JPH03103187A - Production of isomaltulose - Google Patents
Production of isomaltuloseInfo
- Publication number
- JPH03103187A JPH03103187A JP24007789A JP24007789A JPH03103187A JP H03103187 A JPH03103187 A JP H03103187A JP 24007789 A JP24007789 A JP 24007789A JP 24007789 A JP24007789 A JP 24007789A JP H03103187 A JPH03103187 A JP H03103187A
- Authority
- JP
- Japan
- Prior art keywords
- isomaltulose
- culture
- sucrose
- genus
- negative
- 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.)
- Pending
Links
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
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- 239000005720 sucrose Substances 0.000 claims description 31
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- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 abstract 1
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- 238000006243 chemical reaction Methods 0.000 description 8
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- NMXLJRHBJVMYPD-IPFGBZKGSA-N trehalulose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(O)CO[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 NMXLJRHBJVMYPD-IPFGBZKGSA-N 0.000 description 4
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- 235000005822 corn Nutrition 0.000 description 3
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- 229910052757 nitrogen Inorganic materials 0.000 description 3
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- 238000011084 recovery Methods 0.000 description 3
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- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- 230000008025 crystallization Effects 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明はイソマルチユロース(別名/パラチノース)の
製造法に関する。更に詳細には、本発明は蔗糖からイソ
マルチユロースを効率よく、大量に生産しうる方法に関
する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing isomaltulose (also known as palatinose). More specifically, the present invention relates to a method for efficiently producing isomaltulose in large quantities from sucrose.
[従来技術及び発明が解決しようとする課題]従来、糖
転換能を有する微生物、なかでも蔗糖から有用な二糖を
生或する微生物として、例えば、蔗糖をインマルチ二ロ
ースに主に転換する能力をもつロイコノストック・メセ
ンテロイデス(Leuconostoc mesen
teroideS)、プロタミノバクター・ルブラム(
P r o t aminobacter rubr
um)、セラチア・マルセツセンス(Serratia
marcescens)、セラチア●プリムチ力(
Serratia plymuthica)、エルウ
イニア◆カロトポラ(Erwinia caroto
vora)、エルウイニア・カロトポラ・アトロセプチ
カ(Erwinia carotovora va
r.atroseptica)、エルウイニア・デイソ
ルベンス(Erwinia dissolvens)
、エルウイニア・ラホンテイシ(Erwinia r
hapontici)等が知られている。しかしながら
、これらのイソマルチユロース生産性菌株はイソマルチ
ユロースの生戊時に一般にトレハルロース、フルクトー
ス、グルコース等のかなりの副生を伴い、蔗糖からのイ
ソマルチユロースの収率が低下するという欠点がある。[Prior Art and Problems to be Solved by the Invention] Conventionally, microorganisms that have the ability to convert sugars, especially microorganisms that can produce useful disaccharides from sucrose, have been known to have the ability to mainly convert sucrose to inmartinose. Leuconostoc mesenteroides (Leuconostoc mesen)
teroideS), Protaminobacter rubrum (
Pro t aminobacter rubr
um), Serratia marcetuscens
marcescens), Serratia Primchyriki (
Serratia plymuthica), Erwinia carotopora (Erwinia caroto)
vora), Erwinia carotopora atroseptica (Erwinia carotovora va)
r. atroseptica), Erwinia dissolvens
, Erwinia Rahonteisi
hapontici) etc. are known. However, these isomaltulose-producing strains generally produce a considerable amount of by-products such as trehalulose, fructose, and glucose during isomaltulose production, resulting in a drawback that the yield of isomaltulose from sucrose is reduced.
そこで、本発明者らは、この課題を解決し、イソマルチ
ユロースの収率を向上させるべく、さらに優れた糖転換
能を有し、その他の糖類の副生が少なく且つイソマルチ
ユロースの生戊比率の高い新規な微生物を探索すべく多
くの菌株を土壌や植物より常法により分離し、その糖転
換能について検討を加えた。Therefore, in order to solve this problem and improve the yield of isomaltulose, the present inventors have aimed to improve the yield of isomaltulose, which has even better sugar conversion ability, produces less by-products of other sugars, and improves the yield of isomaltulose. In order to search for new microorganisms with a high ratio, many strains were isolated from soil and plants using conventional methods, and their sugar conversion ability was investigated.
[課題を解決するための手段1
その結果、エンテロバクター属に属する或る種の菌株、
例えば、工冫テロバクター・エスピー(Enterob
acter sp.)No.7及びラネラ属に属する
或る種の菌株、例えば、ラネラ・エスピー(Rahna
lla sp.)No.2が優レタクルコシルトラン
ス7エラーゼ生産能を有しており、蔗糖をイソマルチユ
ロースに転換するのに有用な優れた糖転換能を示し、そ
の他の糖類の副生が少なく、イソマルチユロースの生成
比率の高い極めて有用な菌株であることを見い出し、本
発明を完戊するに至った。[Means for solving the problem 1 As a result, a certain strain belonging to the genus Enterobacter,
For example, Enterobacter sp.
acter sp. ) No. 7 and certain strains belonging to the genus Ranella, such as Rahna sp.
lla sp. ) No. 2 has an excellent ability to produce retacreucosyltrans7-erase, and shows excellent sugar conversion ability useful for converting sucrose into isomaltulose, with little by-product of other sugars, and is able to produce isomaltulose. We have discovered that this is an extremely useful strain with a high production rate, and have completed the present invention.
かくして、本発明によれば、エンテロバクター属又はラ
ネラ属に属するグルコシルトランスフエラーゼ生産性微
生物の菌体又はその処理物の存在下に蔗糖をイソマルチ
ユロースに転換することを特徴とする,fソマルチュロ
ースの製造方法が提供される。Thus, according to the present invention, f-someltulose is characterized in that sucrose is converted to isomaltulose in the presence of cells of glucosyltransferase-producing microorganisms belonging to the genus Enterobacter or the genus Ranella or a processed product thereof. A manufacturing method is provided.
また、本発明によれば、エンテロバクター属又はラネラ
属に属するグルコシルトランスフエラーゼ生産性微生物
を蔗糖を含有する培地で培養し、培地からイソマルチユ
ロースを回収することを特徴とするイソマルチユロース
の製造方法が提供される。Further, according to the present invention, a glucosyltransferase-producing microorganism belonging to the genus Enterobacter or the genus Ranella is cultured in a medium containing sucrose, and the isomaltulose is recovered from the medium. A manufacturing method is provided.
本発明は、エンテロバクター属又はラ不ラ属に属する微
生物が生産するグルコシルトラジスフエラーゼを利用し
、蔗糖を酵素反応により又は発酵法により、イソマルチ
ユロースに転換するものである。The present invention utilizes glucosyltradispherase produced by a microorganism belonging to the genus Enterobacter or Lafura to convert sucrose into isomaltulose by an enzymatic reaction or a fermentation method.
本発明の方法に使用しうるエンテロバクター属に属する
グルコシルトランスフエラーゼ生産性微生物の具体例に
は、エンテロバクター・エスビーNo.7(FERM
P−10990)等が挙げられ、また、ラネラ属に属
するグルコシルトランスフエラーゼ生産性微生物の具体
例には、ラネラ・エスビーN0.2(FERM P−
10989)等が挙げられるが、本発明は何らこれらに
限られるものではなく、通常のスクリーニング法を用い
ることにより、例えば、蔗糖を含む寒天平板培地に土壌
懸濁液を塗布し、30°Cで24〜48時間培養後、寒
天平板培地上で生育した蔗糖資化性菌を分離し、上記分
離菌を次に蔗糖を含む液体培地で30’C!で72時間
振盪培養後、イソマルチユロースの生戊の有無をペーパ
ークロマトグラフイーで確認することにより、当業者で
あれば本発明の方法に使用しうるエンテロバクター属又
はラネラ属に属tるグルコシルトランスフエラーゼ生産
性徴生物を容易に取得することができる。Specific examples of glucosyltransferase-producing microorganisms belonging to the genus Enterobacter that can be used in the method of the present invention include Enterobacter Sb No. 7 (FERM
P-10990), etc., and specific examples of glucosyltransferase-producing microorganisms belonging to the genus Ranella include Ranella sb N0.2 (FERM P-
10989), but the present invention is not limited to these. For example, by applying a soil suspension to an agar plate medium containing sucrose and incubating at 30°C, the present invention is not limited to these. After culturing for 24 to 48 hours, the sucrose-assimilating bacteria grown on the agar plate medium were isolated, and the isolated bacteria were then incubated at 30'C in a liquid medium containing sucrose. After culturing with shaking for 72 hours, a person skilled in the art can confirm the presence or absence of isomaltulose growth using paper chromatography. Transferase-producing organisms can be easily obtained.
本発明に使用しうる上記のエンテロバクター・エスピー
No.7は、従来の文献には未載の新規な菌株であり、
その菌学的性質を示せば次のとおりである。The above-mentioned Enterobacter sp. 7 is a novel strain that has not been described in conventional literature,
Its mycological properties are as follows.
これらの菌株の同定実験は、主としてM a n ua
l of Methods for Gene
ral Bacteriology(米国微生物学会
編、1981)および「微生物の分類と同定」(長谷川
武治編 東大出版会、1 9 8 5)に準拠して行っ
た。Identification experiments for these strains were mainly carried out using M an ua
Methods for Gene
ral Bacteriology (edited by the American Society for Microbiology, 1981) and "Classification and Identification of Microorganisms" (edited by Takeharu Hasegawa, University of Tokyo Press, 1985).
■ 細胞形態
肉汁寒天、25゜C培養:通常0.7〜l.OXl.5
〜6.0μmの桿菌。単独または直鎖状の二対をなし、
稀に連鎖した細胞も観察される。■Cell morphology Broth agar, culture at 25°C: Usually 0.7-l. OXl. 5
~6.0 μm bacilli. singly or in pairs in a linear chain,
Rarely linked cells are also observed.
多形性なし。運動性あり。鞭毛は周鞭毛。無胞子。No polymorphism. Has mobility. The flagellum is periflagellate. No spores.
非抗酸性。ダラム陰性。Non-acid-fast. Durham negative.
■ 培養的性質
(1) 肉汁寒天平板培養、25゜C形状:円形 大
きさは24時私感でl mmo周縁:全縁
***:凸状(umbonate)
光沢:あり
表面:平滑
色調:半透明、内容は均質なノ《ター質、ク1ノーム色
(2)Davis寒天平板培養、3 0 ’O :生育
なし
(3) 5% !iucrOse nutrient
Bger 培養、30゜C:コロニーは白色、半透
明、表面平滑、wetでmucoidで1よない
(4)Trypt icase Soy 寒天平板
培養、25゜C:黄色色素生戊なし
(5)肉汁寒天斜面培養、25゜C
生育度:良好
形状:糸状
(6)肉汁液体培養、25℃
生育度;良好 全体に生育し混濁
沈渣:少量
着色、脱色:なし
(7)肉汁ゼラチン穿刺培養
15℃で穿刺線に沿って生育。ゼラチンを液化しない。■Culture properties (1) Broth agar plate culture, 25°C Shape: Circular Size is 24 hours according to my personal feeling L mmo rim: Full ridge: Convex (umbonate) Gloss: Yes Surface: Smooth Color tone: Translucent; Contents are homogeneous, tar-like, chromatic in color (2) Davis agar plate culture, 30'O: no growth (3) 5%! iucrOse nutrient
Bger culture, 30°C: Colonies are white, translucent, smooth surface, mucoid level is not 1 when wet (4) Trypticase Soy agar plate culture, 25°C: No yellow pigment production (5) Juicy agar slant culture , 25°C Growth rate: Good Shape: Filamentous (6) Meat juice liquid culture, 25°C Growth rate: Good Grows all over, turbid sediment: Small amount of coloring, decolorization: None (7) Meat juice gelatin puncture culture At 15°C, at puncture line Grow along. Do not liquefy gelatin.
(8) リトマス・ミルク、25℃ リトマス酸性化後に退色。凝固、ペプトン化なし。(8) Litmus milk, 25℃ Fading after litmus acidification. No coagulation or peptonization.
■ 生理学的性質
(1) 硝酸塩の還元性:陽性
(2)脱窒反応:陽性
(3)MRテスト:陽性
(4)VPテスト:陽性
(5)インドールの生成:陰性
(6)硫化水素の生戊(TSI):陰性(7)デンプン
の加水分解:陰性
(8)クエン酸の利用:
シモンズ培地において陰性、
コーザー培地において陰性、
クリステンセン培地において陽a0
(9)無機窒素源の利用:アンモニウム塩および硝酸塩
ともに利用できる。■ Physiological properties (1) Nitrate reduction: Positive (2) Denitrification reaction: Positive (3) MR test: Positive (4) VP test: Positive (5) Indole production: Negative (6) Hydrogen sulfide production TSI: Negative (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Negative in Simmons' medium, Negative in Coser's medium, Positive a0 in Christensen's medium (9) Utilization of inorganic nitrogen sources: Ammonium salts and Both nitrates can be used.
(1 0) 色素の生或:なし
(1 1) ウレアーゼ:陰性
(l2)オキシダーゼ:陰性
(l3)カタラーゼ:陽性
(l4)生育の範囲:pH 4〜8
温度 4〜30℃
(l5)酸素に対する態度:通性嫌気性(16)O−F
テスト(ヒューレインソン法):D−グルコース発酵性
あり。(10) Pigment production: None (11) Urease: negative (l2) Oxidase: negative (l3) Catalase: positive (l4) Growth range: pH 4-8 Temperature 4-30°C (l5) Resistance to oxygen Attitude: Facultative anaerobic (16) O-F
Test (Heureinson method): D-glucose fermentable.
(l7)炭素源からの酸およびガスの生或の有無(ヒュ
ーレイ7ソン法による):
L−アラビノース
D−キシロース
D−グルコース
D−マンノース
D−7ラクトース
D−ガラクトース
麦芽糖
艷 ガス
十 +
+
+ +
+ +
千十
++
++
ショ糖 + +乳糖
トレハロース + +D−ソル
ビット 、++D−マンニット
+ +イノシット 千
十グリセリン +
デンプン 千 十サリシン
+ 十a−メチルーD−グル
コシド
粘液酸
a−D−ガラクツロン酸 + +(l8)
糖類からの酸の生或の有無:
アドニット
ラフイノース
ラムノース 士
(l9)エスクリンの加水分解:陽性
(20)グルコン酸の酸化:陽性
(2l)有機窒素源の利用:
L−グルタミン酸ナトリウム:陽性
L−ヒドロキシプロリン:陰性
(22)アルギニンの分解(ミューラー法による):陰
性
(23) リジンの脱炭酸反応
(ミューラー法による):陰性
(24)オルニチンの脱炭酸反応
(ミューラー法による):陰性
(25)グルタミン酸の脱炭酸反応
(ミューラー法による):陰性
(26) フエニルアラニンの脱アミノ反応:陰性
(27)β−ガラクトシダーゼテスト:陰性(28)D
Nase:陰性
(29) リパーゼ(c o r n
(30)マロン酸の利用:陽性
上述の菌学的性質をもとにして、
のManual of Deteive Ba
cteriology
974)およびManual of
matic Bacteriol
o i 1):陽性
Bergey
r m i n a t
第8版(l
Syste
ogy第9版(l
984)等を参考にして検索し、公知の菌株とその異同
を検討した。(17) Presence or absence of acid and gas production from carbon source (according to Hewley-Sevenson method): L-arabinose D-xylose D-glucose D-mannose D-7 lactose D-galactose maltose gas + + + + + + Thousands + + + + Sucrose + + Lactose trehalose + + D-sorbitol, + + D-mannitol
+ +Inocit 1,000 Glycerin + Starch 1,000 Salicin
+ tena-methyl-D-glucoside mucinic acid a-D-galacturonic acid + + (l8)
Presence or absence of acid production from sugars: Adonitraffinose rhamnose (19) Hydrolysis of esculin: Positive (20) Oxidation of gluconic acid: Positive (2L) Utilization of organic nitrogen source: Monosodium L-glutamate: Positive L-hydroxyproline: Negative (22) Arginine decomposition (by Mueller method): Negative (23) Lysine decarboxylation reaction (by Mueller method): Negative (24) Ornithine decarboxylation reaction (by Mueller method): Negative ( 25) Glutamic acid decarboxylation reaction (by Muller method): Negative (26) Phenylalanine deamination reaction: Negative (27) β-galactosidase test: Negative (28) D
Nase: Negative (29) Lipase (co r n (30) Utilization of malonic acid: Positive Based on the above-mentioned mycological properties,
Bacteriology 974) and Manual of matic Bacteriol o i 1): Positive Bergey Rmin at 8th edition (l Systemology 9th edition (l 984), etc.) to search for known strains and their differences. investigated.
以上の結果、本菌がエンテロバクター属に属すことが示
された。中でも、エンテロバクター・アグロメランス(
E.nterobacter agglomeran
s)に最も近い性質を示した。The above results showed that this bacterium belongs to the genus Enterobacter. Among them, Enterobacter agglomerans (
E. nterobacter agglomeran
s).
しかし、本菌はエンテロバクター・アグロメランスとは
、クエン酸の利用性(シモンズ)、D−グルコースから
のガスの生或、イノシットの発酵性及び利用性、リパー
ゼ(corn oil)の有無等の点で相違が認めら
れること、並びにエンテロバクター属に属する菌株で、
イソマルチユロースを生産する菌株は今迄の文献では認
められないことから、本菌はインマルチュロースを生産
するという特徴を有する新規な菌株であると考えられる
。However, this bacterium is different from Enterobacter agglomerans in terms of the availability of citric acid (Simmons), the production of gas from D-glucose, the fermentability and availability of inocit, and the presence or absence of lipase (corn oil). Differences are observed, and strains belonging to the Enterobacter genus,
Since no strain that produces isomaltulose has been found in the literature to date, this bacterium is considered to be a new strain that has the characteristic of producing inmartulose.
これにより、本発明者らは上記菌株をエンテロバクター
・エスピーNO.7と命名し、茨城県筑波郡谷田部町東
1丁目1番地3号の微生物工業技術研究所、特許微生物
寄託センターに微工研菌寄第10990号(FERM
P−10990)として寄託した。As a result, the present inventors identified the above-mentioned strain as Enterobacter sp. 7, and submitted it to the Institute of Microbial Technology, Patent Microorganism Depositary, No. 1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, with Microbiology Research Institute No. 10990 (FERM).
P-10990).
本発明では、上記菌株のほか同一菌属に属し、糖転換能
を有する菌株やこれらの変異株なども使用することがで
きる。In the present invention, in addition to the above-mentioned bacterial strains, bacterial strains belonging to the same bacterial genus and having sugar conversion ability, and mutant strains thereof can also be used.
また、本発明に使用しうる上記のラネラ・エスピーNo
.2もまた従来の文献には未載の新規な菌株であり、そ
の菌学的性質を示せば次のとおりである。In addition, the above-mentioned Ranella Sp. No. which can be used in the present invention
.. 2 is also a new strain that has not been described in conventional literature, and its mycological properties are as follows.
これらの菌株の同定実験は、前述のエンテロバクター・
エスピーNo.7の場合と同様に、主としてManua
l of Methods forGenera
l Bacteriology(米国微生物学会編、
1981)および「微生物の分類と同定」(長谷川武治
編 東大出版会、1985)に準拠して行った。Identification experiments for these strains were carried out using the aforementioned Enterobacter spp.
SP No. As in the case of 7, mainly Manua
l of Methods for Genera
l Bacteriology (edited by the American Society for Microbiology,
1981) and "Classification and Identification of Microorganisms" (edited by Takeharu Hasegawa, University of Tokyo Press, 1985).
■ 細胞形態
肉汁寒天、25°C培養二通常1.0〜1.5X2.0
〜5.0μmの桿菌。単独または直鎖状の二対をなし、
稀に連鎖した細胞も観察される。■Cell morphology Broth agar, cultured at 25°C, usually 1.0-1.5X2.0
~5.0 μm bacilli. singly or in pairs in a linear chain,
Rarely linked cells are also observed.
多形性なし。運動性あり(微弱)。鞭毛は周鞭毛。No polymorphism. Motile (weak). The flagellum is periflagellate.
無胞子。非抗酸性。ダラム陰性。No spores. Non-acid-fast. Durham negative.
■ 培養的性質
(1) 肉汁寒天平板培養、25゜C形状:円形 大
きさは24時間で2 mlllo周縁:全縁
***:扁平状
光沢:あり
表面:平滑
色調:半透明、内容は均質なバター質、クリーム色
(2)サブロー寒天平板培養、30゜C:コロニーはク
リーム色、大型(48時間で5mm以上)
(3) トリプシン ダイジェスト 寒天平板培養、3
0°C:コロニーはm u c o i d(4) 5
% sucrose nutrient ager
培養、30゜C:コロニーは白色、半透明、表面平滑
、mucoid
(5)Trypt icase Soy 寒天平板
培養、25゜C:コロニーの大きさは2“4時間で1m
m前後、黄色色素生成なし
(6)肉汁寒天斜面培養、25゜C
生育度:良好
形状248時間でEohinulate(7)肉汁液体
培養、25℃
生育度:良好 全体に生育し混濁
沈渣:少量
着色、脱色:なし
(8)肉汁ゼラチン穿刺培養
15°Cで穿刺線に沿って生育。ゼラチンを液化しない
。■Culture properties (1) Broth agar plate culture, 25°C Shape: Round Size: 2ml in 24 hours Periphery: Full ridge: Flat Gloss: Yes Surface: Smooth Color tone: Translucent, content: homogeneous butter (2) Sabouraud agar plate culture, 30°C: Colonies are cream colored, large (more than 5 mm in 48 hours) (3) Trypsin digest Agar plate culture, 3
0°C: Colony is mucoid(4) 5
% sucrose nutrient ager
Culture, 30°C: Colonies are white, translucent, smooth surface, mucoid (5) Trypt case soy Agar plate culture, 25°C: Colony size is 2" 1 m in 4 hours
Around m, no yellow pigment formation (6) Meat juice agar slant culture, 25°C Growth rate: Good shape Eohinulate in 248 hours (7) Meat juice liquid culture, 25°C Growth rate: Good Grows throughout, turbid sediment: Small amount of coloring, Decolorization: None (8) Meat juice gelatin puncture culture Grows along the puncture line at 15°C. Do not liquefy gelatin.
30℃でゼラチンを液化しない。Do not liquefy gelatin at 30°C.
(9) リトマス・ミルク、25℃ リトマス酸性化後に退色。凝固、ペプトン化なし。(9) Litmus milk, 25℃ Fading after litmus acidification. No coagulation or peptonization.
■ 生理学的性質
(1)硝酸塩の還元性:陽性
(2)脱窒反応:陽性
(3)’MRテスト:陽性
(4)VPテスト:陽性
(5)インドールの生rR=陰性
(6)硫化水素の生戊(TSI):陰性(7)デンプン
の加水分解:陰性
(8) クエン酸の利用:
シモンズ培地において陰性、
コーザー培地において陽性、
クリステンセン培地において陽性。■ Physiological properties (1) Reducibility of nitrate: positive (2) Denitrification reaction: positive (3) 'MR test: positive (4) VP test: positive (5) Indole raw rR = negative (6) Hydrogen sulfide TSI: Negative (7) Hydrolysis of starch: Negative (8) Utilization of citric acid: Negative in Simmons' medium, positive in Coser's medium, positive in Christensen's medium.
(9)無機窒素源の利用:アンモニウム塩および硝酸塩
ともに利用できる。(9) Use of inorganic nitrogen sources: Both ammonium salts and nitrates can be used.
(IO)色素の生戒:なし
(11) ウレアーゼ:陰性
(12)オキシダーゼ:陰性
(l3)カタラーゼ:陽性
(l4)生育の範囲:pH 6〜8
温度 4〜30゜C
(l5)酸素に対する態度二通性嫌気性(1 6)O−
Fテスト(ヒューレイフソン法):D−グルコース発酵
性あり。(IO) Pigment life policy: None (11) Urease: negative (12) Oxidase: negative (l3) Catalase: positive (l4) Growth range: pH 6-8 Temperature 4-30°C (l5) Attitude towards oxygen Bifaculty anaerobic (1 6) O-
F test (Heureifson method): D-glucose fermentable.
(l7)炭素源からの酸およびガスの生戒の有無(ヒュ
ーレイフソン法による):
漿
ガス
L−アラビノース
D−キシロース
D−グルコース
D−マンノース
D−フラクトース
D−ガラクトース
麦芽糖
シヨ糖
乳糖
トレハロース
D−ソルビット
D−マンニット
イノシット
グリセリン
デンプン
ズルシトール
D−アラビトール
粘液酸
(l8)
糖類からの酸の生或の有無:
+
+
+
アドニット
ラフイノース +
ラムノース +
(l9)エスクリンの加水分4解:陽性(20)アルギ
ニンの分解(ミューラー法による):陰性
(2l) リジンの脱炭酸反応
(ミューラー法による):陰性
(22)オルニチンの脱炭酸反応
(ミューラー法による):陰性
(23)グルタミン酸の脱炭酸反応
(ミューラー法による):陰性
(24) フエニルアラニンの脱アミノ反応:陰性
(25) β−ガラクトシダーゼテスト:陽性(26)
DNase+陰性
(27) リパーゼ(corn oil):陽性(2
8)マロン酸の利用:陽性
IV DNAのG−C比(Tm法による):54.1
%
上述の菌学的性質をもとにして、前述のエンテロバクタ
ー・エスビーNo.7の場合と同様に、Bergeyの
Manual of Determinative
Bacteriology 第8版(1974)
およびManual of Systematic
Bacteriology第9版(1984)等を
参考にして検索し、公知の菌株とその異同を検討した。(17) Presence or absence of acid and gas intake from carbon sources (according to Huleifson method): Serum gas L-arabinose D-xylose D-glucose D-mannose D-fructose D-galactose maltose sucrose lactose trehalose D- Sorbit D-Mannitinositol Glycerin Starch Lucitol D-Arabitol Mucic acid (l8) Presence or absence of acid production from saccharides: + + + Adonitraffinose + Rhamnose + (l9) Hydrolysis of esculin : Positive (20) Decomposition of arginine (by Mueller's method): Negative (2L) Decarboxylation of lysine (by Mueller's method): Negative (22) Decarboxylation of ornithine (by Mueller's method): Negative (23) Decomposition of glutamic acid Decarboxylation reaction (by Muller method): Negative (24) Phenylalanine deamination reaction: Negative (25) β-galactosidase test: Positive (26)
DNase + negative (27) Lipase (corn oil): positive (2
8) Utilization of malonic acid: Positive IV DNA GC ratio (by Tm method): 54.1
% Based on the above-mentioned mycological properties, the above-mentioned Enterobacter S.B. No. 7, Bergey's Manual of Determinative
Bacteriology 8th edition (1974)
and Manual of Systematic
We searched with reference to Bacteriology, 9th edition (1984), etc., and examined known bacterial strains and their differences.
以上の結果、本菌がラネラ属に属すことが示された。現
在、ラネラ属の中には一菌種(R a h nella
aquatilis)のみが帰属させられている。The above results showed that this bacterium belongs to the genus Ranella. Currently, there is only one bacterial species within the genus Rah nella.
aquatilis) has been assigned.
本菌はこのラネラ・アクアテイリスに最も近い性質を示
した。しかし、本菌はラ不ラ・アクアテイリスとは、フ
エニルアラニンの脱アミノ反応、及びグリセリン、イノ
シット、D−キシロース等の発酵性及び利用性の点で相
違が認められること、並びにラネラ属に属する菌株で、
イソマルチユロースを生産する菌株は今迄の文献では認
められないことから、本菌はイソマルチユロースを生産
するという特徴を有する新規な菌株であると考えられる
。This bacterium showed properties most similar to Ranella aquateilis. However, this bacterium is different from Lafura aquateilis in terms of the deamination reaction of phenylalanine and the fermentability and utilization of glycerin, inosit, D-xylose, etc., and it belongs to the genus Ranella. With bacterial strains,
Since no strain producing isomaltulose has been found in the literature to date, this bacterium is considered to be a novel strain having the characteristic of producing isomaltulose.
これにより、本発明者らは上記菌株をラネラ・エスビー
No.2と命名し、茨城県筑波郡谷田部町東l丁目工番
3号の微生物工業技術研究所、特許微生物寄託センター
に微工研菌寄第10989号(FERM P−109
89)として寄託した。As a result, the present inventors identified the above-mentioned bacterial strain as Ranella SB No. 2, and submitted No. 10989 (FERM P-109) to the Patent Microorganism Depositary Center, Institute of Microbial Technology, No. 3, Higashi l-chome, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture.
89).
本発明では、上記菌株のほか同一菌属に属し、糖転換能
を有する菌株やこれらの変異株なども使用することがで
きる。In the present invention, in addition to the above-mentioned bacterial strains, bacterial strains belonging to the same bacterial genus and having sugar conversion ability, and mutant strains thereof can also be used.
本発明の方法に用いる微生物の培養は、それ自体既知の
方法により、蔗糖を主たる炭素源として含有する培地で
好適に行うことができる。培地は合戊培地又は天然培地
のいずれであってもよく、含有せしめうる窒素源として
は、例えば硝酸塩、アンモニウム塩などの無機窒素化合
物又は尿素、コーン・スチープ・リカー、カゼイン加水
分解物、ベプトン、酵母エキス、肉エキス、アミノ酸液
などの有機窒素含有物等が用いられ、また、無機塩類と
して、例えばカリウム塩、ナトリウム塩等を少量添加す
ることができる。培地中の蔗糖濃度は一般に2〜40%
範囲内とすることができるが、菌の生育及び増殖等の面
から、2〜20%の範囲内であることが望ましい。The microorganism used in the method of the present invention can be suitably cultured in a medium containing sucrose as the main carbon source by a method known per se. The medium may be either a synthetic medium or a natural medium, and examples of nitrogen sources that can be contained include inorganic nitrogen compounds such as nitrates and ammonium salts, or urea, corn steep liquor, casein hydrolyzate, beptone, Organic nitrogen-containing substances such as yeast extract, meat extract, and amino acid solution are used, and small amounts of inorganic salts such as potassium salts and sodium salts can be added. Sucrose concentration in the medium is generally 2-40%
Although it can be within this range, it is preferably within the range of 2 to 20% from the viewpoint of bacterial growth and proliferation.
培養は通常、温度25〜30℃、pH5.0〜7.0の
範囲内で通性嫌気的に又は好気的条件下で行うことがで
きる。培養方式は回分培養又は半回分培養のいずれでも
よい。Cultivation can usually be carried out under facultative anaerobic or aerobic conditions at a temperature of 25 to 30°C and a pH of 5.0 to 7.0. The culture method may be either batch culture or semi-batch culture.
培養時間は、培地中の蔗糖濃度によって異なるが、大体
16〜48時間程度が適当である。The culture time varies depending on the sucrose concentration in the medium, but approximately 16 to 48 hours is appropriate.
培養後、菌体を培養液から分離する。分離はそれ自体既
知の方法、例えば遠心分離法、濾過法等により行うこと
ができる。遠心法で菌体を分離する場合は、培養液に各
種の凝集剤を添加して、菌体を凝集させることにより分
離を容易かつ能率的にすることができる。After culturing, the bacterial cells are separated from the culture solution. Separation can be carried out by methods known per se, such as centrifugation, filtration, and the like. When separating bacterial cells by centrifugation, the separation can be made easier and more efficient by adding various flocculants to the culture solution to flocculate the bacterial cells.
菌体を除去した培養上清液は、加熱、濾過、イオン交換
樹脂処理等の処理を行い、次いで、例えば70〜80重
量%の固形分まで蒸発濃縮後、ゆっくり撹拌しながら徐
々に例えば約20゜Cまで冷却するとイソマルチユロー
スの結晶が析出する。The culture supernatant from which the bacterial cells have been removed is subjected to treatments such as heating, filtration, and ion exchange resin treatment, and then evaporated and concentrated to a solid content of, for example, 70 to 80% by weight. When cooled to °C, isomaltulose crystals precipitate.
得られる結晶は、例えばバスケット型遠心分離機にかけ
ることにより母液から分離することができる。モラセス
(廃蜜)は、必要により再結晶化工程に送り、再利用す
ることができる。The resulting crystals can be separated from the mother liquor, for example, by applying them to a basket centrifuge. Molasses (waste honey) can be sent to a recrystallization process and reused if necessary.
一方、培養液から分離した菌体はグルコシルトランスフ
エラーゼを含有しており、菌体そのもの又は菌体破砕物
いわゆる粗酵素又はこれを精製した精製酵素などの菌体
処理物は、蔗糖をイソマルチユロースに転換するための
酵素反応に利用することができる。その場合菌体又はそ
の処理物はそのまま又は固定化担体で包括し、さらに必
要に応じて架橋剤を用いて架橋処理を行った後に使用す
ることができ、特にこれら菌体又はその処理物は固定化
して用いるのが望ましい。その固定化はそれ自体既知の
方法で行うことができ、例えば、菌体又はその処理物を
アクリルアミド系単量体に包括し重合を行う方法;菌体
又はその処理物を担体で包括し、架橋剤等で処理する方
法、たとえば、キトサン・グルタルアルデヒド法、カラ
卓−ナン・グルタルアルデヒド法、アルギン酸・グルタ
ルアルデヒド法などを利用することができる。On the other hand, the bacterial cells isolated from the culture solution contain glucosyltransferase, and the bacterial cells themselves or the bacterial cell fragments, so-called crude enzymes, or the purified enzymes obtained by purifying the same, can be used to convert sucrose into isomalutyltransferase. It can be used in enzymatic reactions to convert it into loose. In that case, the bacterial cells or their processed products can be used as they are, or they can be wrapped in an immobilized carrier, and if necessary, they can be used after crosslinking using a crosslinking agent. It is desirable to use it as a standard. The immobilization can be carried out by methods known per se, such as a method in which the bacterial cells or their processed product are wrapped in an acrylamide monomer and polymerized; a method in which the bacterial cells or their processed product are wrapped in a carrier and cross-linked. For example, a chitosan/glutaraldehyde method, a Karataku-nan glutaraldehyde method, an alginic acid/glutaraldehyde method, etc. can be used.
以上に述べた適宜固定化されていてもよい菌体又はその
処理物を用いる蔗糖の転換反応は通常、蔗糖を基質とし
てlO〜75重量%、好ましくは30〜50重量%の濃
度で含有する水性溶媒中に、上記菌体又はその処理物を
加え、約25〜約3000の範囲内の温度で約20〜約
40時間程度インキユベートすることにより行うことが
できる。The above-mentioned sucrose conversion reaction using optionally immobilized bacterial cells or a processed product thereof is usually carried out using an aqueous solution containing sucrose as a substrate at a concentration of 10 to 75% by weight, preferably 30 to 50% by weight. This can be carried out by adding the above-mentioned bacterial cells or a treated product thereof to a solvent and incubating at a temperature within the range of about 25 to about 3,000 ℃ for about 20 to about 40 hours.
生戊スるイソマルチユロースはそれ自体既知の方法、例
えば脱塩、脱色、濃縮、結晶、分蜜等の手段により反応
液から分離し及び/又は精製することができる。The raw isomaltulose can be separated from the reaction solution and/or purified by methods known per se, such as desalting, decolorization, concentration, crystallization, and honeycombing.
次に実施例により本発明をさらに具体的に説明する。Next, the present invention will be explained in more detail with reference to Examples.
実施例l
砂糖2 0 g/ dQ,酵母エキス0.5g/dα及
びK2H P 040 .4 3g/dQの組或を有し
、pH6.0に調整した培地lOOm0.を含むIQ容
量三角フラスコにエンテロバクター・エスビーNO.7
を接種し、25℃24時間振盪培養する。次にこの培養
物全量を同組戊の培地IQに接種し、同温度で24時間
振盪培養する。培養終了液は菌体を遠心分離機で除去し
、上溝を回収する。上溝中の糖組戊比は、HPLCで測
定した結果、イソマルチユロース90%、トリハルロー
ス7.5%、7ルクトース2.5%で、その他の糖は検
出されなかった。Example l Sugar 20 g/dQ, yeast extract 0.5 g/dα and K2H P040. 4 A medium containing 3 g/dQ and adjusted to pH 6.0 lOOm0. Enterobacter S.B. NO. 7
was inoculated and cultured with shaking at 25°C for 24 hours. Next, the entire amount of this culture is inoculated into the same culture medium IQ, and cultured with shaking at the same temperature for 24 hours. After the culture is completed, the bacterial cells are removed using a centrifuge, and the upper layer is collected. As a result of HPLC measurement, the sugar composition ratio in the superior groove was 90% isomaltulose, 7.5% trihalulose, 2.5% 7-luctose, and no other sugars were detected.
この上溝液はイオン交換樹脂IRl20B及びIRA−
4 1 1(オルガノ社製)のモベットに通液脱塩後、
活性炭で脱色して、糖濃度75重量%に濃縮すると、イ
ソマルチユロースが晶析する。結晶の回収率は蔗糖当た
り74%であった。This upper groove liquid contains ion exchange resins IRl20B and IRA-
After desalting by passing the liquid through a Mobetto 411 (manufactured by Organo),
When decolorized with activated carbon and concentrated to a sugar concentration of 75% by weight, isomaltulose crystallizes. The recovery rate of crystals was 74% based on sucrose.
実施例2
砂糖20g/dL酵母エキスl . O g/ dQ,
コーン・スチープ・リカ−3.0g/dQ及びK,HP
0.0.2g/d(2の組成を有し、pH6.04m調
整した培地50ml2を含む500mQ容量坂口フラス
コにエンテロバクター・エスピーNo.7を接種し30
゜C24時間振盪培養する。次に、この培養物に蔗糖1
0gを加え、30゜Cでさらに24時間静置培養をする
。続いて実施例lに記述したと同様の処理を行った。そ
の結果、結晶イソマルチユロースは蔗糖当たり79%の
回収であった。Example 2 Sugar 20g/dL yeast extract l. Og/dQ,
Corn Steep Liquor - 3.0g/dQ and K, HP
Enterobacter sp.
Culture with shaking at °C for 24 hours. Next, add 1 sucrose to this culture.
0g was added and the culture was continued for another 24 hours at 30°C. Subsequently, the same treatment as described in Example 1 was carried out. As a result, the recovery of crystalline isomaltulose was 79% based on sucrose.
実施例3
エンテロバクター・エスピーNo.7を実施例2に記述
したと同様の培地の中で、30℃24時間培養し菌体ス
ラリーをloOm(2得た。Example 3 Enterobacter sp. no. 7 was cultured at 30° C. for 24 hours in the same medium as described in Example 2 to obtain a bacterial cell slurry loOm (2).
この菌体スラリーと4%κ一カラギーナンを同量混合し
たものを3%KCQ溶液に滴下することにより菌体を包
括する。包括菌体は、更にポリエチレンイミン及びグル
グルアルデヒド溶液で架橋し、菌体を固定化した。この
固定化菌体200m(2を、300mQ容量のカラムに
充填し、40%蔗糖液をS.V.=0.2 〜0.3、
25〜30゜O−t’通液したところ蔗糖液は、イソマ
ルチユロース90%、トレハルロース4%、フルクトー
ス4%及ヒクルコース2%の比率で転換された。A mixture of equal amounts of this bacterial cell slurry and 4% κ-carrageenan is added dropwise to a 3% KCQ solution to enclose the bacterial cells. The enclosing bacterial cells were further crosslinked with a polyethyleneimine and grugulaldehyde solution to immobilize the bacterial cells. Fill a column with a capacity of 300 mQ with 200 m of these immobilized bacterial cells, and add a 40% sucrose solution with S.V. = 0.2 to 0.3.
When the solution was passed through the solution at 25-30° O-t', the sucrose solution was converted to a ratio of 90% isomaltulose, 4% trehalulose, 4% fructose and 2% vesicle.
実施例4
砂糖20g/d(2,酵母エキス0.5g/dQ及びK
2HP0.0.43g/d2の組戊を有し、pH6.0
に調整した培地100ml2を含むlQ容量三角フラス
コにラネラ・エスピーNO.2を接種し、25℃24時
間振盪培養する。次にこの培養物全量を同組戊の培地1
12に接種し、同温度で24時間振盪培養する。培養終
了液は菌体を遠心分離機で除去し、上清を回収する。上
清中の糖組或比は、HPLCで測定した結果、イソマル
チユロース95%、トレハルロース3%、フルクトース
2%で、その他の糖は検出されなかった。この上溝液は
イオン交換樹脂IRl2OB及びI RA−4 1 1
(オルガノ社製)のモベットに通液脱塩後、活性炭で脱
色して、糖濃度75重量%に濃縮すると、イソマルチユ
ロースが晶析する。結晶の回収率は蔗糖当たり78%で
あった。Example 4 Sugar 20g/d (2, yeast extract 0.5g/dQ and K
2HP0.0.43g/d2 composition, pH6.0
Ranella sp. NO. 2 and cultured with shaking at 25°C for 24 hours. Next, the entire amount of this culture was added to the medium 1 of the same group.
12 and cultured with shaking at the same temperature for 24 hours. Cells are removed from the culture solution using a centrifuge, and the supernatant is collected. The sugar composition ratio in the supernatant was determined by HPLC and was 95% isomaltulose, 3% trehalulose, and 2% fructose, and no other sugars were detected. This upper groove liquid contains ion exchange resins IRl2OB and IRA-4 1 1
After desalting, the solution is passed through a mobetto (manufactured by Organo), decolorized with activated carbon, and concentrated to a sugar concentration of 75% by weight to crystallize isomaltulose. The recovery rate of crystals was 78% based on sucrose.
実施例5
砂糖2 0 g/ dQ,酵母エキスl . O g/
dQ,コーン・スチーブ− リカ−3.0g/dQ及
びK2HP0.0.2g/d(2の組戊を有し、pH6
.0に調整した培地50+nQを含む500m+2容量
坂口フラスコにラネラ・エスビーNo.2を接種し30
℃2′4時間振盪培養する。次に、この培養物に蔗糖l
ogを加え、30°Cでさらに24時間静置培養をする
。Example 5 Sugar 20 g/dQ, yeast extract l. Og/
dQ, corn stew liquor - 3.0 g/dQ and K2HP 0.0.2 g/d (having a composition of 2, pH 6
.. A 500 m + 2 capacity Sakaguchi flask containing 50 + nQ of medium adjusted to 0. Inoculate 2 and 30
Culture with shaking at ℃2'4 hours. Next, add sucrose to this culture.
og was added and cultured at 30°C for an additional 24 hours.
統いて実施例lに記述したと同様の処理を行った。The same treatment as described in Example 1 was carried out.
その結果、結晶イソマルチユロースは蔗糖当たり83%
の回収であった。As a result, crystalline isomaltulose was 83% based on sucrose.
was recovered.
実施例6
ラ不ラ・エスピーNo.2を実施例5に記述したと同様
の培地の中で、30℃24時間培養し菌体スラリーを1
0 0’m(2得た。この菌体スラリーと4%κ一カ
ラギーナンを同量混合したものを3%KCQ溶液に滴下
することにより菌体を包括する。Example 6 Lafura SP No. 2 was cultured at 30°C for 24 hours in the same medium as described in Example 5, and the bacterial cell slurry was
A mixture of equal amounts of this bacterial slurry and 4% κ-carrageenan was added dropwise to a 3% KCQ solution to enclose the bacterial cells.
包括菌体は、更にポリエチレンイミン及びグルタルアル
デヒド溶液で架橋し、菌体を固定化した。The enclosing bacterial cells were further crosslinked with a polyethyleneimine and glutaraldehyde solution to immobilize the bacterial cells.
この固定化菌体200mGを、300+nQ容量のカラ
ムに充填し、40%蔗糖液をS.V.=0.2 〜0.
3、25〜30゜Cで通液したところ蔗糖液は、イソマ
ルチ二ロース90%、トレハルロース5%、フルクトー
ス3%及びグルコース2%の比率で転換された。200 mG of the immobilized cells were packed into a column with a capacity of 300+nQ, and a 40% sucrose solution was added to the S. V. =0.2 ~0.
3. When passed at 25-30°C, the sucrose solution was converted at a ratio of 90% isomaltitylose, 5% trehalulose, 3% fructose, and 2% glucose.
手続補正書(自発) 平戒l年12月28日Procedural amendment (voluntary) December 28, 2017
Claims (1)
ルトランスフエラーゼ生産性微生物の菌体又はその処理
物の存在下に蔗糖をイソマルチユロースに転換すること
を特徴とするイソマルチユロースの製造方法。 2、エンテロバクター属又はラネラ属に属するグルコシ
ルトランスフエラーゼ生産性微生物を蔗糖を含有する培
地で培養し、培地からイソマルチユロースを回収するこ
とを特徴とするイソマルチユロースの製造方法。 3、エンテロバクター・エスピーNo.7株。 4、ラネラ・エスピーNo.2株。[Scope of Claims] 1. An isomaltulose characterized by converting sucrose into isomaltulose in the presence of cells of a glucosyltransferase-producing microorganism belonging to the genus Enterobacter or the genus Ranella or a processed product thereof. manufacturing method. 2. A method for producing isomaltulose, which comprises culturing a glucosyltransferase-producing microorganism belonging to the genus Enterobacter or the genus Ranella in a medium containing sucrose, and recovering isomaltulose from the medium. 3. Enterobacter sp. no. 7 stocks. 4. Ranella SP No. 2 stocks.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24007789A JPH03103187A (en) | 1989-09-18 | 1989-09-18 | Production of isomaltulose |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24007789A JPH03103187A (en) | 1989-09-18 | 1989-09-18 | Production of isomaltulose |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH03103187A true JPH03103187A (en) | 1991-04-30 |
Family
ID=17054152
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP24007789A Pending JPH03103187A (en) | 1989-09-18 | 1989-09-18 | Production of isomaltulose |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH03103187A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH099958A (en) * | 1995-06-28 | 1997-01-14 | Suedzucker Ag Mannheim Ochsenfurt | Sucrose metabolism mutant |
-
1989
- 1989-09-18 JP JP24007789A patent/JPH03103187A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH099958A (en) * | 1995-06-28 | 1997-01-14 | Suedzucker Ag Mannheim Ochsenfurt | Sucrose metabolism mutant |
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