JP2681495B2 - Bifidobacteria protoplast regeneration medium and regeneration method using the medium - Google Patents
Bifidobacteria protoplast regeneration medium and regeneration method using the mediumInfo
- Publication number
- JP2681495B2 JP2681495B2 JP63228342A JP22834288A JP2681495B2 JP 2681495 B2 JP2681495 B2 JP 2681495B2 JP 63228342 A JP63228342 A JP 63228342A JP 22834288 A JP22834288 A JP 22834288A JP 2681495 B2 JP2681495 B2 JP 2681495B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- protoplasts
- bifidobacterium
- regeneration
- protoplast
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 210000001938 protoplast Anatomy 0.000 title claims description 41
- 241000186000 Bifidobacterium Species 0.000 title claims description 14
- 238000011069 regeneration method Methods 0.000 title claims description 9
- 230000008929 regeneration Effects 0.000 title claims description 7
- 239000002609 medium Substances 0.000 claims description 11
- 241001608472 Bifidobacterium longum Species 0.000 claims description 10
- 241000186148 Bifidobacterium pseudolongum Species 0.000 claims description 10
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 10
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 10
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 10
- 230000001172 regenerating effect Effects 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 229940009291 bifidobacterium longum Drugs 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 230000003204 osmotic effect Effects 0.000 claims description 3
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 229960003067 cystine Drugs 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 239000012137 tryptone Substances 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000186016 Bifidobacterium bifidum Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229940002008 bifidobacterium bifidum Drugs 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 235000015140 cultured milk Nutrition 0.000 description 2
- 108010060371 endo-N-acetylmuramidase Proteins 0.000 description 2
- 210000000416 exudates and transudate Anatomy 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 235000015193 tomato juice Nutrition 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 description 1
- 229960000511 lactulose Drugs 0.000 description 1
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- -1 maltotriose trisaccharide Chemical class 0.000 description 1
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、人の健康維持に関与するとされる腸内細菌
であつて、発酵乳の製造にも利用されるビフィズス菌の
プロトプラストの融合のための再生に用いる培地、及び
該培地を用いたビフィドバクテリウム・シュードロンガ
ム、或いはビフィドバクテリウム・ロンガムのプロトプ
ラストの再生方法に関する。TECHNICAL FIELD The present invention relates to a fusion of bifidobacteria protoplasts, which are intestinal bacteria that are said to be involved in the maintenance of human health, and which are also used in the production of fermented milk. The present invention relates to a medium used for regeneration and a method for regenerating Bifidobacterium pseudolongum or Bifidobacterium longum protoplasts using the medium.
従来技術 現在までに、色々な細菌類のプロトプラスト化が試み
られており、ビフィズス菌のプロトプラスト化について
も以下のように報告されている。2. Description of the Related Art Up to the present, attempts have been made to transform various bacteria into protoplasts, and the following reports on the protoplast formation of bifidobacteria.
例えば、エクステルカーテルらは、ビフィドバクテリ
ウム・ビフィダム・バー・ペンシルバニクス（Bifidoba
cterium bifidum var.pennsylvanicus）に対し、ショ糖
存在下で、リゾーチームを作用させることにより、プロ
トプラストを調製したと報告している〔ビオヒミカ・エ
ト・ビオフイジカ・アクタ（Biochimica et Biophysica
Acta）219,141-154,（1970）〕。For example, Extelcartel and colleagues have published Bifidoba bifidum bar Pennsylvanics.
cterium bifidum var.pennsylvanicus), it was reported that protoplasts were prepared by reacting lysozyme in the presence of sucrose [Biochimica et Biophysica (Biochimica et Biophysica).
Acta) 219, 141-154, (1970)].
ウエダらは、ビフィドバクテリウム・ビフィダムにエ
ンド−Ｎ−アセチルムラミダーゼを作用させて、プロト
プラストを調製したと報告している〔ジヤーナル・オブ
・ジエネラル・アンド・アプライド・ミクロバイオロジ
（Journal of General and Applied microbiology）29,
507（1983）〕。Ueda et al. Reported that endo-N-acetylmuramidase was allowed to act on Bifidobacterium bifidum to prepare protoplasts [Journal of General and Applied Microbiology]. and Applied microbiology) 29,
507 (1983)].
森下らは、ビフィドバクテリウム属に属する微生物に
対し、ショ糖、乳糖、マルトース、ラクチュロース、Ｎ
−アセチルムラミダーゼを作用させることにより、プロ
トプラストを製造する方法を特許出願している（特開昭
59-135883）。Morishita et al. Have demonstrated that sucrose, lactose, maltose, lactulose, N
-A patent application has been filed for a method for producing protoplasts by acting acetylmuramidase (Japanese Patent Laid-Open Publication No. Sho.
59-135883).
ナカイらは、ビフィドバクテリウム・ビフィダムにつ
いて、ショ糖及びペニシリンＧを含有する培地で嫌気的
に生育させて、プロトプラストを調製したと報告してい
る〔ジヤーナル・オブ・ジエネラル・アンド・アプライ
ド・ミクロバイオロジイ（Journalof General and Appl
ied microbiology）30,187（1984）〕。Nakai et al. Reported that Bifidobacterium bifidum was prepared anaerobically in a medium containing sucrose and penicillin G to prepare protoplasts [Jearnal of General and Applied Microbe]. Biology (Journal of General and Appl
ied microbiology) 30,187 (1984)].
小此木らは、ビフィドバクテリウム属に属する微生物
に対し、ラフィノース、メレジトース、あるいはマルト
トリオースの三糖類及びマグネシウムイオンの存在下
で、Ｎ−アセチルムラミダーゼを作用させることによ
り、プロトプラストを製造する方法を特許出願している
〔特開昭61-280267〕。Kokonoki et al., A method for producing a protoplast by reacting a microorganism belonging to the genus Bifidobacterium with raffinose, melezitose, or maltotriose trisaccharide and magnesium ion in the presence of N-acetylmuramidase. Has been filed as a patent application [JP-A-61-280267].
しかし、プロトプラスト化に当つては、それぞれの菌
種毎に、最適の条件設定が必要であり、また、プロトプ
ラストの再生についても同様に、最適の条件設定が必要
であり、したがつて、高収率でビフィズス菌のプロトプ
ラストを製造し、かつ高率でビフィズス菌のプロトプラ
ストを再生する方法については未だ確立されておらず、
ビフィズス菌の育種及び改良に関しては、他の微生物に
比べて著しく遅れているという問題があつた。However, when converting to protoplasts, it is necessary to set the optimum conditions for each bacterial species, and also for the regeneration of protoplasts, it is necessary to set the optimum conditions, and therefore high yields are obtained. The method for producing Bifidobacteria protoplasts at a high rate and regenerating Bifidobacteria protoplasts at a high rate has not yet been established,
Regarding the breeding and improvement of Bifidobacterium, there was a problem that it was significantly delayed compared to other microorganisms.
因に、ビフィズス菌は発酵乳、乳酸菌飲料、菓子など
の食品、整腸剤、栄養剤あるいは飼料などに広く利用さ
れている微生物である。Bifidobacteria are microorganisms widely used for fermented milk, lactic acid beverages, foods such as confectionery, intestinal regulators, nutritional supplements and feeds.
発明が解決しようとする課題 本発明は、叙上の状況に鑑みなされたものであつて、
ビフィズス菌のプロトプラストが高頻度で再生させるた
めに用いる再生用培地を提供することを課題とする。ま
た、本発明はビフィドバクテリウム・シュードロンガ
ム、或いはビフィドバクテリウム・ロンガムを対象と
し、そのプロトプラストを調製し、プロトプラストが高
頻度で再生させるための再生方法を提供することを課題
とする。すなわち、本発明は、上記ビフィズス菌のプロ
トプラストを高頻度で再生することにより、その融合を
可能にするものである。Problem to be Solved by the Invention The present invention has been made in view of the above situation,
An object of the present invention is to provide a regenerating medium used for regenerating protoplasts of Bifidobacterium at a high frequency. Further, the present invention is directed to Bifidobacterium pseudolongum, or Bifidobacterium longum, to prepare a protoplast thereof, and to provide a regeneration method for protoplasts to be regenerated at high frequency. . That is, the present invention enables the fusion by proliferating the Bifidobacterium protoplasts at a high frequency.
課題を解決するための手段 本発明の特徴は、ビフィズス菌のプロトプラストを再
生するに適した、下記に示した基本組成をもつ再生用MN
M-G培地にある。Means for Solving the Problems A feature of the present invention is that MN for regeneration having a basic composition shown below, which is suitable for regenerating protoplasts of Bifidobacterium.
Located in MG medium.
MNM-G培地の基本組成：（/l） K2HPO4 2.5g CH3COONa 10 ｇ トリプトン 10 ｇ 酵母エキス 5.0g システィン・HCl・H2O 0.4g グルコース 5.0g 寒天 15 ｇ CaCl2・2H2O 5 mM MgCl2・6H2O 5 mM pH6.5 更に、本発明の特徴は、ビフィドバクテリウム・シュ
ードロンガム、或いは、ビフィドバクテリウム・ロンガ
ムを、ラフィノースを浸透圧安定剤（Osmotic stabiliz
er）として添加した上記のMNM-G培地に塗沫して嫌気培
養することにある。Basic composition of MNM-G medium: (/ l) K 2 HPO 4 2.5g CH 3 COONa 10 g Tryptone 10 g Yeast extract 5.0 g Cystine ・ HCl ・ H 2 O 0.4 g Glucose 5.0 g Agar 15 g CaCl 2・ 2H 2 O 5 mM MgCl 2 .6H 2 O 5 mM pH 6.5 Furthermore, the feature of the present invention is that Bifidobacterium pseudolongum or Bifidobacterium longum is added to raffinose as an osmotic stabilizer (Osmotic stabiliz
er) is applied to the above MNM-G medium and anaerobically cultured.
本発明では、ビフィドバクテリウム・シュードロンガ
ム（以下B.シュードロンガムと略記する）、或いは、ビ
フィドバクテリウム・ロンガム（以下B.ロンガムと略記
する）のプロトプラストは、ビフィズス菌の公知のプロ
トプラスト化の方法により調製し得るが、本発明者らが
開発した方法に従つて調製することが好ましく、安定な
プロトプラストが得られる。In the present invention, Bifidobacterium pseudolongum (hereinafter abbreviated as B. pseudolongum), or Bifidobacterium longum (hereinafter abbreviated as B. longum) protoplast is a known bifidobacteria. Although it can be prepared by the method of protoplast formation, it is preferable to prepare it according to the method developed by the present inventors, and stable protoplasts can be obtained.
なお、B.シュードロンガムのプロトプラストの調製法
に関しては、特開平2-195872号公報に、B.ロンガムのプ
ロトプラストの調製法に関しては、特開平2-167070号公
報に、それぞれ詳しく記載されている。Incidentally, for the preparation method of B. pseudolongum protoplasts, JP-A 2-195872, for the preparation method of B. longum protoplasts, JP-A 2-167070, is described in detail respectively. .
本発明は、上述のようにして調製したB.シュードロン
ガム、或いは、B.ロンガムのプロトプラストを、ラフィ
ノースを好ましくは0.2〜0.3Mの濃度に添加した前記のM
NM-G培地を再生培地として用いて培養する。The present invention, B. pseudolongum prepared as described above, or B. longum protoplasts, raffinose is preferably added to the concentration of 0.2 ~ 0.3M M above.
Culture using NM-G medium as regeneration medium.
ラフィノースを添加したMNM-G培地に、特開平2-19587
2号公報で開示した方法で調製したB.シュードロンガム
のプロトプラスト懸濁液、或いは、特開平2-167070号公
報で開示した方法で調製したB.ロンガムのプロトプラス
ト懸濁液を高張緩衝液で希釈したものを塗沫し、28〜30
℃で嫌気培養し、６日乃至７日後、プロトプラスト再生
頻度（FR）を、有効プロトプラスト出現頻度（FAP）及
び浸透圧安定細胞の出現頻度（FOS）とともに計算す
る。Raffinose-added MNM-G medium was added to Japanese Patent Application Laid-Open No. 2-19587.
Protoplast suspension of B. pseudolongum prepared by the method disclosed in JP 2, or B. Longum protoplast suspension prepared by the method disclosed in JP-A 2-167070 with a hypertonic buffer. Apply diluted product and apply 28-30
After 6 to 7 days of anaerobic culture at 0 ° C., the protoplast regeneration frequency (FR) is calculated together with the frequency of effective protoplasts (FAP) and the frequency of osmotically stable cells (FOS).
上記高張緩衝液には30mMトリス−HCl（pH8.0）、3mH
MgCl2・6H2O、0.3Mラフィノースを含むNo.3THMRバッフ
ァーを用いるとよい。The above hypertonic buffer contains 30 mM Tris-HCl (pH 8.0), 3 mH
It is recommended to use No.3 THMR buffer containing MgCl 2 · 6H 2 O and 0.3 M raffinose.
寒天としては、バクト寒天（Bacto agar）（DIFCO LA
BORATORIES（米国）製、商標名）を用いるとよい。For agar, Bacto agar (DIFCO LA)
BORATORIES (USA), trade name) may be used.
以下に実施例を示して本発明を具体的に説明する。 Hereinafter, the present invention will be described specifically with reference to examples.
実施例１ 供試菌株としてB.シュードロンガムSBT 2908（FERM P
-10138号）を用い、該菌株を40％のトマトジュース浸出
液を含むGAM培地に37℃の温度に一夜培養した。Example 1 As a test strain, B. pseudolongum SBT 2908 (FERM P
-10138) was used to cultivate the strain in GAM medium containing 40% tomato juice exudate at a temperature of 37 ° C. overnight.
次いで、培養により得られた菌体を、3mMの塩化マグ
ネシウムと0.3Mのラフィノースを含む30mMのTris-HCl
（pH8.0）に懸濁させた。この菌体懸濁液に800μg/mlの
リゾチームと500μg/mlのα−アミラーゼを同時に添加
してプロトプラスト化を行つた。Then, the cells obtained by the culture were treated with 30 mM Tris-HCl containing 3 mM magnesium chloride and 0.3 M raffinose.
It was suspended in (pH 8.0). To this cell suspension, 800 μg / ml lysozyme and 500 μg / ml α-amylase were added at the same time for protoplast formation.
プロトプラストの形成は90％以上であつた。 The formation of protoplasts was over 90%.
上記のように形成したプロトプラストの懸濁液を、ラ
フィノースを0.2Mの濃度に添加した前記組成のMNM-G培
地に塗沫し、28℃で７日間嫌気培養を行つた。The protoplast suspension formed as described above was smeared on the MNM-G medium having the above composition to which raffinose was added at a concentration of 0.2 M, and anaerobic culture was carried out at 28 ° C. for 7 days.
再生頻度の計算結果を以下に示す。 The calculation result of the reproduction frequency is shown below.
実施例２ 供試菌株としてB.ロンガム SBT 2933-R（FERMP-8743
号）を用い、実施例１と同様に、該菌株を40％のトマト
ジュース浸出液を含むGAM培地に37℃の温度に一夜培養
した。 Example 2 B. longum SBT 2933-R (FERMP-8743 as a test strain
No.), the strain was cultured overnight at 37 ° C. in a GAM medium containing 40% tomato juice exudate as in Example 1.
次いで、培養により得られた菌体を、3mMのMgCl2と0.
3Mのラフィノースを含む30mMのトリス−HClバッフアー
（pH8.0）に懸濁させた。得られた菌体懸濁液にリゾチ
ームを2mg/mlの濃度に添加して１時間処理した後、アク
チナーゼＥを1mg/mlの濃度に添加して30分間処理した。Then, the bacterial cells obtained by culturing were treated with 3 mM MgCl 2 and 0.
The cells were suspended in 30 mM Tris-HCl buffer (pH 8.0) containing 3M raffinose. Lysozyme was added to the obtained bacterial cell suspension at a concentration of 2 mg / ml and treated for 1 hour, and then actinase E was added to a concentration of 1 mg / ml and treated for 30 minutes.
プロトプラストの形成は90％以上であつた。 The formation of protoplasts was over 90%.
上記のように処して形成したプロトプラストの懸濁液
を、ラフィノースを0.2Mの濃度に添加した前記組成のMN
M-G培地に塗沫し、28℃で７日間嫌気培養を行つた。The suspension of protoplasts formed as described above was treated with MN of the above composition to which raffinose was added to a concentration of 0.2M.
It was smeared on MG medium and anaerobically cultured at 28 ° C for 7 days.
再生頻度の計算結果を以下に示す。 The calculation result of the reproduction frequency is shown below.
発明の効果 以上述べたとおり、本発明によると、FAPはプロトプ
ラストを調製する際の細胞壁溶解酵素によるいずれの作
用時間でも99％以上であり、したがつて、上記溶解酵素
の作用時間を0.5時間乃至1.0時間に設定すればプロトプ
ラスト化が十分進行し、10-2の高頻度で再生することが
可能となる。 EFFECTS OF THE INVENTION As described above, according to the present invention, FAP is 99% or more in any action time by the cell wall lysing enzyme when preparing protoplasts, and thus the action time of the lytic enzyme is 0.5 hours to If it is set to 1.0 hour, protoplast formation will proceed sufficiently and it will be possible to reproduce at a high frequency of 10 -2 .
Claims (3)
た、下記に示した基本組成を特徴とするビフィズス菌プ
ロトプラストの再生用MNM-G培地。 MNM-G培地の基本組成：（/l） K2HPO4 2.5g CH3COONa 10 ｇ トリプトン 10 ｇ 酵母エキス 5.0g システィン・HCl・H2O 0.4g グルコース 5.0g 寒 天 15 ｇ CaCl2・2H2O 5 mM MgCl2・6H2O 5 mM pH6.5 1. An MNM-G medium for regenerating Bifidobacterium protoplasts, which is suitable for regeneration of Bifidobacterium protoplasts and has the basic composition shown below. Basic composition of MNM-G medium: (/ l) K 2 HPO 4 2.5g CH 3 COONa 10 g Tryptone 10 g Yeast extract 5.0 g Cystine ・ HCl ・ H 2 O 0.4 g Glucose 5.0 g Agar 15 g CaCl 2・ 2H 2 O 5 mM MgCl 2 · 6H 2 O 5 mM pH6.5
或いは、ビフィドバクテリウム・ロンガムのプロトプラ
ストを、請求項（１）に記載の再生用MNM-G培地に浸透
圧安定剤としてラフィノースを添加した培地に塗沫し
て、嫌気培養することを特徴とする上記プロトプラスト
の再生方法。2. A medium in which Bifidobacterium pseudolongum or protoplasts of Bifidobacterium longum are added to the regenerating MNM-G medium according to claim 1 as raffinose as an osmotic pressure stabilizer. The method for regenerating protoplasts described above, which comprises anaerobically culturing the cells on the surface.
添加する請求項（２）に記載のプロトプラストの再生方
法。3. The method for regenerating protoplasts according to claim 2, wherein raffinose is added to the medium at a concentration of 0.2 to 0.3M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63228342A JP2681495B2 (en) | 1988-09-14 | 1988-09-14 | Bifidobacteria protoplast regeneration medium and regeneration method using the medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63228342A JP2681495B2 (en) | 1988-09-14 | 1988-09-14 | Bifidobacteria protoplast regeneration medium and regeneration method using the medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0276572A JPH0276572A (en) | 1990-03-15 |
| JP2681495B2 true JP2681495B2 (en) | 1997-11-26 |
Family
ID=16874961
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63228342A Expired - Fee Related JP2681495B2 (en) | 1988-09-14 | 1988-09-14 | Bifidobacteria protoplast regeneration medium and regeneration method using the medium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2681495B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2076560A1 (en) * | 1991-08-29 | 1993-03-01 | Kazuya Ogino | Dye-containing polarizing film |
| JPH088858B2 (en) * | 1991-09-10 | 1996-01-31 | 栃木県 | Separation and selection method of symbiotic microorganisms used for disease control of dicotyledonous plants |
| CN114181836A (en) * | 2021-11-25 | 2022-03-15 | 南京农业大学 | Preparation and regeneration method of musk mold protoplast |
-
1988
- 1988-09-14 JP JP63228342A patent/JP2681495B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0276572A (en) | 1990-03-15 |
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