JPS59192086A - Bacillus licheniformis mn001 - Google Patents
Bacillus licheniformis mn001Info
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- JPS59192086A JPS59192086A JP58065200A JP6520083A JPS59192086A JP S59192086 A JPS59192086 A JP S59192086A JP 58065200 A JP58065200 A JP 58065200A JP 6520083 A JP6520083 A JP 6520083A JP S59192086 A JPS59192086 A JP S59192086A
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Abstract
Description
【発明の詳細な説明】
本発明は、バチルス・リケニホルミス（Ｂａｃｉｌｌｕ
ｓｌｉｃｈｅｎｉｆｏｒｍｉｓ ） ＭＮ−００１に関
するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of Bacillus licheniformis (Bacillus licheniformis).
slicheniformis) MN-001.
更に詳細には、本発明は、プロテアーゼ、セルラーゼを
菌体外に多量生成する飼料添加に好適なバチルス・リケ
ニホルミス ＭＮ−００１に関するものである。More specifically, the present invention relates to Bacillus licheniformis MN-001, which produces large amounts of protease and cellulase extracellularly and is suitable for addition to feed.
現在、配合飼料は、自家製飼料を主体とするものから、
近代的工場で生産される配合飼料への転換がすすみ、全
飼料に占める配合飼料の比率は極めて高い値となってい
る。Currently, compound feeds range from mainly homemade feeds to
The shift to formula feed produced in modern factories has progressed, and the ratio of formula feed to total feed has become extremely high.
これらの配合飼料においては、主原料である穀類、油粕
類、魚粉、油脂類の他に、栄養補給、消化吸収改善、成
長促進、疾病防止等々の目的で、アミノ酸類、ミネラル
類、ビタミン類、抗生物質、酵素類、生菌剤等が副原料
として用いられている。In addition to the main ingredients, such as grains, oil cake, fishmeal, and fats and oils, these compounded feeds also contain amino acids, minerals, vitamins, Antibiotics, enzymes, probiotics, etc. are used as auxiliary raw materials.
しかしながらこのうち抗生物質についてはその残留移行
によシ、人間にアレルギーや、腸内細菌の変化を引き起
こす恐れがあるため使用上の規制が強められ、使用範囲
が限定、縮少される方向にある。また酵素類については
、リパーゼ剤、アミラーゼ剤、セルラーゼ剤、プロテア
ーゼ剤、混合酵素剤等について、生菌剤については、納
豆菌、乳酸菌、酪酸菌、ビフィズス菌等について検討さ
れているが、その効果がまちまちであり常に一定した効
果は期待できないのが現状である。However, due to the risk of residual migration of antibiotics, which may cause allergies or changes in intestinal bacteria in humans, restrictions on the use of antibiotics are being tightened, and the range of use is being limited or reduced. . Regarding enzymes, lipase agents, amylase agents, cellulase agents, protease agents, mixed enzyme agents, etc. are being investigated, and regarding viable bacterial agents, Bacillus natto, lactic acid bacteria, butyric acid bacteria, bifidobacteria, etc. are being studied, but their effects are being investigated. The current situation is that it is not possible to always expect a constant effect as the results vary.
本発明者らは、生体にきわめて安全なバチルス属の菌の
なかに酵素生産性のよりすぐれた変異株が見出せるので
はないかとの想定のもとに、自然界から多くのバチルス
属の菌を分離し、更に変異操作を行った結果、プロテア
ーゼ、セルラーゼの菌体外生産能が著しるしくすぐれた
バチルス・リケニホルミス ＭＮ−００１を単離獲得す
るに至った。The present inventors isolated many bacteria of the genus Bacillus from nature, based on the assumption that mutant strains with better enzyme productivity could be found among bacteria of the genus Bacillus, which are extremely safe for living organisms. However, as a result of further mutational manipulation, Bacillus licheniformis MN-001 was isolated and obtained which has a remarkable ability to produce protease and cellulase outside the cell.
ことに単離されたバチルス・リケニホルミスＭＮ−００
１は菌学的性質においてはバチルス・リケニホルミスと
嫌気的条件下で生育する点及びその他の性質でよく一致
しているが、プロテアーゼ、セルラーゼの生産性が顕著
である点で全く相違しており、バチルス・リケニホルミ
スの変異株と認められたものである。Particularly isolated Bacillus licheniformis MN-00
1 has the same mycological properties as Bacillus licheniformis in that it grows under anaerobic conditions and other properties, but it is completely different in that it has remarkable productivity of protease and cellulase. It was recognized as a mutant strain of Bacillus licheniformis.
バチルス・リケニホルミス ＭＮ−００１は微工研にＭ
Ｎ−００１の表示の下にＦＥＲＭ ＢＰ−２６６として
寄託されている。Bacillus licheniformis MN-001 is M
Deposited as FERM BP-266 under the designation N-001.
次に、バチルス・リケニホルミス ＭＮ−００１の菌学
的性質を示す。Next, the mycological properties of Bacillus licheniformis MN-001 will be shown.
Ａ、形態 （１）巾０．５〜０８μｍ１長さ２〜６μｍの桿菌。A, form (1) Bacillus with a width of 0.5 to 08 μm and a length of 2 to 6 μm.
（２） ダラム染色：陽性。(2) Durham staining: Positive.
（３）楕円状の内生胞子を形成。(3) Forms oval endospores.
（４）運動性：有。(4) Motility: Yes.
Ｂ、生育
（１）普通寒天培地：非常に良く生育する。色素の生成
なし。B. Growth (1) Ordinary agar medium: Very good growth. No pigment formation.
（２）糖蜜、硫酸アンモニウムを主体とした培地：非常
に良く生育する。色素の生成なし。(2) Medium mainly composed of molasses and ammonium sulfate: Grows very well. No pigment formation.
（３）食塩液体培地 ５％：生育する。(3) Salt liquid medium 5%: Grows.
７％：生育する。7%: Grows.
１０チ：生育する。10chi: Grow.
Ｃ６生理学的性質
（１） カタラーゼ生成 ＋（２
）嫌気的条件下での生育 ＋（３１ＶＰ反応
＋（４）高温での生育状態
５０°Ｃ＋
５５゛Ｃ−
（５）糖類から酸生成の有無
グルコース ＋
Ｌ−アラビノース ＋
Ｄ−キシロース ＋
マンニット ＋
Ｄ−タガトース ＋（６） アル
ギニンジヒドロラーゼ ＋（７）レシチナーゼ
〜（８）デンプンの加水分解性
＋（９）ゼラチンの加水分解性 十
Ｑ（１） ＤＮＡの加水分解性 ＋（１１
）クエン酸塩の利用性 十ｕり プロピ
オン酸塩の利用性 ＋（ｌ四 硝酸還元性
＋（１４）インドール生成
−（１９フェニルアラニンの脱アミノ化
−〇〇 特異的性質ニ
プロテアーゼ（バチルス−リケニホルミス ＩＦＯ１２
１９９に比
較して約１０倍菌体外に
生産する）
セルラーゼ （バチルス・リケニホルミス ＩＦＯ１２
１９９に比
較して約１５倍菌体外に
生産する）
の菌体外生産能が著しるしく高い。C6 physiological properties (1) Catalase production + (2
) Growth under anaerobic conditions + (31VP reaction + (4) Growth state at high temperature
50°C + 55°C- (5) Presence or absence of acid production from sugars Glucose + L-arabinose + D-xylose + Mannitol + D-tagatose + (6) Arginine dihydrolase + (7) Lecithinase
~(8) Hydrolyzability of starch
+(9) Hydrolyzability of gelatin 10Q(1) Hydrolyzability of DNA +(11
) Utilization of citrate Utilization of propionate + (l4 Nitrate reducing ability
+(14) Indole generation
-(19 Deamination of phenylalanine
-〇〇 Specific properties Niprotease (Bacillus licheniformis IFO12
cellulase (Bacillus licheniformis IFO12)
It has a significantly higher extracellular production capacity (about 15 times that of 199).
本発明のバチルス・リケニホルミス ＭＮ−００１を培
養するには、通常の醗酵法に準じて、液体培養、又は固
体培養する事によって行なえばよい。Bacillus licheniformis MN-001 of the present invention may be cultured by liquid culture or solid culture according to a conventional fermentation method.
培地としては天然培地、半合成培地、合成培地等いずれ
でも使用する事ができる。培地の組成は、（１）炭素源
としては、グルコース、サッカロース、デキストリン、
グリセリン、デンプン、糖蜜等を、（１１）窒素源とし
ては、ペプトン、肉エキス、酵母エキス、乾燥酵母、大
豆粕、尿素、チオ尿素、アンモニウム塩、硝酸塩、その
他有機あるいは無機窒素化合物を、冊無機塩としては、
マグネシウム、マンガン、カリウム、カルシウム、鉄等
の燐酸塩、硝酸塩、炭酸塩、塩化物などが用いられる。As the medium, any of natural medium, semi-synthetic medium, synthetic medium, etc. can be used. The composition of the medium is as follows: (1) Carbon sources include glucose, sucrose, dextrin,
(11) Nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean meal, urea, thiourea, ammonium salts, nitrates, and other organic or inorganic nitrogen compounds. As salt,
Phosphates, nitrates, carbonates, chlorides, etc. of magnesium, manganese, potassium, calcium, iron, etc. are used.
寸だアミノ酸類、ビタミン類、核酸およびその関連化合
物を添加してもよい。培養温度は、通常の醗酵法に準す
るが、たとえば２０°Ｃ〜５０°Ｃで好適に培養できる
。培養期間も通常の醗酵法に準するがたとえば１２時間
〜５日で好適に培養できる。Amino acids, vitamins, nucleic acids and related compounds may also be added. The culture temperature is based on the usual fermentation method, but it can be suitably cultured at, for example, 20°C to 50°C. The culturing period is also similar to that of ordinary fermentation methods, but for example, 12 hours to 5 days is suitable for culturing.
ここに得られた培養物は、そのままもしくは洗滌をくり
かえして菌体のみにして凍結乾燥、噴霧乾燥するなどに
よシ製剤化することができる。The culture thus obtained can be prepared as it is or by repeated washing to obtain only bacterial cells and freeze-drying, spray-drying, etc. to form a preparation.
動物用飼料又は飼料添加物とする場合は、たとえば、培
養液をそのまま、あるいは、小麦粉、デンプン、デキス
トリン等や、飼料用原料の穀類、脱脂米ぬか等の糟糠類
、油粕等の適当な希釈剤を用い、製剤化する事ができる
。又培養液から菌体のみを洗滌分離し、同様に製剤化し
て、用いてもよい。When used as animal feed or feed additives, for example, the culture solution may be used as is, or an appropriate diluent such as wheat flour, starch, dextrin, etc., grains as raw materials for feed, bran such as defatted rice bran, oil cake, etc. It can be used and formulated into formulations. Alternatively, only the bacterial cells may be washed and separated from the culture solution, formulated in the same manner, and used.
このようにして得られたバチルス・リケニホルミス Ｍ
Ｎ−００１製剤は動物用飼料又は飼料添加物として、肉
用牛、乳用牛、子牛、豚、子豚、羊、山羊、馬、ウサギ
、犬、猫等の家畜類、および肉用鶏、採卵鶏、雛、種鶏
、アヒル、鵞鳥、七面鳥、うずら、子馬等の家禽類等に
適宜与えて有効である。また投与量は対象動物によシ又
対象動物の日冷により、又飼料の他の成分によシ変わシ
うるもので一概には規定できないが通常バチルス・リケ
ニホルミス ＭＮ−００１菌体が飼料１ ｋｇ当り１０
２〜１０１５個、望ましくは１０４〜１０１２個、最も
望ましくは１０５〜１０１０個になるごとく添加すると
よい。The thus obtained Bacillus licheniformis M
The N-001 formulation can be used as animal feed or feed additive for livestock such as beef cattle, dairy cattle, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, and chickens for meat. It is effective when given appropriately to poultry such as egg-laying hens, chicks, breeding hens, ducks, geese, turkeys, quail, and foals. The dosage cannot be determined unconditionally as it may vary depending on the target animal, depending on the animal's diurnal cooling, or depending on other ingredients of the feed, but usually Bacillus licheniformis MN-001 cells are added to 1 kg of the feed. Hit 10
It is preferable to add 2 to 1015 pieces, preferably 104 to 1012 pieces, most preferably 105 to 1010 pieces.
次に本発明の試験例、実施例、参考例を示す。Next, test examples, examples, and reference examples of the present invention will be shown.
試験例
１、 バチルス・リケニホルミス ＴＦＯ１２１９９２
、バチルス・リケニホルミスＭＮ−００１、Ｆｌ、Ｍ
ＢＰ−２６６
上記１．２の各菌株を、５００ ｍｌ 坂ロフラスコに
糖蜜 ２ｇ、硫酸アンモニウム １ 、！ｉ’、 Ｋ、
ＨＰｏ、 ０．ＩＩ、酵母エキス００５Ｉを添加し、水
道水を１００ｍＡ入れ、０．ＩＮ ＨＣ７と０．ＩＮ
ＮａＯＨでｐＨを８に調整した後、１２１℃で１５分間
殺菌した培地に接種し、６７°Ｃで２４時間振とう培養
し、それぞれの菌体外酵素（プロテアーゼ、アミラーゼ
、セルラーゼ）を測定した。Test Example 1, Bacillus licheniformis TFO121992
, Bacillus licheniformis MN-001, Fl, M
BP-266 Each strain in 1.2 above was placed in a 500 ml Sakalo flask with 2 g of molasses and 1 portion of ammonium sulfate! i', K,
HPo, 0. II, yeast extract 005I was added, tap water was added at 100 mA, and 0. IN HC7 and 0. IN
After adjusting the pH to 8 with NaOH, it was inoculated into a medium that had been sterilized at 121°C for 15 minutes, cultured with shaking at 67°C for 24 hours, and the levels of each extracellular enzyme (protease, amylase, cellulase) were measured.
測定方法は培養Ｆ液を粗酵素液として用い、アミラーゼ
活性測定には、基質としてデンプンを用いたジニトロサ
リチル酸法、セルラーセ活性測定には、基質としてカル
ボキシメチルセルロースを用いたジニトロサリチル酸法
、プロテア−上宿性測定には、基質としてミルクカゼイ
ンを用いた銅Ｆｏｌｉｎ法を採用し、菌体外生産能を比
較した。The measurement method is to use culture F solution as a crude enzyme solution, and to measure amylase activity, use the dinitrosalicylic acid method using starch as a substrate.To measure cellulase activity, use the dinitrosalicylic acid method using carboxymethyl cellulose as a substrate. The copper Folin method using milk casein as a substrate was used to measure the cellulose production ability, and the extracellular production ability was compared.
なお、表中の数字はＩＦＯ１２１９９の値を１とした時
の相対値である。Note that the numbers in the table are relative values when the value of IFO12199 is set to 1.
結果は次の第１表に示される。The results are shown in Table 1 below.
第１表
実施例
ジャーファーメンタ−に試験例と同じ組成の培地１１を
入れ、殺菌後あらかじめ前培養をしたＢａｃｉｌｌｕｓ
Ｉｉｃｈｅｎｉｆｏｒｍｉｓ ＭＮ−００１、Ｆ
ＩＲＭＢＰ−２６６を接種し、２４時間振とり培養した
。Table 1 Examples A medium 11 having the same composition as the test example was placed in a jar fermenter, and Bacillus was precultured after sterilization.
Iicheniformis MN-001, F
IRMBP-266 was inoculated and cultured with shaking for 24 hours.
遠心分離により培養源から菌体を分離後、デンプン１ｋ
ｇを添加し乾燥させＪ３ａｃｉｌｌｕｓ Ｉｉｃｈｅｎ
ｉｆｏｒｍｉｓＭＮ−００１胞子製剤を作製した。この
製剤の生菌数は１０８個／、！９であった。After separating the bacterial cells from the culture source by centrifugation, 1k starch
Add J3acillus Iichen and dry.
An iformisMN-001 spore preparation was prepared. The number of viable bacteria in this preparation is 108/! It was 9.
参考例
ブロイラー１２０羽を用い（雄雌各４０羽）各群４０羽
（雄雌２０羽）ずつの２区に分け、第２表に示した基本
飼料に対照区（デンプン添加区）実施例で得たＭＮ−０
０１製剤添加区（デンプン１ｇ当９１０８個の菌数を含
有）を設け、８週間の飼養試験を実施した。結果を第３
表に示した。Reference Example: Using 120 broiler chickens (40 males and 40 females each), they were divided into two groups of 40 birds each (20 males and females), and the control group (starch added group) was fed with the basic feed shown in Table 2. Obtained MN-0
A group containing 01 formulation (containing 9108 bacteria per gram of starch) was established, and an 8-week feeding test was conducted. 3rd result
Shown in the table.
第 ２ 表 （基本飼料の組成）
第３表
以上のように、ＭＮ−００１製剤は、動物に対して優れ
た効果を発揮する。Table 2 (Composition of basic feed) As shown in Table 3 and above, the MN-001 formulation exhibits excellent effects on animals.
また菌体外酵素産生能の優れたＭＮ−００１製剤は動物
用飼料又は飼料添加物のみならず、魚類等の飼料用、食
品用、医薬品用、化粧品用、酵素製造用等の用途にも有
効である。In addition, the MN-001 preparation, which has excellent extracellular enzyme production ability, is effective not only as animal feed or feed additive, but also for use in fish feed, food, pharmaceuticals, cosmetics, enzyme production, etc. It is.
代理人 弁理士 戸 １）親 列Agent Patent attorney 1) Parent row
Claims (1)
ミス ＭＮ−００１゜ Ａ、形態 （１）巾０．５〜０．８μｍ１長さ２〜３μｍの桿菌。 （２）ダラム染色：陽性。 （３）楕円状の内生胞子を形成。 （４）運動性：有。 Ｂ、生育 （１）普通寒天培地：非常に良く生育する。色素の生成
なし。 （２）糖蜜、硫酸アンモニウムを主体とした培地：非常
に良く生育する。色素の生成なし。 （３）食塩液体培地 ５％：生育する。 ７チ：生育する。 １０％：生育する。 Ｃ０生理学的性質 （１） カタラーゼ生成 ＋（２）
嫌気的条件下での生育 ＋（３）ＶＰ反応
＋（４）高温での生育状態 ５
０°Ｃ＋ ５５°Ｃ− （５）糖類から酸生成の有無 グＪレコース ＋ Ｌ−アラビノース ＋ Ｄ−キシロース 士 マンニット ＋ Ｄ＝タガトース 十（６） アル
ギニンジヒドロラーゼ′ 十（カ レシチナーゼ
−（８）デンプンの加水分解性
＋（９）ゼラチンの加水分解性
十ＱＩＤＮＡの加水分解性 十αυ クエ
ン酸塩の利用性 ＋σつ プロピオン酸塩
の利用性 十（ｌ尋 硝酸還元性
十α→ インドール生成
−（へ） フェニルアラニンの脱アミノ化 −（１
６１％異的性質； プロテアーゼ、セルラーゼの菌体外生産能力が著しるし
く高い。[Scope of Claims] (1) Bacillus licheniformis MN-001°A having the following mycological properties, form (1) A rod having a width of 0.5 to 0.8 μm and a length of 2 to 3 μm. (2) Durham staining: positive. (3) Forms oval endospores. (4) Motility: Yes. B. Growth (1) Ordinary agar medium: Very good growth. No pigment formation. (2) Medium mainly composed of molasses and ammonium sulfate: Grows very well. No pigment formation. (3) Saline liquid medium 5%: Grows. 7chi: Grow. 10%: Grows. C0 physiological properties (1) Catalase production + (2)
Growth under anaerobic conditions + (3) VP reaction
+(4) Growth conditions at high temperatures 5
0°C+ 55°C- (5) Presence or absence of acid production from sugars Glucose + L-arabinose + D-xylose Mannitol + D=tagatose 10 (6) Arginine dihydrolase' 10 (karecitinase - (8) Hydrolyzability of starch
+(9) Hydrolyzability of gelatin
10QI DNA hydrolysis 10αυ Citrate utilization +σ 1 Propionate utilization 10(1 fathom) Nitrate reduction
10α → indole production
-(to) Deamination of phenylalanine -(1
61% heterogeneity: The ability to produce protease and cellulase outside the cell is extremely high.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58065200A JPS59192086A (en) | 1983-04-15 | 1983-04-15 | Bacillus licheniformis mn001 |
| GB08406605A GB2138023A (en) | 1983-03-29 | 1984-03-14 | Bacillus licheniformis used in animal feeds |
| DE19843410771 DE3410771A1 (en) | 1983-03-29 | 1984-03-23 | ANIMAL FEED ADDITIVE, FOOD AND MICROORGANISM USED IN IT |
| PH30448A PH19390A (en) | 1983-03-29 | 1984-03-27 | Animal feed additive,feed and microorganism employed therefor |
| KR1019840001623A KR880001275B1 (en) | 1983-03-29 | 1984-03-29 | Manufacturing process for making animal feeds |
| FR8404894A FR2543409A1 (en) | 1983-03-29 | 1984-03-29 | FOOD ADDITIVE AND FOOD FOR ANIMALS CONTAINING BACILLUS LICHENIFORMIS AND NOVEL STRAIN OF THIS MICROORGANISM |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58065200A JPS59192086A (en) | 1983-04-15 | 1983-04-15 | Bacillus licheniformis mn001 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59192086A true JPS59192086A (en) | 1984-10-31 |
| JPH0379986B2 JPH0379986B2 (en) | 1991-12-20 |
Family
ID=13280030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP58065200A Granted JPS59192086A (en) | 1983-03-29 | 1983-04-15 | Bacillus licheniformis mn001 |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59192086A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1093803A2 (en) * | 1996-10-23 | 2001-04-25 | Sanofi-Synthelabo | Cosmetic composition comprising antagonist of the neuropeptide Y receptor |
| KR100443267B1 (en) * | 2002-07-22 | 2004-08-04 | 한국생명공학연구원 | Novel Bacillus licheniformis H-3 strain having capability of food waste decomposition |
-
1983
- 1983-04-15 JP JP58065200A patent/JPS59192086A/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1093803A2 (en) * | 1996-10-23 | 2001-04-25 | Sanofi-Synthelabo | Cosmetic composition comprising antagonist of the neuropeptide Y receptor |
| EP1093803A3 (en) * | 1996-10-23 | 2004-03-31 | Sanofi-Synthelabo | Cosmetic composition comprising an antagonist of the neuropeptide Y receptor |
| KR100443267B1 (en) * | 2002-07-22 | 2004-08-04 | 한국생명공학연구원 | Novel Bacillus licheniformis H-3 strain having capability of food waste decomposition |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0379986B2 (en) | 1991-12-20 |
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