶出又は過分解が起
こる場合もあるため、処理時間は１時間以下、処理温度は１３０℃以下であることが好ま
しい。
The chemical treatment is a treatment in which woody biomass is immersed in an aqueous solution of a chemical such as acid or alkali to make it suitable for enzymatic saccharification treatment. The chemicals used for the chemical treatment are not particularly limited. For example, at least one selected from alkali metal or alkaline earth metal hydroxides, sulfuric acid, dilute sulfuric acid sulfides, carbonates or sulfites. Is. As the chemical treatment, an alkali treatment obtained by immersing in an aqueous solution of one or more kinds of chemicals selected from sodium hydroxide, calcium hydroxide, sodium sulfide, sodium carbonate, calcium carbonate, sodium sulfite and the like is suitable. Further, chemical treatment with an oxidizing agent such as ozone or chlorine dioxide is also possible.
The chemical treatment is preferably performed as a post-treatment of the pretreatment in combination with the mechanical treatment. The amount of chemicals used for chemical treatment can be arbitrarily adjusted according to the situation, but from the viewpoint of reducing chemical costs and from the viewpoint of preventing yield loss due to cellulose elution and overdegradation, It is desirable that it is 50 mass parts or less with respect to 100 mass parts of absolutely dry.
The immersion time and the treatment temperature of the chemical in the chemical treatment can be arbitrarily set depending on the raw materials and chemicals to be used, but a treatment time of 30 minutes to 1 hour and a treatment temperature of 80 to 130 ° C. are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing time is preferably 1 hour or less and the processing temperature is 130 ° C. or less.
糖化及び発酵処理に適した前処理が施されている木質バイオマスに対しては、リグノセ
ルロース原料を含む懸濁液の調製に使用する前に、殺菌処理を行ってもよい。木質バイオ
マス原料中に雑菌が混入していると、酵素による糖化を行う際に雑菌が糖を消費して生成
物の収量が低下してしまうという問題が発生する。殺菌処理は、酸やアルカリなど、菌の
生育困難なｐＨに原料を晒す方法でも良いが、高温下で処理する方法でも良く、両方を組
み合わせても良い。酸、アルカリ処理後の原料については、中性付近、もしくは、糖化・
発酵工程に適したｐＨに調整した後に原料として使用することが好ましい。また、高温殺
菌した場合も、室温もしくは糖化・発酵工程に適した温度まで降温させてから原料として
使用することが好ましい。このように、温度やｐＨを調整してから原料を送り出すことで
、好適ｐＨ、好適温度外に酵素が晒されて、失活することを防ぐことができる。
Woody biomass that has been subjected to pretreatment suitable for saccharification and fermentation treatment may be sterilized before being used for preparing a suspension containing a lignocellulose raw material. When miscellaneous bacteria are mixed in the woody biomass raw material, a problem arises that when the saccharification is performed by the enzyme, the miscellaneous bacteria consume sugar and the yield of the product is reduced. The sterilization treatment may be a method in which the raw material is exposed to a pH at which bacteria are difficult to grow, such as acid or alkali, but may be a method in which the raw material is treated at a high temperature, or a combination of both. For raw materials after acid or alkali treatment, neutral or saccharification /
It is preferable to use it as a raw material after adjusting to a pH suitable for the fermentation process. In addition, even when pasteurized at high temperature, it is preferably used as a raw material after being cooled to room temperature or a temperature suitable for the saccharification / fermentation process. Thus, by feeding out the raw material after adjusting the temperature and pH, it is possible to prevent the enzyme from being exposed to the outside of the preferred pH and the preferred temperature and being deactivated.
（広葉樹クラフトパルプ）
本発明の特に好ましい態様においては、広葉樹クラフトパルプを、木質バイオマスとし
て使用することができる。該パルプを製造するための原料として使用する木材チップとし
ては、ユーカリ、オーク、アカシア、ビーチ、タンオーク、オルダー等の広葉樹材であれ
ば特に限定されない。また、使用する広葉樹材に多少の針葉樹材を含まれていても構わな
い。
上記の木材チップをクラフト蒸解処理に供することによって、広葉樹未漂白クラフト
パルプ（ＬＵＫＰ）を得ることができる。次いで、酸素脱リグニン工程により酸素脱リグ
ニンパルプを得ることができる。さらに、酸素脱リグニンパルプを漂白処理に供すること
によって、広葉樹漂白クラフトパルプ（ＬＢＫＰ）を得ることができる。
(Hardwood kraft pulp)
In a particularly preferred embodiment of the present invention, hardwood kraft pulp can be used as woody biomass. The wood chip used as a raw material for producing the pulp is not particularly limited as long as it is a hardwood material such as eucalyptus, oak, acacia, beach, tan oak, and alder. Moreover, some softwoods may be included in the hardwood used.
Hardwood unbleached kraft pulp (LUKP) can be obtained by subjecting the wood chips to kraft cooking. Subsequently, an oxygen delignified pulp can be obtained by an oxygen delignification step. Furthermore, a hardwood bleached kraft pulp (LBKP) can be obtained by subjecting the oxygen delignified pulp to a bleaching treatment.
クラフト蒸解は公知の方法により行うことができる。例えば、木材をクラフト蒸解する
場合、クラフト蒸解液の硫化度は５〜７５％、好ましくは２０〜３５％であり、有効アル
カリ添加率は絶乾木材質量当たり５〜３０質量％、好ましくは１０〜２５質量％であり、
蒸解温度は１４０〜１７０℃である。しかし、クラフト蒸解の条件はこれらに限定される
ものではない。また、クラフト蒸解方式は、連続蒸解法あるいはバッチ蒸解法のどちらで
もよく、連続蒸解釜を用いる場合は、蒸解白液を分割で添加する蒸解法でもよく、その方
式は特に限定されない。
蒸解に際して使用する蒸解液には、蒸解助剤が添加されてもよい。例として、公知の環
状ケト化合物、例えばベンゾキノン、ナフトキノン、アントラキノン、アントロン、フェ
ナントロキノン及び前記キノン系化合物のアルキル、アミノ等の核置換体が挙げられる。
或いは前記キノン系化合物の還元型であるアントラヒドロキノンのようなヒドロキノン系
化合物が挙げられる。さらにはディールスアルダー法によるアントラキノン合成法の中間
体として得られる安定な化合物である９，１０−ジケトヒドロアントラセン化合物等が挙
げられる。これらから選ばれた１種又は２種以上が添加されてもよい。添加率は特に限定
されないが、一般的には、木材チップの絶乾質量当たり０．００１〜１．０質量％である
。
Kraft cooking can be performed by a known method. For example, when kraft cooking wood, the sulfidity of the kraft cooking solution is 5 to 75%, preferably 20 to 35%, and the effective alkali addition rate is 5 to 30% by weight, preferably 10 to 10% by weight of the absolutely dry wood. 25% by weight,
The cooking temperature is 140-170 ° C. However, the conditions for kraft cooking are not limited to these. The kraft cooking method may be either a continuous cooking method or a batch cooking method, and when a continuous cooking kettle is used, it may be a cooking method in which cooking white liquor is added in portions, and the method is not particularly limited.
A cooking aid may be added to the cooking liquid used for cooking. Examples include known cyclic keto compounds, for example, benzoquinone, naphthoquinone, anthraquinone, anthrone, phenanthroquinone, and nuclear substitutes such as alkyl and amino of the quinone compounds.
Alternatively, a hydroquinone compound such as anthrahydroquinone, which is a reduced form of the quinone compound, may be mentioned. Furthermore, the 9,10-diketohydroanthracene compound which is a stable compound obtained as an intermediate of the anthraquinone synthesis method by Diels Alder method, etc. are mentioned. One or more selected from these may be added. Although an addition rate is not specifically limited, Generally, it is 0.001-1.0 mass% per the absolute dry mass of a wood chip.
クラフト蒸解法により得られた未漂白化学パルプは、所望により、洗浄工程を経て、公
知の酸素脱リグニン法により脱リグニンすることができる。酸素脱リグニン法に用いるア
ルカリとしては苛性ソーダあるいは酸化されたクラフト白液を使用することができる。酸
素ガスとしては、深冷分離法からの酸素、ＰＳＡ（ＰＲＥＳＳＵＲＥ Ｓｗｉｎｇ Ａｄｓ
ｏｒｐｔｉｏｎ）からの酸素、ＶＳＡ（Ｖａｃｕｕｍ Ｓｗｉｎｇ Ａｄｓｏｒｐｔｉｏｎ
）からの酸素等が使用できる。
酸素脱リグニン工程では、前記酸素ガスとアルカリが中濃度ミキサーにおいて中濃度の
パルプスラリーに添加され、混合が十分に行われた後、加圧下でパルプ、酸素及びアルカ
リの混合物を一定時間保持できる反応塔へ送られ、脱リグニンされる。酸素ガスの添加率
は特に限定されないが、絶乾パルプ質量当たり０．５〜３質量％であり、アルカリ添加率
は０．５〜４質量％である。また、反応温度は８０〜１２０℃で、反応時間は１５〜１０
０分であり、パルプ濃度は８〜１５質量％であるが、これらの条件は特に限定されない。
The unbleached chemical pulp obtained by the kraft cooking method can be delignified by a known oxygen delignification method through a washing step if desired. As the alkali used in the oxygen delignification method, caustic soda or oxidized kraft white liquor can be used. Examples of oxygen gas include oxygen from a cryogenic separation method, PSA (PRESSURE Swing Ads).
oxygen, VSA (Vacuum Swing Adsorption)
Oxygen etc. from) can be used.
In the oxygen delignification step, the oxygen gas and alkali are added to a medium concentration pulp slurry in a medium concentration mixer, and after sufficiently mixed, a reaction that can hold a mixture of pulp, oxygen and alkali for a certain period of time under pressure It is sent to the tower and delignified. Although the addition rate of oxygen gas is not specifically limited, it is 0.5-3 mass% with respect to the absolute dry pulp mass, and an alkali addition rate is 0.5-4 mass%. Moreover, reaction temperature is 80-120 degreeC, and reaction time is 15-10.
Although it is 0 minutes and the pulp concentration is 8 to 15% by mass, these conditions are not particularly limited.
酸素脱リグニンを施されたパルプは洗浄工程へ送ることができる。酸素脱リグニン後の
洗浄工程で使用する洗浄機、及び多段漂白工程中の洗浄に使用する洗浄機は、特に限定さ
れるものではない。例えば、プレッシャーディフューザー、ディフュージョンウオッシャ
ー、加圧型ドラムウオッシャー、水平長網型ウオッシャー、プレス洗浄機等を挙げること
ができる。
Pulp that has been subjected to oxygen delignification can be sent to the washing step. The washing machine used in the washing process after oxygen delignification and the washing machine used for washing during the multi-stage bleaching process are not particularly limited. For example, a pressure diffuser, a diffusion washer, a pressure drum washer, a horizontal long washer, a press washer, and the like can be given.
上記の通り脱リグニン処理されたパルプは多段漂白工程へ供することができる。多段漂
白工程は、二酸化塩素（Ｄ）、アルカリ（Ｅ）、酸素（Ｏ）、過酸化水素（Ｐ）、オゾン
（Ｚ）といった公知のＥＣＦ漂白法を組合せて行うことができる。また、多段漂白工程中
に、高温酸処理段（Ａ）や酸洗浄段、酵素処理段、高温二酸化塩素漂白段、過硫酸や過酢
酸等による過酸漂白段を導入することもできる。多段漂白工程中には、エチレンジアミン
テトラ酢酸（ＥＤＴＡ）やジエチレントリアミンペンタ酢酸（ＤＴＰＡ）等によるキレー
ト剤処理段等を導入することもできる。
The pulp that has been delignified as described above can be subjected to a multistage bleaching step. The multi-stage bleaching step can be performed by combining known ECF bleaching methods such as chlorine dioxide (D), alkali (E), oxygen (O), hydrogen peroxide (P), and ozone (Z). Further, during the multi-stage bleaching step, a high-temperature acid treatment stage (A), an acid washing stage, an enzyme treatment stage, a high-temperature chlorine dioxide bleaching stage, a peracid bleaching stage using persulfuric acid or peracetic acid can be introduced. A chelating agent treatment stage with ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or the like can be introduced into the multistage bleaching process.
上記により、本発明の特に好ましい態様において原料として使用できる広葉樹漂白クラ
フトパルプ（ＬＢＫＰ）を得ることができる。
From the above, hardwood bleached kraft pulp (LBKP) that can be used as a raw material in a particularly preferred embodiment of the present invention can be obtained.
＜乳酸菌＞
本発明には、Ｄ−乳酸生産菌が用いられる。本発明に用いられるＤ−乳酸生産菌は、糖
類（六炭糖、五炭糖）を発酵して、Ｄ−乳酸を製造できるものであれば特に限定はされな
い。Ｄ−乳酸生産菌としては、例えば、ラクトバシラス属（Ｌａｃｔｏｂａｃｉｌｌｕｓ
）、ビフィドバクテリウム属（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍ）、エンテロコッカス属
（Ｅｎｔｅｒｏｃｏｃｃｕｓ）、ラクトコッカス属（Ｌａｃｔｏｃｏｃｃｕｓ）、ペデ
ィオコッカス属（Ｐｅｄｉｏｃｏｃｃｕｓ）、リューコノストック属（Ｌｅｕｃｏｎｏｓ
ｔｏｃ）、又はスポロラクトバシラス属（Ｓｐｏｌｏｌａｃｔｏｂａｃｉｌｌｕｓ属）に
属する細菌を挙げることができる。しかしながら、これらに限定されない。具体的には、
ラクトバシラス・デルブルキ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ）
、ラクトバシラス・プランタルム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）
、ロイコノストック・メセンテロイデス（Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏ
ｉｄｅｓ）などを挙げることができる。しかしながらこれらに限定されない。また、遺伝
子組換え技術を用いて作製した遺伝子組換え微生物（細菌等）を用いることもできる。遺
伝子組換え微生物としては、六炭糖又は五炭糖を発酵してＤ−乳酸を生産できる微生物を
特に制限なく用いることができる。
<Lactic acid bacteria>
In the present invention, D-lactic acid-producing bacteria are used. The D-lactic acid-producing bacterium used in the present invention is not particularly limited as long as it can produce D-lactic acid by fermenting saccharides (hexose, pentose). Examples of D-lactic acid-producing bacteria include Lactobacillus (Lactobacillus).
), Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconos
toc), or bacteria belonging to the genus Sporolactobacillus. However, it is not limited to these. In particular,
Lactobacillus delbrueckii
, Lactobacillus plantarum
Leuconostoc mesenteroides (Leuconostoc mesentero)
ides). However, it is not limited to these. Moreover, genetically modified microorganisms (bacteria etc.) produced using genetic recombination techniques can also be used. As the genetically modified microorganism, a microorganism capable of producing D-lactic acid by fermenting hexose or pentose can be used without particular limitation.
微生物は固定化して用いても良い。微生物を固定化しておくと、次工程で微生物を分離
して再回収するという工程を省くことができるため、少なくとも回収工程に要する負担を
軽減することができ、微生物のロスが軽減できるというメリットがある。また、凝集性の
ある微生物を選択することにより微生物の回収を容易にすることができる。
Microorganisms may be immobilized and used. By immobilizing microorganisms, the process of separating and re-recovering microorganisms in the next process can be omitted, so that at least the burden required for the recovery process can be reduced and the loss of microorganisms can be reduced. is there. Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.
＜糖化処理、及び発酵処理＞
本発明の実施態様においては、酵素による糖化とＤ−乳酸生産菌を用いた発酵とを順次
行ってもよく、また木質バイオマスに酵素及びＤ−乳酸生産菌を同時に作用させて糖化及
び発酵を併行して行ってもよい。なお、本明細書では、本発明の実施態様のうち、糖化及
び発酵を併行して行う態様を例に、Ｄ−乳酸生産菌による発酵工程を説明することがある
。しかしながらその説明は、特に記載した場合を除き、酵素による糖化とＤ−乳酸生産菌
を用いた発酵とを順次行う場合の発酵工程にも当てはまる。
<Saccharification treatment and fermentation treatment>
In the embodiment of the present invention, enzymatic saccharification and fermentation using a D-lactic acid-producing bacterium may be sequentially performed, and saccharification and fermentation are performed simultaneously by causing an enzyme and a D-lactic acid-producing bacterium to act simultaneously on woody biomass. You may do it. In addition, in this specification, the fermentation process by a D-lactic acid production microbe may be demonstrated to the example which carries out saccharification and fermentation simultaneously among the embodiments of this invention. However, the explanation also applies to the fermentation process in the case where saccharification by an enzyme and fermentation using a D-lactic acid-producing bacterium are sequentially performed unless otherwise specified.
Ｄ−乳酸生産菌が用いられ、原料木質バイオマスから酵素の作用で生成された糖類が、
Ｄ−乳酸生産菌によりＤ−乳酸に変換される。このようなＤ−乳酸生産菌が用いられる工
程において使用される液を培地という。なお、本発明において、培地に含まれる成分濃度は、特に記載した場合を除き、培養開始時の濃度（初濃度）をいう。
D-lactic acid producing bacteria are used, and saccharides produced by the action of enzymes from raw wood biomass are
Converted to D-lactic acid by D-lactic acid producing bacteria. A liquid used in a process in which such a D-lactic acid-producing bacterium is used is called a culture medium. In the present invention, the concentration of a component contained in a medium refers to the concentration at the start of culture (initial concentration) unless otherwise specified.
（原料バイオマスの量）
糖化工程又は併行糖化発酵工程で用いる木質バイオマスの懸濁濃度は、典型的には、１
．０質量％以上とすることができる。好ましくは１．５質量％以上とすることができ、より好ましくは３．０質量％以上とすることができ、さらに好ましくは４．５質量％以上とすることができ、さらに好ましくは５．０質量％以上とすることができ、特に好ましくは７．０質量％以上とすることができる。
(Amount of raw material biomass)
The suspension concentration of woody biomass used in the saccharification process or the concurrent saccharification and fermentation process is typically 1
. It can be 0 mass% or more. Preferably it can be 1.5% by mass or more, more preferably 3.0% by mass or more, still more preferably 4.5% by mass or more, still more preferably 5.0% by mass. It can be set to mass% or more, and particularly preferably set to 7.0 mass% or more.
（ビタミン類）
本発明において、培地にシアノコバラミン（ビタミンＢ１２）を添加する。また、シアノコバラミンとリボフラビン（ビタミンＢ２）を併用して添加することもできる。培地に添加するシアノコバラミンの濃度（終濃度）は、０．１ｍｇ／Ｌ以上が好ましい。また、シアノコバラミンとリボフラビンを併用して培地に添加する場合、培地に添加するシアノコバラミンの濃度（終濃度）は、０．１ｍｇ／Ｌ以上が好ましく、リボフラビンの濃度（終濃度）は、０．２５ｍｇ／Ｌ以下が好ましい。前記の濃度範囲のシアノコバラミン、またはリボフラビンを培地に添加することによりＤ−乳酸の光学純度、及びＤ−乳酸の生産性を高めることができる。生産するＤ−乳酸の光学純度は、９８％ee以上が好ましく、９９％ee以上がさらに好ましい。
(Vitamins)
In the present invention, cyanocobalamin (vitamin B12) is added to the medium. Moreover, cyanocobalamin and riboflavin (vitamin B2) can be used in combination. The concentration (final concentration) of cyanocobalamin added to the medium is preferably 0.1 mg / L or more. When cyanocobalamin and riboflavin are added to the medium in combination, the concentration of cyanocobalamin added to the medium (final concentration) is preferably 0.1 mg / L or more, and the concentration of riboflavin (final concentration) is 0.25 mg / liter. L or less is preferable. The optical purity of D-lactic acid and the productivity of D-lactic acid can be increased by adding cyanocobalamin or riboflavin in the above concentration range to the medium. The optical purity of the produced D-lactic acid is preferably 98% ee or higher, more preferably 99% ee or higher.
（ペプトン、牛肉エキス、酵母エキス）
本発明の好ましい態様においては、Ｄ−乳酸生産菌を培養するための培地は、酵母エキス、ペプトン（ポリペプトンも含む）、及び牛肉エキスからなる群より選択されるいずれか一つ以上を含んでいてもよい。酵母エキスとしては、例えば、市販のイーストイクスト（Difco Laboratories） 酵母エキスＬ（ＭＣフードスペシャリティーズ株式会社）、酵母エキスＳＬ−Ｗ（ＭＣフードスペシャリティーズ株式会社）、リボネックスＢ２−Ｐ（サッポロビール株式会社）、リボネックスＮ７−Ｐ（サッポロビール株式会社）、ミーストＰ１Ｇ（アサヒフードアンドヘルスケア）、ミーストＰ２Ｇ（アサヒフードアンドヘルスケア）、ミーストＡＰ−１１２２（アサヒフードアンドヘルスケア）、酵味（酵味ペースト、酵味粉末：ＭＣフードスペシャリティーズ株式会社）など特に限定なく用いることができる。酵母エキスを用いる場合、種々のものを選択できるが、サッカロマイセス属に属する酵母(例えば、サッカロマイセス・セレビシエ、サッカロマイセス・ロゼイ、サッカロマイセス・ウバルム、サッカロマイセス・シバリエリに属する酵母)を用いることができる。又はキャンディダ属に属する酵母（例えば、キャンディダ・ユティルスに属する酵母）を用いることができる。ビール酵母又はパン酵母であるサッカロマイセス・セレビシエに分類される酵母から得られたエキスを用いることができる。前記酵母エキスの中で、ビール酵母由来の酵母エキスを用いるのが好ましい。また、ビール酵母由来の酵母エキスの中で、酵素によって分解された分解物を含有する酵母エキスを用いるのが好ましい。ビール酵母由来の酵母エキスの中で、酵素によって分解された分解物（アミノ酸、ペプチド、核酸）を含有する酵母エキスとして、例えば、酵味（酵味ペースト、酵味粉末）が挙げられる。ペプトンとしては、例えば、ハイポリペプトン（日本製薬株式会社）が挙げられる。
(Peptone, beef extract, yeast extract)
In a preferred embodiment of the present invention, the medium for culturing the D-lactic acid-producing bacterium contains any one or more selected from the group consisting of yeast extract, peptone (including polypeptone), and beef extract. Also good. Examples of yeast extract include commercially available yeast extract (Difco Laboratories) yeast extract L (MC Food Specialties Co., Ltd.), yeast extract SL-W (MC Food Specialties Co., Ltd.), Ribonex B2-P (Sapporo Beer Co., Ltd.) Company), Ribonex N7-P (Sapporo Beer Co., Ltd.), Mist P1G (Asahi Food and Healthcare), Mist P2G (Asahi Food and Healthcare), Mist AP-1122 (Asahi Food and Healthcare), Fermentation (Fermentation) Taste paste, fermentation powder: MC Food Specialties Co., Ltd.) and the like can be used without particular limitation. When using yeast extract, various types can be selected, but yeast belonging to the genus Saccharomyces (for example, yeast belonging to Saccharomyces cerevisiae, Saccharomyces rosei, Saccharomyces ubalum, Saccharomyces siberi) can be used. Alternatively, yeast belonging to the genus Candida (for example, yeast belonging to Candida utils) can be used. Extracts obtained from yeast classified as Saccharomyces cerevisiae, which are beer yeast or baker's yeast, can be used. Among the yeast extracts, it is preferable to use a yeast extract derived from brewer's yeast. Moreover, it is preferable to use the yeast extract containing the degradation product decomposed | disassembled with the enzyme in the yeast extract derived from beer yeast. Among yeast extracts derived from brewer's yeast, examples of yeast extracts containing degradation products (amino acids, peptides, nucleic acids) degraded by enzymes include fermentation taste (fermented paste, fermented powder). Examples of peptone include high-poly peptone (Nippon Pharmaceutical Co., Ltd.).
ペプトン、牛肉エキス、及び酵母エキスからなる群より選択されるいずれか一つ以上を
用いる場合、培地におけるその濃度（複数用いる場合は、各々の濃度）は適宜とすること
ができる。典型的には、０．１〜１０質量％とすることができ、好ましくは０．２〜７．
５質量％とすることができ、より好ましくは０．４〜５．０質量％とすることができる。
この範囲であれば、Ｄ−乳酸生産菌の増殖及び発酵が十分に達成され、かつ経済的でもあ
る。
When any one or more selected from the group consisting of peptone, beef extract, and yeast extract is used, the concentration in the medium (each concentration in the case of using a plurality thereof) can be appropriately determined. Typically, it can be 0.1-10 mass%, Preferably it is 0.2-7.
It can be 5 mass%, More preferably, it can be 0.4-5.0 mass%.
Within this range, the growth and fermentation of D-lactic acid-producing bacteria are sufficiently achieved and economical.
（培地中の他成分）
本発明においては、培地中にマグネシウム、アルミニウム、マンガン、鉄、ストロンチウム、バリウムなどの成分（以下、「金属イオン」という。）を添加することができる。
(Other ingredients in the medium)
In the present invention, components such as magnesium, aluminum, manganese, iron, strontium, barium (hereinafter referred to as “metal ions”) can be added to the medium.
培地中にマグネシウムを添加する場合のマグネシウム濃度は６５００ｍｇ／Ｌ以下、好ましくは１０００ｍｇ／Ｌ以下、より好ましくは５００ｍｇ／Ｌ以下、さらに好ましくは１８０ｍｇ／Ｌ以下、さらに好ましくは１００ｍｇ／Ｌ以下、特に好ましくは７５ｍｇ／Ｌ以下とすることができる。培地中のマグネシウム濃度の下限値は、３２ｍｇ／Ｌ以上、好ましくは３５ｍｇ／Ｌ以上、より好ましくは４０ｍｇ／Ｌ以上、さらに好ましくは４５ｍｇ／Ｌ以上、特に好ましくは４７．５ｍｇ／Ｌ以上とすることができる。前記濃度より低いと、用いる乳酸菌によっては、乳酸菌の増殖や発酵効率が低下するため好ましくない。 When adding magnesium to the medium, the magnesium concentration is 6500 mg / L or less, preferably 1000 mg / L or less, more preferably 500 mg / L or less, further preferably 180 mg / L or less, more preferably 100 mg / L or less, particularly preferably. Can be 75 mg / L or less. The lower limit of the magnesium concentration in the medium is 32 mg / L or more, preferably 35 mg / L or more, more preferably 40 mg / L or more, still more preferably 45 mg / L or more, and particularly preferably 47.5 mg / L or more. Can do. When the concentration is lower than the above-mentioned concentration, depending on the lactic acid bacterium used, the growth and fermentation efficiency of the lactic acid bacterium are not preferable.
培地中にアルミニウムを添加する場合の培地中のアルミニウム濃度の下限値は、０．４１ｍｇ／Ｌ以上、好ましくは０．８２ｍｇ／Ｌ以上とすることができる。培地中に鉄を添加する場合の培地中の鉄濃度の下限値は、０．３３ｍｇ／Ｌ以上、好ましくは０．６６ｍｇ／Ｌ以上とすることができる。培地中にストロンチウムを添加する場合の培地中のストロンチウム濃度の下限値は、０．１５ｍｇ／Ｌ以上、好ましくは０．２９ｍｇ／Ｌ以上とすることができる。培地中にマンガンを添加する場合の培地中のマンガン濃度の下限値は、０．１３ｍｇ／Ｌ以上、好ましくは０．２６ｍｇ／Ｌ以上とすることができる。培地中にバリウムを添加する場合の培地中のバリウム濃度の下限値は、０．０９ｍｇ／Ｌ以上、好ましくは０．１８ｍｇ／Ｌ以上とすることができる。
いずれの場合も、培養や発酵を阻害しない限り、培地中の上限値は適宜、適切な値に設定とすることができるが、典型的には、５０ｍｇ／Ｌ以下であり、好ましくは２５ｍｇ／Ｌ以下とすることができる。
When aluminum is added to the medium, the lower limit of the aluminum concentration in the medium can be 0.41 mg / L or more, preferably 0.82 mg / L or more. When iron is added to the medium, the lower limit value of the iron concentration in the medium can be 0.33 mg / L or more, preferably 0.66 mg / L or more. When strontium is added to the medium, the lower limit of the strontium concentration in the medium can be 0.15 mg / L or more, preferably 0.29 mg / L or more. When manganese is added to the medium, the lower limit of the manganese concentration in the medium can be 0.13 mg / L or more, preferably 0.26 mg / L or more. When barium is added to the medium, the lower limit value of the barium concentration in the medium can be 0.09 mg / L or more, preferably 0.18 mg / L or more.
In any case, the upper limit value in the medium can be appropriately set to an appropriate value as long as the culture and fermentation are not inhibited, but is typically 50 mg / L or less, preferably 25 mg / L. It can be as follows.
前記、培地中の金属イオンの量及び濃度は、培地中に含まれるあらゆる形態の金属イオンの総和の量及び濃度である。培地に含まれる原料であって金属イオンを含有するのは、主として、木質バイオマス、酵母エキス、添加される金属イオンであるため、培地中の金属イオン濃度は、これらの原料にそれぞれ含まれる総和として計算することもできる。前記金属イオンは、金属を含む化合物の形態（金属塩などの形態）で培地に添加することもできる。なお、培地中の金属イオンの量及び濃度は、当業者に周知の方法により、測定することができ、また用いる原料中のそれらの量から、計算値として求めることができる。 The amount and concentration of metal ions in the medium are the total amount and concentration of all forms of metal ions contained in the medium. The raw materials contained in the medium that contain metal ions are mainly woody biomass, yeast extract, and added metal ions, so the concentration of metal ions in the medium is the sum of these raw materials. It can also be calculated. The metal ions can be added to the medium in the form of a compound containing a metal (a form such as a metal salt). The amount and concentration of metal ions in the medium can be measured by methods well known to those skilled in the art, and can be determined as a calculated value from those amounts in the raw material used.
（塩基の存在）
本発明の好ましい態様においては、糖化及び発酵を塩基の存在下においてｐＨ４．０〜
７．０で行うことができる。ｐＨは、より好ましくはｐＨ４．５〜５．８であり、さらに
好ましくｐＨ４．６〜５．６であり、さらに好ましくはｐＨ４．９〜５．５である。
(Presence of base)
In a preferred embodiment of the invention, saccharification and fermentation are carried out in the presence of a base at a pH of 4.0 to 4.0.
7.0 can be performed. The pH is more preferably pH 4.5 to 5.8, further preferably pH 4.6 to 5.6, and still more preferably pH 4.9 to 5.5.
使用する塩基の種類は、培養液のｐＨをｐＨ４．０〜７．０に調節できるものであれば
特に限定されず、無機塩基でも有機塩基でもよい。無機塩基としては、例えば、炭酸カル
シウム、炭酸ナトリウム、炭酸水素ナトリウム、水酸化カリウム、水酸化ナトリウム、水
酸化カルシウム、水酸化マグネシウムなどが挙げられる。有機塩基としては、例えば、モ
ノエタノールアミン、トリエタノールアミン、ジイソプロパノールアミン、トリイソプロ
パノールアミン、リジンなどが挙げられる。塩基は好ましくは、上記の中でも無機塩基で
あり、特に好ましくは炭酸カルシウムである。
The type of base used is not particularly limited as long as the pH of the culture solution can be adjusted to pH 4.0 to 7.0, and may be an inorganic base or an organic base. Examples of the inorganic base include calcium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydroxide, sodium hydroxide, calcium hydroxide, magnesium hydroxide and the like. Examples of the organic base include monoethanolamine, triethanolamine, diisopropanolamine, triisopropanolamine, and lysine. The base is preferably an inorganic base among the above, and particularly preferably calcium carbonate.
糖化及び発酵を併行して行う際、木質バイオマスに酵素及びＤ−乳酸生産菌を同時に作
用させる方法は、培養液のｐＨをｐＨ４．０〜７．０に調節できる限り特に限定されない
。但し、本発明の好ましい態様によれば、培養容器の底部から、塩基を含む第１層、木質
バイオマスを含む第２層、並びに酵素及びＤ−乳酸生産菌を含む第３層を、この順番で配
置して、糖化及び発酵を併行して行うことができる。上記の通り、第１層〜第３層をこの
順番で配置することにより、培養液のｐＨをｐＨ４．０〜７．０に調節するという塩基の
作用を上手く達成することができる。
When performing saccharification and fermentation in parallel, the method of allowing an enzyme and a D-lactic acid-producing bacterium to simultaneously act on woody biomass is not particularly limited as long as the pH of the culture solution can be adjusted to pH 4.0 to 7.0. However, according to a preferred embodiment of the present invention, from the bottom of the culture vessel, the first layer containing the base, the second layer containing the woody biomass, and the third layer containing the enzyme and D-lactic acid-producing bacteria are in this order. It can be arranged to perform saccharification and fermentation in parallel. As above-mentioned, by arrange | positioning a 1st layer-a 3rd layer in this order, the effect | action of the base of adjusting the pH of a culture solution to pH 4.0-7.0 can be achieved well.
（嫌気・好気条件）
糖化及び発酵は、嫌気条件下又は好気条件下の何れで行ってもよいが、好ましくは嫌気
条件下で行うことができる。糖化及び発酵を、嫌気条件下で行うことにより、好気条件下
で行う場合と比較してより高い光学純度のＤ−乳酸を製造することが可能になる。嫌気条
件としては、例えば、糖化及び発酵処理中に培養液の回転振とうは行わないという方法が
挙げられる。この場合でも１日に１回〜数回程度培養液を攪拌することはできる。一方、好気条件としては、培養液の回転振とうしながら糖化及び発酵処理を行う方法が挙げられる。培養液を回転振とうする際の回転速度は特に限定されないが、例えば、１０〜２００ｒｐｍが好ましい。
(Anaerobic / aerobic conditions)
Saccharification and fermentation may be performed under anaerobic conditions or aerobic conditions, but can be preferably performed under anaerobic conditions. By performing saccharification and fermentation under anaerobic conditions, it becomes possible to produce D-lactic acid with a higher optical purity than when it is performed under aerobic conditions. Examples of the anaerobic condition include a method in which the culture solution is not rotated and shaken during the saccharification and fermentation treatment. Even in this case, the culture solution can be stirred once to several times a day. On the other hand, examples of the aerobic condition include a method of performing saccharification and fermentation treatment while rotating and shaking the culture solution. Although the rotation speed at the time of carrying out rotation shaking of a culture solution is not specifically limited, For example, 10-200 rpm is preferable.
（糖化酵素）
糖化で使用する酵素は、セルロース分解酵素であれは、セロビオヒドロラーゼ活性、エ
ンドグルカナーゼ活性、ベータグルコシダーゼ活性を有する、所謂セルラーゼと総称され
る酵素である。各セルロース分解酵素は、夫々の活性を有する酵素を適宜の量で添加して
も良いが、市販されているセルラーゼ製剤は、上記の各種のセルラーゼ活性を有すると同
時に、ヘミセルラーゼ活性も有しているものが多いので市販のセルラーゼ製剤を用いれば
良い。
(Saccharifying enzyme)
Enzymes used for saccharification are cellulolytic enzymes, which are enzymes collectively called cellulases having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity. Each cellulolytic enzyme may be added with an appropriate amount of an enzyme having the respective activity. However, commercially available cellulase preparations have the above-mentioned various cellulase activities and also have hemicellulase activity. Since many products are available, a commercially available cellulase preparation may be used.
市販のセルラーゼ製剤としては、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属、アクレ
モニウム（Ａｃｒｅｍｏｎｉｕｍ）属、アスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属、
ファネロケエテ（Ｐｈａｎｅｒｏｃｈａｅｔｅ）属、トラメテス（Ｔｒａｍｅｔｅｓ）属
、フーミコラ（Ｈｕｍｉｃｏｌａ）属、バシラス（Ｂａｃｉｌｌｕｓ）属などに由来する
セルラーゼ製剤がある。このようなセルラーゼ製剤の市販品としては、全て商品名で、例
えば、セルロイシンＴ２（エイチピィアイ社製）、メイセラーゼ（明治製菓社製）、ノボ
ザイム１８８（ノボザイム社製）、ジェネンコア社製のセルラーゼ製剤等が挙げられる。
Commercially available cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus,
There are cellulase preparations derived from the genus Phanerocheete, the genus Trametes, the genus Humicola, the genus Bacillus and the like. Examples of such commercially available cellulase preparations are all trade names, for example, cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), cellulase preparation manufactured by Genencor, etc. Is mentioned.
原料固形分１００質量部に対するセルラーゼ製剤の使用量は、０．５〜１００質量部が
好ましく、１〜５０質量部が特に好ましい。
0.5-100 mass parts is preferable and, as for the usage-amount of the cellulase formulation with respect to 100 mass parts of raw material solid content, 1-50 mass parts is especially preferable.
（培養条件）
糖化及び発酵処理の温度は、酵素及び発酵の至適温度の範囲内であれば特に制限はなく
、通例２５℃〜５０℃が好ましく、３７℃〜５０℃がさらに好ましい。培養は、連続式が
好ましいが、フェドバッチ式、バッチ方式でも良い。培養時間は、酵素濃度などによっても異なるが、フェドバッチ式、及びバッチ式の場合は１０〜２４０時間が好ましく、１５〜１６０時間がさらに好ましい。連続式の場合は、平均滞留時間が、１０〜１５０時間が好ましく、１５〜１００時間がさらに好ましい。
(Culture conditions)
The temperature for saccharification and fermentation treatment is not particularly limited as long as it is within the optimum temperature range for the enzyme and fermentation, and is usually preferably 25 ° C to 50 ° C, more preferably 37 ° C to 50 ° C. The culture is preferably continuous, but may be fed batch or batch. The culture time varies depending on the enzyme concentration and the like, but is preferably 10 to 240 hours and more preferably 15 to 160 hours in the case of the fed-batch type and the batch type. In the case of a continuous type, the average residence time is preferably 10 to 150 hours, and more preferably 15 to 100 hours.
＜他の工程＞
本発明の実施態様においては、Ｄ−乳酸の回収工程を含んでいてもよい。上記の発酵により生産されたＤ−乳酸は、発酵液から公知の方法により分離・精製することにより回収することができる。例えば、発酵液を、遠心分離や濾過等によって不溶な物質（菌体など）を除去した後、イオン交換樹脂などで脱塩し、その溶液から、結晶化やカラムクロマトグラフィー等の常法に従って所望の乳酸を分離・精製することができる。
<Other processes>
In the embodiment of the present invention, a step of recovering D-lactic acid may be included. D-lactic acid produced by the above fermentation can be recovered from the fermentation broth by separation and purification by a known method. For example, after removing insoluble substances (such as bacterial cells) from the fermentation broth by centrifugation, filtration, etc., desalting with an ion exchange resin, etc., and then using the solution according to conventional methods such as crystallization and column chromatography Of lactic acid can be separated and purified.
＜本発明の製造方法で得られるＤ−乳酸＞
本発明の製造方法によれば、光学純度の高いＤ−乳酸を製造することができる。具体的
には本発明の製造方法によれば、光学純度が９８％ee以上、より好ましくは光学純度が９９％ee以上のＤ−乳酸を製造することができる。乳酸の光学純度は、本技術分野で知られた種々の手段で測定することができる。本発明でのＤ−乳酸の光学純度は、特に示した場合を除き、実施例の項中の式により算出した値をいう。
<D-lactic acid obtained by the production method of the present invention>
According to the production method of the present invention, D-lactic acid with high optical purity can be produced. Specifically, according to the production method of the present invention, it is possible to produce D-lactic acid having an optical purity of 98% ee or higher, more preferably an optical purity of 99% ee or higher. The optical purity of lactic acid can be measured by various means known in the art. The optical purity of D-lactic acid in the present invention refers to a value calculated by the formula in the section of Examples, unless otherwise indicated.
本発明の方法を用いて産生したＤ−乳酸は、例えば、ポリＤ−乳酸や、ポリＬ−乳酸と
ポリＤ−乳酸とのステレオコンプレックスを製造するための原料として使用することがで
きる。ポリＬ−乳酸とポリＤ−乳酸とのステレオコンプレックスは、耐熱性が高い生分解
性プラスチックとなり得る。Ｄ−乳酸には、農業中間体としての用途もあることが知られ
ている。
D-lactic acid produced using the method of the present invention can be used as a raw material for producing, for example, poly-D-lactic acid or a stereocomplex of poly-L-lactic acid and poly-D-lactic acid. A stereocomplex of poly L-lactic acid and poly D-lactic acid can be a biodegradable plastic having high heat resistance. It is known that D-lactic acid also has uses as an agricultural intermediate.
本発明の効果を以下の実施例等を挙げて具体的に説明する。但し、本発明の範囲はこれらの実施例等によって制限されない。 The effects of the present invention will be specifically described with reference to the following examples. However, the scope of the present invention is not limited by these examples.
［製造例１］
＜広葉樹クラフトパルプ（ＬＢＫＰ）の製造＞
広葉樹混合木材チップ（ユーカリ７０％、アカシア３０％）を用い、液比４、硫化度２８％、有効アルカリ添加率１７％（Ｎａ２Ｏとして）となるように調製した蒸解白液に木材チップに加えた後、蒸解温度１６０℃にて２時間クラフト蒸解を行なった。クラフト蒸解終了後、黒液を分離し、得られたチップを解繊後、遠心脱水と水洗浄を３回繰り返し、次いでスクリーンにより未蒸解物を除き、蒸解未漂白パルプ（ＬＵＫＰ）を得た。この未漂白パルプ絶乾質量に対して、ＮａＯＨを２．０質量％添加し、酸素ガスを注入し、１００℃で６０分間酸素脱リグニン処理を行ない、酸素脱リグニンパルプ（ＬＯＫＰ）を得た。続いて、酸素脱リグニンパルプを、Ｄ−Ｅ−Ｐ−Ｄの４段漂白処理に供した。漂白時のパルプ濃度は全て１０質量％に調製し、最初の二酸化塩素処理（Ｄ）は、対絶乾パルプの二酸化塩素添加率１．０質量％、７０℃、４０分間処理を行ない、イオン交換水にて洗浄、脱水した。次いで、パルプ絶乾質量に対してＮａＯＨ添加率を１質量％として、７０℃、９０分間のアルカリ抽出処理（Ｅ）を行ない、イオン交換水にて洗浄、脱水した。次いで、パルプ絶乾質量に対して過酸化水素添加率を０．２質量％、ＮａＯＨ添加率を０．５質量％とし、７０℃、１２０分間の過酸化水素処理（Ｐ）を行ない、イオン交換水にて洗浄、脱水した。次いで、パルプ絶乾質量に対して二酸化塩素添加率を０．２質量％とし、７０℃、１２０分間の二酸化塩素処理（Ｄ）を行ない、イオン交換水にて洗浄、脱水後、白色度８５％の漂白パルプ（ＬＢＫＰ）を得た。
[Production Example 1]
<Manufacture of hardwood kraft pulp (LBKP)>
Using hardwood mixed wood chips (eucalyptus 70%, acacia 30%), the liquid ratio, sulfidity 28%, effective alkali addition rate 17% (as Na 2 O), and cooking white liquor to wood chips After the addition, kraft cooking was performed at a cooking temperature of 160 ° C. for 2 hours. After completion of the kraft cooking, the black liquor was separated, and the obtained chips were defibrated. Then, centrifugal dehydration and water washing were repeated three times, and then the uncooked material was removed with a screen to obtain a cooked unbleached pulp (LUKP). With respect to the absolute dry mass of unbleached pulp, 2.0% by mass of NaOH was added, oxygen gas was injected, and oxygen delignification treatment was performed at 100 ° C. for 60 minutes to obtain oxygen delignified pulp (LOKP). Subsequently, the oxygen delignified pulp was subjected to a four-stage bleaching process of D-E-P-D. The pulp concentration at the time of bleaching is adjusted to 10% by mass, and the first chlorine dioxide treatment (D) is performed by treating the dry dry pulp with chlorine dioxide at a rate of 1.0% by mass at 70 ° C. for 40 minutes. Washed with water and dehydrated. Next, an alkali extraction treatment (E) was performed at 70 ° C. for 90 minutes with an NaOH addition rate of 1% by mass with respect to the absolute dry mass of the pulp, washed with ion-exchanged water, and dehydrated. Next, the hydrogen peroxide addition rate is 0.2% by mass and the NaOH addition rate is 0.5% by mass with respect to the absolute dry mass of the pulp, and hydrogen peroxide treatment (P) is performed at 70 ° C. for 120 minutes to perform ion exchange. Washed with water and dehydrated. Next, the chlorine dioxide addition rate is 0.2% by mass with respect to the absolute dry mass of the pulp, and the chlorine dioxide treatment (D) is performed at 70 ° C. for 120 minutes, washed with ion-exchanged water, dehydrated, and the whiteness is 85%. Of bleached pulp (LBKP) was obtained.
＜Ｄ−乳酸生産菌の前培養＞
−８０℃で凍結保存したＤ−乳酸生産菌であるLactobacillus delbrueckii subsp. delbrueckii NBRC3202株［独立行政法人製品評価技術基盤機構（千葉県木更津市かずさ鎌足２−５−８）から入手可能］を用いた。解凍後、オートクレーブにより滅菌した前培養培地（培地組成は下記表１参照、オートクレーブ前に７０％硫酸でｐＨを5.5に調整）１０ｍｌに一白金耳接種し、３７℃、４８-７２時間静置培養を行ない、前々培養液を調製した。さらに、滅菌処理した前培養培地３０ｍｌに、前々培養液を０．９ｍｌ接種した。これを３７℃、４８時間静置培養を行ない、前培養液（以下、「乳酸菌前培養液」という。
）を調製した。
<Pre-culture of D-lactic acid producing bacteria>
Lactobacillus delbrueckii subsp. Delbrueckii NBRC3202 strain [available from Kazusa-Kamazu 2-5-8, Kisarazu City, Chiba Prefecture] It was. After thawing, preculture medium sterilized by autoclaving (see Table 1 below for medium composition, pH adjusted to 5.5 with 70% sulfuric acid before autoclaving) 10 ml of platinum ear inoculation and stationary culture at 37 ° C for 48-72 hours The culture solution was prepared in advance. Furthermore, 0.9 ml of the previous culture solution was inoculated into 30 ml of the sterilized preculture medium. This was subjected to stationary culture at 37 ° C. for 48 hours and referred to as a preculture solution (hereinafter referred to as “lactic acid bacteria preculture solution”).
) Was prepared.
＜酵味ペースト＞
ビール酵母を１００％使用した酵素分解型酵母エキス(ＭＣフードスペシャリティーズ株式会社製)。固形分６１〜６７％、固形分当たりの全窒素５．５％以上、ｐＨ４．８〜６．０。
<Fermentation paste>
Enzymatic degradation yeast extract (manufactured by MC Food Specialties Co., Ltd.) using 100% brewer's yeast. 61-67% solids, 5.5% or more total nitrogen per solid, pH 4.8-6.0.
＜併行糖化発酵＞
次に、培養容器（２５０ｍｌの滅菌フィルター付きキャップのプラスチック製三角フラスコ）にｐＨ調整剤として炭酸カルシウム６ｇ、ＬＢＫＰ７．５ｇを添加した後、オートクレーブで滅菌した。
次に窒素源として、酵味ペースト（固形分８０ｇ／Ｌに希釈後、７０％硫酸でｐＨを5.5に調整し、オートクレーブ滅菌）の濃度が０．７質量％になるようにクリ−ンベンチ内で添加した。その後、ダニスコジャパン社製セルラーゼ製剤２ｍｌ、乳酸菌前培養液２ｍｌを添加し、蒸留水で全量が１００ｍｌになるように培地（以下、「培地Ａ」という。）を調製した。
<Concurrent saccharification and fermentation>
Next, 6 g of calcium carbonate and 7.5 g of LBKP were added as pH adjusters to a culture container (250 ml plastic conical flask with a sterilizing filter cap), and then sterilized by an autoclave.
Next, in the clean bench, the concentration of the fermented paste (diluted to 80 g / L solid content, adjusted to pH 5.5 with 70% sulfuric acid and sterilized by autoclave) as the nitrogen source is 0.7% by mass. Added. Thereafter, 2 ml of cellulase preparation manufactured by Danisco Japan Co., Ltd. and 2 ml of pre-culture solution of lactic acid bacteria were added, and a medium (hereinafter referred to as “medium A”) was prepared with distilled water so that the total amount became 100 ml.
前記培養容器（フラスコ）のキャップに付属している滅菌フィルター上に切り込みを入れたビニールテープを張り、発酵により生産される乳酸と炭酸カルシウムが反応して発生する炭酸ガスをフラスコ外へ放出した。そして外部からの空気流入を極力抑制するように施し、フラスコに該キャップで締め、外部との通気を遮断した。 A cut vinyl tape was put on a sterilizing filter attached to the cap of the culture vessel (flask), and carbon dioxide generated by the reaction of lactic acid and calcium carbonate produced by fermentation was released out of the flask. And it applied so that the air inflow from the outside might be suppressed as much as possible, and it tightened with the cap to the flask, and blocked | blocking ventilation | gas_flowing with the exterior.
前記培養容器を４８℃で３日間培養した。培養の最初の1日間は静置し、残り２日間は回転振とうさせた。培養液のｐＨ、グルコース、Ｄ−乳酸、Ｌ−乳酸を経時的に測定した。尚、グルコース及び乳酸は下記の方法で分析した。結果を表２に示す。 The culture vessel was cultured at 48 ° C. for 3 days. The culture was allowed to stand for the first day of the culture, and was subjected to rotary shaking for the remaining 2 days. The pH, glucose, D-lactic acid, and L-lactic acid of the culture solution were measured over time. Glucose and lactic acid were analyzed by the following method. The results are shown in Table 2.
＜グルコース及び乳酸の分析＞
培養開始直後、１日目、２日目、３日目の培養液を２ｍｌ採取し、遠心分離後、上清を０．２μｍのディスクフィルター（DISMIC 13HP020AN、ADVANTEC社製）でろ過し、脱塩水で２０倍希釈し、グルコース及び乳酸定量用試料（以下、「定量用試料」という。）とした。
<Analysis of glucose and lactic acid>
Immediately after the start of culture, 2 ml of the culture solution of the first day, the second day, and the third day is collected. After centrifugation, the supernatant is filtered through a 0.2 μm disk filter (DISMIC 13HP020AN, manufactured by ADVANTEC), and desalted water The sample was diluted 20-fold with a glucose and lactic acid quantitative sample (hereinafter referred to as “quantitative sample”).
（グルコースの定量）
前記定量用試料３００μｌを専用セルに採取し、バイオセンサー（BF-5／王子計測機器社製）のオートサンプラーにセットして自動計測した。尚、電極は、プレナ電極交換用グルコース電極EDO05-0003／王子計測機器社製）を使用した。
(Quantification of glucose)
300 μl of the sample for quantification was collected in a dedicated cell, set in an autosampler of a biosensor (BF-5 / Oji Scientific Instruments) and automatically measured. As the electrode, a planar electrode replacement glucose electrode EDO05-0003 / manufactured by Oji Scientific Instruments) was used.
（乳酸の定量）
前記定量用試料１ｍｌをマイクロバイアルに採取してオートサンプラーにセットし、高速液体クロマトグラフＨＰＬＣ（alliance・2695／Waters社製）を用い、以下の条件で測定した。
カラム：住友分析センター社製SUMICHIRAL OA-5000（内径4.6ｍｍ、カラム長25.0ｃｍ）温度：３０℃
移動相：２ｍＭ ＣｕＳＯ４・７Ｈ２Ｏの水−２-プロパノール混液（９８：２）溶液
流速：１．０ｍｌ／ｍｉｎ
検出波長：２５４ｎｍ
(Quantification of lactic acid)
1 ml of the sample for quantification was collected in a micro vial, set in an autosampler, and measured using a high performance liquid chromatograph HPLC (alliance 2695 / Waters) under the following conditions.
Column: SUMICHIRAL OA-5000 manufactured by Sumitomo Analysis Center Co., Ltd. (inner diameter: 4.6 mm, column length: 25.0 cm) Temperature: 30 ° C.
Mobile phase: 2 mM CuSO 4 · 7H 2 O in water-2-propanol (98: 2) Solution flow rate: 1.0 ml / min
Detection wavelength: 254 nm
＜光学純度の測定＞
Ｄ−乳酸の光学純度を次式で計算した。
光学純度（％ｅｅ） ＝ （Ｄ−Ｌ）／（Ｄ＋Ｌ）×１００
ここで、ＤはＤ−乳酸濃度、ＬはＬ−乳酸濃度を表す。
<Measurement of optical purity>
The optical purity of D-lactic acid was calculated by the following formula.
Optical purity (% ee) = (D−L) / (D + L) × 100
Here, D represents the D-lactic acid concentration, and L represents the L-lactic acid concentration.
Ｄ−乳酸量は培養４５時間で７３．２ｇ/Ｌ、光学純度は９８．３％ｅｅであった。 The amount of D-lactic acid was 73.2 g / L after 45 hours of culture, and the optical purity was 98.3% ee.
［製造例２］
製造例１の培地Ａに対して、シリンジフィルター（25 mm GD/X PES 0.2 μm、GEヘルスケア・ジャパン社製）で滅菌したシアノコバラミン（ビタミンＢ１２、和光純薬工業製）を終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 2]
The final concentration of cyanocobalamin (vitamin B12, manufactured by Wako Pure Chemical Industries, Ltd.) sterilized with a syringe filter (25 mm GD / X PES 0.2 μm, manufactured by GE Healthcare Japan) is 0. The test was conducted in the same manner as in Production Example 1 except that it was added in a clean bench to 1 mg / L. The results are shown in Table 2.
［製造例３］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 3]
A test was conducted in the same manner as in Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 0.25 mg / L. The results are shown in Table 2.
［製造例４］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 4]
A test was conducted in the same manner as in Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.5 mg / L. The results are shown in Table 2.
［製造例５］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 5]
A test was conducted in the same manner as in Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 1.0 mg / L. The results are shown in Table 2.
［製造例６］
製造例１の培地Ａに対して、シリンジフィルター（25 mm GD/X PES 0.2 μm、GEヘルスケア・ジャパン社製）で滅菌したリボフラビン（ビタミンＢ２、和光純薬工業製）を終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 6]
Riboflavin (vitamin B2, manufactured by Wako Pure Chemical Industries, Ltd.) sterilized with a syringe filter (25 mm GD / X PES 0.2 μm, manufactured by GE Healthcare Japan, Inc.) with respect to the medium A of Production Example 1 has a final concentration of 0. The test was conducted in the same manner as in Production Example 1 except that it was added in a clean bench to 1 mg / L. The results are shown in Table 2.
［製造例７］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．１ｍｇ／Ｌ、さらにリボフラビンを終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 7]
Same as Production Example 1 except that cyanocobalamin was added to Medium A in Production Example 1 at a final concentration of 0.1 mg / L and riboflavin was added in the clean bench to a final concentration of 0.1 mg / L. The method was tested. The results are shown in Table 2.
［製造例８］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．２５ｍｇ／Ｌ、さらにリボフラビンを終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 8]
Same as Production Example 1 except that cyanocobalamin was added to Medium A in Production Example 1 at a final concentration of 0.25 mg / L and riboflavin was added in the clean bench to a final concentration of 0.1 mg / L. The method was tested. The results are shown in Table 2.
［製造例９］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．５ｍｇ／Ｌ、さらにリボフラビンを終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 9]
Similar to Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 at a final concentration of 0.5 mg / L and riboflavin was added in the clean bench to a final concentration of 0.1 mg / L. The method was tested. The results are shown in Table 2.
［製造例１０］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が１．０ｍｇ／Ｌ、さらにリボフラビンを終濃度が０．１ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 10]
Same as Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 at a final concentration of 1.0 mg / L and riboflavin was added in the clean bench to a final concentration of 0.1 mg / L. The method was tested. The results are shown in Table 2.
［製造例１１］
製造例１の培地Ａに対して、リボフラビンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 11]
A test was conducted in the same manner as in Production Example 1 except that riboflavin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.25 mg / L. The results are shown in Table 2.
［製造例１２］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．１ｍｇ／Ｌ、リボフラビンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 12]
The same as Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 0.1 mg / L and riboflavin was 0.25 mg / L. The method was tested. The results are shown in Table 2.
［製造例１３］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．２５ｍｇ／Ｌ、リボフラビンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 13]
The same as Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.25 mg / L and riboflavin was 0.25 mg / L. The method was tested. The results are shown in Table 2.
［製造例１４］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．５ｍｇ／Ｌ、リボフラビンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 14]
Similar to Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 0.5 mg / L and riboflavin was 0.25 mg / L. The method was tested. The results are shown in Table 2.
［製造例１５］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が１．０ｍｇ／Ｌ、リボフラビンを終濃度が０．２５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 15]
The same as in Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 1.0 mg / L and riboflavin was 0.25 mg / L. The method was tested. The results are shown in Table 2.
［製造例１６］
製造例１の培地Ａに対して、リボフラビンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 16]
A test was conducted in the same manner as in Production Example 1 except that riboflavin was added to Medium A in Production Example 1 in a clean bench so that the final concentration was 0.5 mg / L. The results are shown in Table 2.
［製造例１７］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．１ｍｇ／Ｌ、リボフラビンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 17]
The same as Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 0.1 mg / L and riboflavin was 0.5 mg / L. The method was tested. The results are shown in Table 2.
［製造例１８］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．２５ｍｇ／Ｌ、リボフラビンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 18]
The same as in Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.25 mg / L and riboflavin was 0.5 mg / L. The method was tested. The results are shown in Table 2.
［製造例１９］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．５ｍｇ／Ｌ、リボフラビンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 19]
The same as Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.5 mg / L and riboflavin was 0.5 mg / L. The method was tested. The results are shown in Table 2.
［製造例２０］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が１．０ｍｇ／Ｌ、リボフラビンを終濃度が０．５ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 20]
The same as in Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 1.0 mg / L and riboflavin was 0.5 mg / L. The method was tested. The results are shown in Table 2.
［製造例２１］
製造例１の培地Ａに対して、リボフラビンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 21]
A test was conducted in the same manner as in Production Example 1 except that riboflavin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 1.0 mg / L. The results are shown in Table 2.
［製造例２２］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．１ｍｇ／Ｌ、リボフラビンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 22]
The same as Production Example 1 except that cyanocobalamin was added to the medium A of Production Example 1 in a clean bench so that the final concentration was 0.1 mg / L and riboflavin was 1.0 mg / L. The method was tested. The results are shown in Table 2.
［製造例２３］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．２５ｍｇ／Ｌ、リボフラビンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 23]
The same as Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.25 mg / L and riboflavin was 1.0 mg / L. The method was tested. The results are shown in Table 2.
［製造例２４］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が０．５ｍｇ／Ｌ、リボフラビンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 24]
The same as Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 0.5 mg / L and riboflavin was 1.0 mg / L. The method was tested. The results are shown in Table 2.
［製造例２５］
製造例１の培地Ａに対して、シアノコバラミンを終濃度が１．０ｍｇ／Ｌ、リボフラビンを終濃度が１．０ｍｇ／Ｌになるようにクリ−ンベンチ内で添加した以外は製造例１と同様の方法で試験した。結果を表２に示す。
[Production Example 25]
The same as Production Example 1 except that cyanocobalamin was added to the culture medium A of Production Example 1 in a clean bench so that the final concentration was 1.0 mg / L and riboflavin was 1.0 mg / L. The method was tested. The results are shown in Table 2.
表２に示すように、シアノコバラミン単独、またはシアノコバラミン及びリボフラビンを併用した場合、高い光学純度のＤ−乳酸を高い生産効率で製造することができた。 As shown in Table 2, when cyanocobalamin alone or cyanocobalamin and riboflavin were used in combination, D-lactic acid with high optical purity could be produced with high production efficiency.
本発明により、光学純度の高いＤ−乳酸を高い生産効率で生産することが可能となるため、Ｄ−乳酸を製造する際の製造コストの低減が可能となる。 According to the present invention, it is possible to produce D-lactic acid with high optical purity with high production efficiency, and thus it is possible to reduce the production cost when producing D-lactic acid.
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