JP2008167705A - Thermophilic l-lactic acid-producing microorganism and method for producing l-lactic acid solution - Google Patents

Thermophilic l-lactic acid-producing microorganism and method for producing l-lactic acid solution Download PDF

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JP2008167705A
JP2008167705A JP2007005115A JP2007005115A JP2008167705A JP 2008167705 A JP2008167705 A JP 2008167705A JP 2007005115 A JP2007005115 A JP 2007005115A JP 2007005115 A JP2007005115 A JP 2007005115A JP 2008167705 A JP2008167705 A JP 2008167705A
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lactic acid
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bacillus coagulans
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Kenji Sakai
謙二 酒井
Masako Iwatani
正子 岩谷
Satoshi Yoshino
諭 吉野
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Oita University
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<P>PROBLEM TO BE SOLVED: To provide an improved method for producing L-lactic acid, by which the lactic acid can directly be fermented from a polysaccharide such as starch as a raw material, while omitting a saccharification treatment process, because a conventional method for producing the lactic acid from garbage requires a treatment for saccharifying a polysaccharide such as starch with an amylase into glucose of monosaccharide at a high cost. <P>SOLUTION: This method for producing the L-lactic acid is characterized by directly fermenting a culture medium containing cellobiose, starch with a new separated and identified heat-resistant microorganism: Bacillus coagulans M21, having an ability for directly fermenting the starch into the lactic acid. <P>COPYRIGHT: (C)2008,JPO&INPIT

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本発明は、新規に分離され，高い生産性によって多糖であるセロビオース、スターチからのＬ−乳酸発酵生産に利用しうる耐熱性微生物Bacillus coagulansと同定された好熱性Ｌ−乳酸生産菌とこれを用いてＬ−乳酸液を製造する方法に関するものである。 The present invention relates to a thermophilic L-lactic acid-producing bacterium identified as a thermostable microorganism Bacillus coagulans, which is newly isolated and can be used for L-lactic acid fermentation production from cellobiose and starch which are polysaccharides with high productivity. The present invention relates to a method for producing an L-lactic acid solution.

乳酸は種々の食品添加物や工業用原料として重要な物質である。特に乳酸を化学的に重合することで得られる高分子ポリ乳酸はバイオマス由来プラスチック素材として近年大きく注目されている。３炭素化合物である乳酸の２位の炭素はメチル基、水酸基、カルボキシル基と結合する不斉炭素であり、その立体配座からＤ−乳酸およびＬ−乳酸の２種類の光学異性体（鏡像異性体）が存在する。世界的には現在、全生産量の90％が発酵法により、残り10％が化学的合成法により生産されている。 Lactic acid is an important substance as various food additives and industrial raw materials. In particular, polymer polylactic acid obtained by chemically polymerizing lactic acid has attracted much attention in recent years as a biomass-derived plastic material. The carbon at the 2-position of lactic acid, which is a three-carbon compound, is an asymmetric carbon bonded to a methyl group, a hydroxyl group, and a carboxyl group, and from its conformation, two kinds of optical isomers (enantiomers) of D-lactic acid and L-lactic acid. Body). Worldwide, 90% of the total production is currently produced by fermentation and the remaining 10% is produced by chemical synthesis.

発酵菌として使用される微生物には、Ｌ−乳酸を主に生成する多くのラクトバチルス属の常温細菌（Lactobacillus rhamnosus，Lactococcus lactisなど）や、カビの一種Rhizopus oryzaeなどが用いられる事が総説にまとめられている（非特許文献１、非特許文献２）。また、Bacillus属の細菌を用いることによるＬ−乳酸の生産方法に関してBacillus coagulansJCM2257,Bacillus cereusJCM2152,Bacillus subtilisJCM1465,及び昆虫毒素などを生産するBacillus thuringiensis,Bacillus larvae,Bacillus lentimorbus,Bacillus popilliae,Bacillus shphaericusなどの微生物に関して特許が取得されている（特許文献１）。これとは別に、Bacillus coagulansを用いて膜分離を行いながら50℃にて連続的に乳酸を生産する方法が特許化されている（特許文献２）。これらの方法により光学純度[（Ｌ−乳酸 - Ｄ−乳酸）/（Ｌ−乳酸 ＋ Ｄ−乳酸）×100(％)]が約90〜98％の乳酸が生産されていた。 For the microorganisms used as fermenting bacteria, it is summarized in the review that many room temperature bacteria of the genus Lactobacillus that mainly produce L-lactic acid (Lactobacillus rhamnosus, Lactococcus lactis, etc.) and the mold Rhizopus oryzae are used. (Non-patent document 1, Non-patent document 2). Further, regarding the production method of L-lactic acid by using bacteria belonging to the genus Bacillus, Bacillus coagulans JCM2257, Bacillus cereus JCM2152, Bacillus subtilis JCM1465, and Bacillus thuringiensis, Bacillus larvae, Bacillus lentimorbus, Bacillus lollimorbus, Bacillus popilliae, Bacillus shphaeric, etc. that produce insect toxins and the like. Has been acquired (Patent Document 1). Apart from this, a method for producing lactic acid continuously at 50 ° C. while performing membrane separation using Bacillus coagulans has been patented (Patent Document 2). By these methods, lactic acid having an optical purity of [(L-lactic acid-D-lactic acid) / (L-lactic acid + D-lactic acid) × 100 (%)] of about 90 to 98% was produced.

１９９０年頃より再生可能なバイオマス原料から得られ、生分解性を有するプラスチックとしてポリ乳酸が注目され、その生産量は年々増加している。ポリ乳酸はＬ−またはＤ−乳酸あるいはそれらの混合物を脱水重合して得られるが、原料の光学活性が高い乳酸と光学活性が低い乳酸で、前者からは結晶性が高く堅いプラスチックが、後者からは結晶性が低く解けやすいポリ乳酸が得られる。例えば堅さが要求されるエンジニアリングプラスチックなどではポリＬ−乳酸が主に用いられている。即ち光学純度の異なる乳酸はポリ乳酸プラスチックに求められる物性に応じた原料として使い分けられる。 Polylactic acid has been attracting attention as a biodegradable plastic obtained from renewable biomass materials since around 1990, and its production volume is increasing year by year. Polylactic acid is obtained by dehydration polymerization of L- or D-lactic acid or a mixture thereof. The raw material is lactic acid having high optical activity and low optical activity, and the former is a hard plastic with high crystallinity, and the latter. Produces a polylactic acid having low crystallinity and easy dissolution. For example, poly L-lactic acid is mainly used in engineering plastics that require firmness. That is, lactic acid having different optical purity can be used as a raw material according to the physical properties required for polylactic acid plastic.

一方、アセトニトリルを出発原料とする化学合成法では光学純度０%のＬ乳酸とＤ乳酸の混合物即ちラセミ乳酸が得られている。また、前述の総説にはＤ−，Ｌ−乳酸を生成するLactobacillus helveticus,Lactobacillus aminovorusによるラセミ乳酸の発酵生産の例も報告されている。ラセミ乳酸やそのエステルなどは食品添加物として用いられるが、このような用途では光学純度の高低はその効能と無関係である。 On the other hand, in a chemical synthesis method using acetonitrile as a starting material, a mixture of L lactic acid and D lactic acid having an optical purity of 0%, ie, racemic lactic acid, is obtained. In the above-mentioned review, examples of fermentative production of racemic lactic acid by Lactobacillus helveticus and Lactobacillus aminovorus producing D-, L-lactic acid are also reported. Racemic lactic acid and its esters are used as food additives, but in such applications, the level of optical purity is unrelated to their efficacy.

これに関連して、糖質を高く含有すると共にその他の栄養成分を同時に含有する等、内容物が複雑で、雑菌、自然乳酸菌が多数生存している生ゴミや農産バイオマスを原料として、設備、エネルギーコストがかからず、かつ高圧蒸気滅菌などによる栄養成分の分解や発酵阻害物質の生成が回避できる非殺菌開放系で、Ｄ−，Ｌ−乳酸（Lactobacillus plantarum）生産菌あるいはＬ−乳酸（Bacillus coagulans）生産菌の選択的増殖を制御することで、廉価でかつ用途に応じた光学純度の乳酸を高収率で生産する方法が申請者らにより現在公開されている（特許文献３）。しかしながら，本法においてB.coagulansを用いた場合、生ゴミや農産バイオマスを酵素アミラーゼを用い、糖化処理を行わねばならず、時間とコストがかかってしまう。 In this connection, the contents are complex, such as containing a high amount of carbohydrates and other nutritional components at the same time, etc., using raw garbage and agricultural biomass with many germs and natural lactic acid bacteria living as raw materials, equipment, It is a non-sterilized open system that does not require energy costs and can avoid the decomposition of nutrients by high-pressure steam sterilization or the like, and the production of fermentation inhibitors. D-, L-lactic acid (Lactobacillus plantarum) -producing bacteria or L-lactic acid (Bacillus A method for producing lactic acid with a low yield and optical purity according to the application in a high yield by controlling the selective growth of coagulans-producing bacteria is currently disclosed by the applicants (Patent Document 3). However, when B. coagulans is used in this method, saccharification treatment must be performed on the garbage and agricultural biomass using the enzyme amylase, which takes time and cost.

J.H.Litchfield著「Microbiological production of lactic acid」Advances in Applied Microbiology 42巻45-95頁１９９６年J.H.Litchfield, "Microbiological production of lactic acid" Advances in Applied Microbiology 42: 45-95 1996 K.Hofvendahl,B.H-Hagerdal著「Factors affecting thefermentativelacticacidproductionfromrenewableresources」EnzymeandMicrobialTechnology２６巻８７−１０７頁２０００年26:87-107(2000).K. Hofvendahl, B. H-Hagerdal, `` Factors affecting thefermentative lactic acid production from renewable resources '', Enzyme and Microbial Technology 26: 87-107 2000 26: 87-107 (2000). K.Sakai,M.Yoshihiro,H.Yamazumi,Y.Tau,M.Mori,M.Moriguchi,Y.Shirai著「SelectiveProliferationofLacticAcidBacteriaAndAccumulationofLacticAcidduringOpenFermentationofKitchenRefusewithIntermittentpHAdjustment.」FoodScienceandTechnologyResearch６巻１４０−１４５頁２０００年K.Sakai, M.Yoshihiro, H.Yamazumi, Y.Tau, M.Mori, M.Moriguchi, Y.Shirai `` SelectiveProliferationofLacticAcidBacteriaAndAccumulationofLacticAcidduringOpenFermentationofKitchenRefusewithIntermittentpHAdjustment. H.Ohara,M.Yahata,USPatent5801025１９９６年MethodforproducingL-lacticacidwithhighopticalpurityusingbacillusstrains(JP7-280660,JP-280661)H.Ohara, M.Yahata, USPatent58010251996MethodforproducingL-lacticacidwithhighopticalpurityusingbacillusstrains (JP7-280660, JP-280661) VanNispen,JoannesG.M.,Jonker,Ronald,USPatent5002881１９８９年Processforthefermentativepreparationoforganicacids.VanNispen, Joannes G.M., Jonker, Ronald, US Patent 5002881 1989 Process for the fermentative preparation of organic acids. 酒井謙二，江崎豊，白井義人，特願2003-306871（２００３年）乳酸液の製造方法Kenji Sakai, Yutaka Esaki, Yoshito Shirai, Japanese Patent Application 2003-306871 (2003) Method for producing lactic acid solution

生ゴミからの乳酸生産は、酵素アミラーゼにより、多糖であるスターチなどの糖源を単糖のグルコースに糖化処理しなければならず、コストがかかってしまう。スターチなどの多糖からの直接乳酸発酵が可能であれば、糖化処理の工程を省くことができる。

現在、報告されているBacillus coagulansでは、スターチからの直接乳酸発酵できるという報告はない。 Lactic acid production from raw garbage requires a sugar source such as starch, which is a polysaccharide, to be saccharified to glucose, which is expensive, using enzyme amylase. If direct lactic acid fermentation from a polysaccharide such as starch is possible, the step of saccharification treatment can be omitted.

In Bacillus coagulans currently reported, there is no report that direct lactic acid fermentation from starch is possible.

新規に分離，同定された耐熱性微生物Bacillus coagulans Ｍ２１を好熱性Ｌ乳酸生産菌とし、これを用い，セロビオース、スターチを含有する培地から直接発酵するＬ−乳酸液製造方法である。
本発明は、前記課題を解決するためになされたものであり，それは次の(1)と(2)のとうりである。
(1). Bacillus coagulans M21の16sリボゾーマルRNA遺伝子の部分配列は、
GGATAACGCCGGGAACCGGGGCTAATACCGGATAGTTTTTCCTCCGCAGGAGGAAAAGGAAAGGCGGCTTCGGCTGCCACTTACAGATGGCCCCGCGGCGCATTAGCTAGTTGGCGGGGTAACGGCCCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCGGGTCGTAAAACTCTGTTGCCGGGGAAGAACAAGTGCCGTTCGAACAGGGCGGCGCCTTGACGGTACCCGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGCGGCTCAAACCGCAAGCGGTCATTGGAAACTGGGAGGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTGGAGACAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCAGAAAACCATTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTT
である耐熱性微生物Bacillus coagulans AB116143 (99.286% identity)からなることを特徴とするＬ−乳酸生産菌。
(2). グルコースとセロビオース、及びスターチを含有する糖濃度2.0〜10％の改変MRS培地に、上記耐熱性微生物Bacillus coagulans M21 を植菌濃度2.4％にして加え，50℃，ｐH7において殺菌することなく直接培養基として発酵することを特徴とするＬ−乳酸液製造方法。
即ち、本発明は、従来方法のように細菌，糸状菌によりデンプン（やセロビオース）を直接発酵してＬ−乳酸を生成するもの、例えば精製デンプンや米などを加熱によりα化の構造変化を起こさせ分解する方法の持つ高粘性、高濃度化による操作性の問題を解決したもので、前記新規な好熱性（耐熱性）を示す微生物により、殺菌系や冷却を必要としない系でのデンプンからの直接発酵を操作性良く可能にした進歩性ある優れた発明である。 This is a method for producing an L-lactic acid solution in which a newly isolated and identified thermostable microorganism, Bacillus coagulans M21, is used as a thermophilic L lactic acid-producing bacterium and is directly fermented from a medium containing cellobiose and starch.
The present invention has been made in order to solve the above-mentioned problems, and it is the following (1) and (2).
(1). The partial sequence of 16s ribosomal RNA gene of Bacillus coagulans M21 is

An L-lactic acid-producing bacterium characterized by comprising the thermostable microorganism Bacillus coagulans AB116143 (99.286% identity).
(2). Add the heat-resistant microorganism Bacillus coagulans M21 to a modified MRS medium containing glucose, cellobiose, and starch at a sugar concentration of 2.0 to 10% at an inoculation concentration of 2.4% and sterilize at 50 ° C and pH7. A method for producing an L-lactic acid solution, characterized in that it is directly fermented as a culture medium.
That is, the present invention is such that, as in the conventional method, starch (or cellobiose) is directly fermented by bacteria or filamentous fungi to produce L-lactic acid, for example, purified starch or rice undergoes a structural change in the pregelatinization by heating. It solves the problem of operability due to the high viscosity and high concentration of the method of letting it break down, and from the starch in a system that does not require sterilization or cooling by the above-mentioned novel thermophilic microorganism (heat resistance) It is an excellent invention with an inventive step that enables direct fermentation of the above with good operability.

本発明の好熱性Ｌ−乳酸生産菌とＬ−乳酸液製造方法は、糖源にグルコースとセロビオース、及びスターチを含有する培地から，新規に分離された前記耐熱性微生物Bacillus coagulansＭ21を一定量植菌し、初期pHを7.0にし、50℃の温度で加温することで多糖からの直接乳酸発酵ができるものである。
これにより従来生ゴミや農産バイオマスを酵素アミラーゼで糖化処理していた工程を省くことができ、乳酸発酵における時間の短縮と低コスト化が期待される。 The thermophilic L-lactic acid-producing bacterium and the L-lactic acid solution production method of the present invention inoculate a predetermined amount of the thermostable microorganism Bacillus coagulans M21 newly isolated from a medium containing glucose, cellobiose and starch as sugar sources. The initial pH is set to 7.0 and heating at a temperature of 50 ° C. enables direct lactic acid fermentation from polysaccharides.
As a result, the process of saccharifying raw garbage and agricultural biomass with enzyme amylase can be omitted, and shortening of the time and cost reduction in lactic acid fermentation are expected.

本発明を実施するための最良の形態は、培地の糖源に多糖であるセロビオース、スターチを用い、この原料を高圧蒸気滅菌し、植菌量を２．４％とし、50℃に保つことで光学純度の高いＬ−乳酸を生産する方法である。
即ち本発明は、上記の手段により多糖であるセロビオースやスターチから乳酸を直接的に発酵でき、かつ９９%以上の高い光学純度の乳酸液が効率よく生産できる効果を得る。
これらの手段と効果は、図１〜図９に示す新規な上記好熱性Ｌ乳酸生産菌の同定と培養実験により得た新知見によってもたらされたものである。
好熱性Ｌ乳酸生産菌即ち耐熱性微生物Bacillus coagulansＭ21はグラム陽性桿菌であり，内生胞子を形成した。また，カタラーゼ活性は陽性であった。以上はBacillus属に属する細菌の性質とよく一致した。
表１には本分離菌耐熱性微生物Bacillus coagulans Ｍ２１の16SリボゾーマルRNA遺伝子の部分配列（○○○ｂｐ）の分析結果を示した。これにより本分離菌の16SリボゾーマルRNA遺伝子の部分配列はBacillus coagulans AB116143 と99.286%%相同であることが分かったのである。
以上のことから本分離菌耐熱性微生物Bacillus coagulans Ｍ２１はBacillus coagulans AB116143と命名されるべきであることが示された。 The best mode for carrying out the present invention is to use cellobiose and starch, which are polysaccharides, as the sugar source of the culture medium, sterilize the raw material by autoclaving, keep the inoculation amount 2.4% and keep it at 50 ° C. This is a method for producing L-lactic acid with high optical purity.
That is, the present invention has an effect that lactic acid can be directly fermented from cellobiose and starch which are polysaccharides by the above-described means, and a lactic acid solution having a high optical purity of 99% or more can be efficiently produced.
These means and effects are brought about by the new knowledge obtained by the identification of the above-mentioned thermophilic L-lactic acid-producing bacteria and culture experiments shown in FIGS.
The thermophilic L-lactic acid-producing bacterium, namely the thermostable microorganism Bacillus coagulans M21, is a Gram-positive gonococcus and formed endospores. Catalase activity was positive. The above agreed well with the characteristics of bacteria belonging to the genus Bacillus.
Table 1 shows the results of analysis of the partial sequence (XXX) of the 16S ribosomal RNA gene of the heat-resistant microorganism Bacillus coagulans M21 of this isolate. This revealed that the partial sequence of the 16S ribosomal RNA gene of this isolate was 99.286% homologous to Bacillus coagulans AB116143.
From the above, it was shown that the heat-resistant microorganism Bacillus coagulans M21 of this isolate should be named Bacillus coagulans AB116143.

Figure 2008167705
図１のグラフには、各pHに調整したSYP培地（１L中にグルコース２０ｇ、ペプトン５ｇ、酵母エキス５ｇ、炭酸カルシウム１０gを含む）を用いて新規Ｌ乳酸生産菌（熱性微生物Bacillus coagulans Ｍ２１）を静置培養した際の、培養時の初発ｐＨと生育度（６１０ｎｍにおける濁度）の関係を示す。
これにより、本菌の最適ｐＨが７．３であり、生育可能ｐＨ範囲が５〜８付近であることが判明したのである。
図２のグラフには、pH７．０に調整したMRS培地中で，温度勾配培養装置を用いて各種温度で新規好熱性Ｌ−乳酸生産菌（耐熱性微生物Bacillus coagulans Ｍ２１）を静置培養した際の、その培養時の温度と生育度（６１０ｎｍにおける濁度）の関係を示す。
これにより、本菌の生育上限温度が、５０℃前後であることが判明したのである。
図３のグラフには、各pHに調整した改変MRS培地（１L中にセロビオース２０ｇ、ペプトン１０ｇ、肉エキス８ｇ、酢酸ナトリウム５ｇ、酵母エキス４ｇ、リン酸２カリウム２ｇ、クエン酸三アンモニウム２ｇ、硫酸マグネシウム０.２ｇ、硫酸マンガン０.０５ｇ、ソルビタンモノオレエート１ｍｌを含む）を用いて新規Ｌ乳酸生産菌（熱性微生物Bacillus coagulans Ｍ２１）と既存株であるBacillus coagulans ＮＢＲＣ１２５８３を静置培養した際の、培養後の乳酸生産量の関係を示す。
図４のグラフは上述の改変ＭＲＳ培地に寒天を添加した場合の比較である。
図５のグラフは、上述の改変ＭＲＳ培地の糖源にスターチを用いた場合の比較である。
図６のグラフは、上述の改変ＭＲＳ培地の糖源にスターチを用いて、さらに寒天を添加した場合の比較である。
Figure 2008167705
 In the graph of FIG. 1, a novel L lactic acid-producing bacterium (thermophilic microorganism Bacillus coagulans M21) is prepared using a SYP medium adjusted to each pH (containing 20 g of glucose, 5 g of peptone, 5 g of yeast extract, and 10 g of calcium carbonate in 1 L). The relationship between the initial pH at the time of culture and the degree of growth (turbidity at 610 nm) during stationary culture is shown.
As a result, it was found that the optimum pH of the present bacterium was 7.3 and the viable pH range was around 5-8.
In the graph of FIG. 2, when a novel thermophilic L-lactic acid-producing bacterium (heat-resistant microorganism Bacillus coagulans M21) is statically cultured at various temperatures in a MRS medium adjusted to pH 7.0. Shows the relationship between the temperature during the culture and the degree of growth (turbidity at 610 nm).
As a result, it was found that the upper limit temperature for growth of this bacterium was around 50 ° C.
The graph of FIG. 3 shows a modified MRS medium adjusted to each pH (20 g cellobiose, 10 g peptone, 8 g meat extract, 5 g sodium acetate, 4 g yeast extract, 2 g dipotassium phosphate, 2 g triammonium citrate, sulfuric acid When 0.2 g of magnesium, 0.05 g of manganese sulfate and 1 ml of sorbitan monooleate are used, a novel L lactic acid-producing bacterium (thermophilic microorganism Bacillus coagulans M21) and the existing strain Bacillus coagulans NBRC12583 are statically cultured. The relationship of the amount of lactic acid production after culture is shown.
The graph of FIG. 4 is a comparison when agar is added to the modified MRS medium described above.
The graph of FIG. 5 is a comparison when starch is used as the sugar source of the modified MRS medium.
The graph in FIG. 6 is a comparison when starch is used as the sugar source of the modified MRS medium and agar is further added.

図３〜６には従来用いられていたBacillus coagulansと新規分離菌即ち耐熱性微生物Bacillus coagulansＭ２１の改変ＭＲＳ培地の発酵結果を比較した。その結果乳酸蓄積量は、Bacilluscoagulansよりも高く、特に培地に寒天を添加した軟寒天培地ではセロビオース、またはスターチを糖源とした場合においても４倍以上に達することがわかったのである。 3 to 6 compare the fermentation results of the modified MRS mediums of Bacillus coagulans and Bacillus coagulans conventionally used, that is, the thermostable microorganism Bacillus coagulans M21. As a result, the accumulated amount of lactic acid was higher than that of Bacillus coagulans, and it was found that the soft agar medium in which agar was added to the medium reached 4 times or more even when cellobiose or starch was used as the sugar source.

図７〜図９のグラフは、上述の改変MRS培地の糖源にグルコース、セロビオース、スターチを糖濃度３．５．７．１０．１５％と含有し、４８時間培養した後の乳酸、Ｄ／Ｌ−乳酸、酢酸、蟻酸の蓄積量を示すグラフ。 The graphs of FIGS. 7 to 9 include glucose, cellobiose and starch in the sugar source of the above-described modified MRS medium at a sugar concentration of 3.5.7.1.15%, and lactic acid, D / The graph which shows the accumulation amount of L-lactic acid, acetic acid, and formic acid.

以上の説明で明らかなように、本発明は、従来のBacillus coagulansでの生ゴミからの乳酸生産において、酵素アミラーゼによる糖化処理を省くことが可能であり、コストを大幅に削減することができる。 As is apparent from the above description, the present invention can omit the saccharification treatment with the enzyme amylase in the production of lactic acid from raw garbage with conventional Bacillus coagulans, and can greatly reduce the cost.

本発明における耐熱性微生物Bacillus coagulans Ｍ２１の、培養時の初発ｐＨと生育度（６１０ｎｍにおける濁度）の関係を示すグラフ。The graph which shows the relationship between the initial pH at the time of culture | cultivation, and the growth degree (turbidity in 610 nm) of the thermostable microorganism Bacillus coagulans M21 in this invention. 本発明における耐熱性微生物Bacillus coagulans Ｍ２１の、培養時の温度と生育度（６１０ｎｍにおける濁度）の関係を示すグラフ。The graph which shows the relationship between the temperature at the time of culture | cultivation, and the growth degree (turbidity in 610 nm) of the thermostable microorganism Bacillus coagulans M21 in this invention. セロビオースを糖源とする液体培地におけるBacillus coagulans NBRC１２５８３とBacillus coagulans Ｍ２１との比較Comparison of Bacillus coagulans NBRC12583 and Bacillus coagulans M21 in liquid medium using cellobiose as a sugar source セロビオースを糖源とする軟寒天培地におけるBacillus coagulans NBRC１２５８３とBacillus coagulans Ｍ２１との比較Comparison of Bacillus coagulans NBRC12583 and Bacillus coagulans M21 in soft agar medium using cellobiose as a sugar source スターチを糖源とする液体培地におけるBacillus coagulans NBRC１２５８３とBacillus coagulans Ｍ２１との比較Comparison of Bacillus coagulans NBRC12583 and Bacillus coagulans M21 in a liquid medium using starch as a sugar source スターチを糖源とする軟寒天培地におけるBacillus coagulans NBRC１２５８３とBacillus coagulans Ｍ２１との比較Comparison of Bacillus coagulans NBRC12583 and Bacillus coagulans M21 in soft agar medium with starch as sugar source 各糖濃度のグルコース軟寒天培地におけるBacillus coagulans Ｍ２１の乳酸生産量Lactic acid production of Bacillus coagulans M21 in glucose soft agar medium with various sugar concentrations 各糖濃度のセロビオース軟寒天培地におけるBacillus coagulans Ｍ２１の乳酸生産量Lactic acid production of Bacillus coagulans M21 in cellobiose soft agar medium with various sugar concentrations 各糖濃度のスターチ軟寒天培地におけるBacillus coagulans Ｍ２１の乳酸生産量Lactic acid production of Bacillus coagulans M21 in starch soft agar medium with various sugar concentrations

符号の説明Explanation of symbols

△ 図１におけるM21・生育度
○ 図１におけるM21・乳酸生産量
△ 図２におけるM21
□ 図３〜図６におけるＬ−乳酸
■ 図３〜図６におけるＤ−乳酸
◆ 図７〜図９における乳酸
■ 図７〜図９におけるＬ−乳酸 △ M21 and growth degree in Fig. 1 M21 and lactic acid production in Fig. 1 M21 in Fig. 2
L-lactic acid in FIGS. 3 to 6 D-lactic acid in FIGS. 3 to 6 Lactic acid in FIGS. 7 to 9 L-lactic acid in FIGS.

Claims (2)

Bacillus coagulans Ｍ２１の16sリボゾーマルRNA遺伝子の部分配列は、
GGATAACGCCGGGAACCGGGGCTAATACCGGATAGTTTTTCCTCCGCAGGAGGAAAAGGAAAGGCGGCTTCGGCTGCCACTTACAGATGGCCCCGCGGCGCATTAGCTAGTTGGCGGGGTAACGGCCCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCGGGTCGTAAAACTCTGTTGCCGGGGAAGAACAAGTGCCGTTCGAACAGGGCGGCGCCTTGACGGTACCCGGCCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGCTTCTTAAGTCTGATGTGAAATCTTGCGGCTCAAACCGCAAGCGGTCATTGGAAACTGGGAGGCTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACCTCCCTGGAGACAGGGCCTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCGAGACCGCGAGGTTAAGCCAATCCCAGAAAACCATTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTT
である耐熱性微生物Bacillus coagulans AB116143 (99.286% identity)からなることを特徴とするＬ−乳酸生産菌。 The partial sequence of the 16s ribosomal RNA gene of Bacillus coagulans M21 is

An L-lactic acid-producing bacterium characterized by comprising the thermostable microorganism Bacillus coagulans AB116143 (99.286% identity). グルコースとセロビオース、スターチを含有する糖濃度2.0〜10％の培地に、上記耐熱性微生物Bacillus coagulans M21を植菌濃度1.0〜10％にして加え，30〜58℃，pH5〜８において殺菌することなく直接培養基として発酵することを特徴とするＬ−乳酸液製造方法。 The above heat-resistant microorganism Bacillus coagulans M21 was added to a medium containing 2.0 to 10% of sugar containing glucose, cellobiose and starch at an inoculation concentration of 1.0 to 10%, and without sterilization at 30 to 58 ° C and pH 5 to 8 A method for producing an L-lactic acid solution, which comprises directly fermenting as a culture medium.
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JP2016518136A (en) * 2013-04-26 2016-06-23 ザイレコ，インコーポレイテッド Biomass treatment to obtain hydroxyl-carboxylic acid
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011000008A (en) * 2009-06-16 2011-01-06 Toyo Seikan Kaisha Ltd Method for detecting bacillus coagulans in test sample specifically, in a short period of time and with high sensitivity
JP2016518136A (en) * 2013-04-26 2016-06-23 ザイレコ，インコーポレイテッド Biomass treatment to obtain hydroxyl-carboxylic acid
CN110643673A (en) * 2019-08-27 2020-01-03 湖北华扬科技发展有限公司 Method for counting bacillus coagulans spores in feeding composite microecological product
CN110643673B (en) * 2019-08-27 2023-08-08 湖北华扬科技发展有限公司 Counting method of bacillus coagulans spores in feeding composite microecological product

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