JPS63214192A - Production of alginic acid oligosaccharide - Google Patents
Production of alginic acid oligosaccharideInfo
- Publication number
- JPS63214192A JPS63214192A JP62048383A JP4838387A JPS63214192A JP S63214192 A JPS63214192 A JP S63214192A JP 62048383 A JP62048383 A JP 62048383A JP 4838387 A JP4838387 A JP 4838387A JP S63214192 A JPS63214192 A JP S63214192A
- Authority
- JP
- Japan
- Prior art keywords
- alginic acid
- alginate
- reaction
- alteromonas
- oligosaccharide
- 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.)
- Granted
Links
- 235000010443 alginic acid Nutrition 0.000 title claims abstract description 59
- 229920000615 alginic acid Polymers 0.000 title claims abstract description 59
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 39
- 229960001126 alginic acid Drugs 0.000 title claims abstract description 36
- 239000000783 alginic acid Substances 0.000 title claims abstract description 36
- -1 alginic acid oligosaccharide Chemical class 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 150000004781 alginic acids Chemical class 0.000 claims abstract description 22
- 150000003839 salts Chemical class 0.000 claims abstract description 12
- 241000590031 Alteromonas Species 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 10
- 238000002835 absorbance Methods 0.000 claims abstract description 9
- 241000589563 Alteromonas sp. Species 0.000 claims abstract description 7
- 238000010438 heat treatment Methods 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 7
- 229940072056 alginate Drugs 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 17
- 241000237502 Ostreidae Species 0.000 claims description 8
- 235000020636 oyster Nutrition 0.000 claims description 8
- 241001474374 Blennius Species 0.000 claims description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 4
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 abstract description 15
- 229940005550 sodium alginate Drugs 0.000 abstract description 15
- 239000000661 sodium alginate Substances 0.000 abstract description 15
- 235000010413 sodium alginate Nutrition 0.000 abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 26
- 229940088598 enzyme Drugs 0.000 description 17
- 230000000694 effects Effects 0.000 description 12
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 11
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 10
- 230000008635 plant growth Effects 0.000 description 10
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 9
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 9
- 230000001737 promoting effect Effects 0.000 description 7
- 241000512259 Ascophyllum nodosum Species 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 244000155437 Raphanus sativus var. niger Species 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 150000002482 oligosaccharides Chemical class 0.000 description 6
- 239000013535 sea water Substances 0.000 description 6
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 238000000354 decomposition reaction Methods 0.000 description 4
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 3
- 108090000856 Lyases Proteins 0.000 description 3
- 102000004317 Lyases Human genes 0.000 description 3
- 241000220259 Raphanus Species 0.000 description 3
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 150000004804 polysaccharides Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 244000233513 Brassica perviridis Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 241001469654 Lawsonia <weevil> Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 125000005613 guluronic acid group Chemical group 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 244000045561 useful plants Species 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はアルギン酸オリゴ糖の製造法に関し、詳しくは
植物の生長促進作用を有するアルギン酸オリゴ糖の工業
的製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing alginic acid oligosaccharides, and more particularly to an industrial method for producing alginic acid oligosaccharides that have a growth-promoting effect on plants.
農作物の生長を促進し、単位面積当りの収ａｍを増し、
さらには作付回数を増やすことによって農作物の増収を
図ることは農業生産上重要な課題である。Promote the growth of agricultural crops, increase the yield per unit area,
Furthermore, increasing the yield of agricultural crops by increasing the number of plantings is an important issue in agricultural production.
この課題について従来より種々の提案がなされ、或種の
農作物では所定の成果が得られているが、その過程でア
ルギン酸を分解して得られるオリゴ糖が植物の生長促進
作用を有していることを見出した（特願昭６ｌ−２４５
４００）が、このアルギン酸オリゴ糖を工業的に製造す
るためには以下のような問題点がある。Various proposals have been made to address this problem, and certain results have been obtained for certain types of agricultural products, but it has been discovered that oligosaccharides obtained by decomposing alginic acid during the process have a growth-promoting effect on plants. (Patent application Sho 6l-245)
400), however, there are the following problems in industrially producing this alginic acid oligosaccharide.
（１） 市販酵素としてアワビの消化管酵素（商品名
：アバロンアセトンパウダー、Ｍｅｒｃｋ社製）が知ら
れているが、これは極めて高価であり、本酵素をアルギ
ン酸オリゴ糖の工業的製造に使用することは困難である
。(1) Abalone gastrointestinal enzyme (trade name: Avalon Acetone Powder, manufactured by Merck) is known as a commercially available enzyme, but this enzyme is extremely expensive and cannot be used for industrial production of alginate oligosaccharide. That is difficult.
（２） アルギン酸マンヌロン酸とグルロン酸から成
る多糖体であるが、これをアルギン酸リアーゼで分解す
ると、分解が進むにしたがって多種多様なオリゴ糖が生
成し、最終的にはその構成糖成分であるマンヌロン酸と
グルロン酸にまで分解される。しかし、これらの単糖類
には植物生長促進作用が認められないので、植物生長作
用からみたアルギン酸の最適分解条件の確立が必要であ
る。(2) Alginic acid is a polysaccharide consisting of mannuronic acid and guluronic acid. When this is decomposed by alginate lyase, a wide variety of oligosaccharides are produced as the decomposition progresses, and finally the constituent sugar component, mannuron, is produced. It is broken down into acid and guluronic acid. However, since these monosaccharides do not have a plant growth promoting effect, it is necessary to establish optimal decomposition conditions for alginic acid from the viewpoint of plant growth effects.
本発明者らは、これらの問題点を解決するために、鋭意
検討した結果、安価な酵素源として牡蠣の消化管酵素を
使用できることおよび新たに褐藻類の溶解菌として海水
中から分離した微生物が効率よくアルギン酸リアーゼを
生産すること、さらにはこれらのアルギン酸リアーゼを
アルギン酸に作用させてアルギン酸オリゴ糖を生成させ
る工程において反応液自体の２３０ｎａ＋の吸光度の推
移と反応生成物の植物生長促進活性に一定の関係があり
、該吸光度の範囲を特定して反応を停止させることによ
って植物生長促進活性の高い反応生成物が得られること
を見出し、本発明を完成するに至った。In order to solve these problems, the present inventors conducted intensive studies and discovered that oyster gastrointestinal enzymes can be used as an inexpensive enzyme source and that microorganisms newly isolated from seawater as brown algae lytic bacteria can be used. In order to efficiently produce alginate lyase, and furthermore, in the process of making alginate lyase act on alginic acid to produce alginate oligosaccharide, it is necessary to maintain a certain level of change in the absorbance of 230na+ of the reaction solution itself and the plant growth promoting activity of the reaction product. The present inventors have discovered that there is a relationship between the two, and that a reaction product with high plant growth promoting activity can be obtained by specifying the range of absorbance and stopping the reaction, leading to the completion of the present invention.
すなわち本発明は、アルギン酸もしくはその塩を含む物
質にアルギン酸リアーゼを作用させてアルギン酸オリゴ
糖を製造するにあたり、反応液の２３ｏｎ−の吸光度（
Ｅｊ二）が３０〜６０となったとき反応を停止すること
を特徴とするアルギン酸オリゴ糖の製造法並びに上記の
如く反応を停止後、反応液のｐＨを１．０〜４．０とし
て加熱処理を行うことを特徴とするアルギン酸オリゴ糖
の製造法を提供するものである。That is, in the present invention, when producing alginate oligosaccharide by causing alginate lyase to act on a substance containing alginic acid or a salt thereof, the absorbance of the reaction solution at 23 on-
2) A method for producing alginic acid oligosaccharides characterized in that the reaction is stopped when Ej becomes 30 to 60, and after stopping the reaction as described above, the reaction solution is heated to a pH of 1.0 to 4.0. The present invention provides a method for producing alginic acid oligosaccharides, which is characterized by carrying out the following steps.
本発明の原料であるアルギン酸もしくはその塩を含む物
質としては市販のアルギン酸、アルギン酸ソーダのほか
、アルギン酸を含有する昆布、カジノ、レソニア、ドル
ベリアなどの海藻類およびシュードモナスなどの微生物
の生産するアルギン酸様多糖質などがあり、アルギン酸
もしくはその塩（特にナトリウム塩、カリウム塩など）
を含有するものは任意に使用できる。Substances containing alginic acid or its salts, which are raw materials for the present invention, include commercially available alginic acid and sodium alginate, as well as alginic acid-containing seaweeds such as kelp, Casino, Lawsonia, and Dolveria, and alginic acid-like polysaccharides produced by microorganisms such as Pseudomonas. Alginic acid or its salts (especially sodium salts, potassium salts, etc.)
Those containing can be used arbitrarily.
次に、本発明に用いる酵素、アルギン酸リアーゼについ
ては、まずアルテロモナス属に属する微生物の産生ずる
酵素がある。アルテロモナス属に属する微生物としては
、たとえば本発明者らによって海水中から分離されたア
ルテロモナス・エスピー（Ａｌｔｅｒｏｓｏｎａｓ ｓ
ｐ、） ＬＢ １０２株がある。Next, regarding the enzyme alginate lyase used in the present invention, there is an enzyme produced by a microorganism belonging to the genus Alteromonas. Examples of microorganisms belonging to the genus Alteromonas include Alteromonas sp., which was isolated from seawater by the present inventors.
p,) There are 102 LB strains.
本国の主要な菌学的性質を以下に示す。The main mycological properties of this country are shown below.
（１） ダラム染色： 陰性
（２）運動性 ： 有り
（３）色素生産 ： なし
く４） Ｈｕｇｈ−Ｌｅｉｆｓｏｎ培地におけるブド
ウ塘の分解形式：酸化型
（５）ゼラチン分解性：陽性
（６）ＤＮアーゼの生産：陽性
以上の性質から本国はアルテロモナス属に属する細菌で
あると考えられる。本国は微生物工業技術研究所にＦＥ
ＲＭ Ｐ−９２１８として寄託されている。本発明に
おいては、本国のほか通常の変異手段を適用して得られ
る変異株であって、上記酵素産生能を有するものも使用
することができる。(1) Durham staining: Negative (2) Motility: Yes (3) Pigment production: Absent 4) Decomposition type of grape tang in Hugh-Leifson medium: Oxidized type (5) Gelatin degradability: Positive (6) DNase Production: Based on the positive or higher characteristics, the origin is considered to be a bacterium belonging to the genus Alteromonas. Home country is FE at Microbial Technology Research Institute
It has been deposited as RM P-9218. In the present invention, not only native strains but also mutant strains obtained by applying conventional mutation methods and having the ability to produce the above-mentioned enzyme can also be used.
本国を用いてアルギン酸リアーゼを生産するには、たと
えばアルギン酸ソーダ０．５％、ペプトン０．５％、酵
母エキス０．２５％、Ｍｇ５Ｏｎ・７Ｈ！００．６４％
、ＮａＣ１０，９６％、海水２０％を含有する培地で本
国を２５℃、２５０ｒｐ饋の条件で２４時間培養すれば
よく、その培養濾液をそのまま酵素液として使用できる
。さらには、溶剤沈澱、硫安塩析、限外濾過、ゲル濾過
等の酵素精製に関する通常の手段ですｎ製したものを酵
素として利用することができる。アルテロモナス属の生
産するアルギン酸リアーゼの作用至適ｐＨは７．０作用
至適温度は４０℃である。To produce alginate lyase using your own country, for example, sodium alginate 0.5%, peptone 0.5%, yeast extract 0.25%, Mg5On.7H! 00.64%
The native culture may be cultured for 24 hours at 25° C. and 250 rpm in a medium containing 10.96% NaC and 20% seawater, and the culture filtrate can be used as it is as an enzyme solution. Furthermore, it is possible to use the enzymes prepared by conventional enzyme purification methods such as solvent precipitation, ammonium sulfate salting out, ultrafiltration, and gel filtration. The optimal pH for alginate lyase produced by Alteromonas is 7.0 and the optimal temperature for action is 40°C.
また、牡蠣の消化管に由来する酵素は、生牡蠣。In addition, enzymes derived from the digestive tract of oysters come from raw oysters.
冷凍牡蠣を原料として原料１部あたり水１部を加え、こ
れをホモゲナイザーで磨砕して得られたものを直接使用
するかあるいは遠心分離等の手段で不溶性残渣を除去し
て得た抽出液を酵素液として用いることができる。牡蠣
消化管のアルギン酸リアーゼの作用至適ｐＨは７．０、
作用至適温度は５０℃である。Use frozen oysters as a raw material, add 1 part of water per 1 part of the raw material, and use the resulting product directly by grinding it with a homogenizer, or use the extract obtained by removing insoluble residues by means such as centrifugation. It can be used as an enzyme solution. The optimal pH for alginate lyase in the oyster digestive tract is 7.0.
The optimum temperature for action is 50°C.
上記アルギン酸リアーゼを用いてアルギン酸もしくはそ
の塩からアルギン酸オリゴＩ！を製造する方法について
、原料としてアルギン酸ソーダを用いる場合を例にして
述べると、アルギン酸ソーダ１〜５部に水を１００部加
えてアルギン酸を熔解後、酵素の至適ｐＨにｐＨを調整
し、アルギン酸リアーゼをアルギン酸ソーダ１ｇ当り１
００〜４０００単位添加して酵素の作用至適温度で２４
〜４８時間反応させることによって目的とするアルギン
酸オリゴ糖を調整することができる。なお、アルギン酸
リアーゼの酵素活性はＰＨ７，０，３０℃で０．２％ア
ルギン酸ソーダ溶液に酵素を作用させたとき、３０分間
に２３ｏｎ−の吸光度をｏ、ｏｏｉ上昇させる酵素力を
１単位として表示した。Alginate Oligo I from alginate or its salt using the above alginate lyase! To describe the method for producing alginate using sodium alginate as a raw material as an example, add 100 parts of water to 1 to 5 parts of sodium alginate to melt the alginic acid, adjust the pH to the optimum pH for the enzyme, and dissolve the alginic acid. 1 lyase per 1g of sodium alginate
Add 00 to 4,000 units and heat at the optimum temperature for enzyme action.
The desired alginate oligosaccharide can be prepared by reacting for ~48 hours. In addition, the enzymatic activity of alginate lyase is expressed as the enzymatic power that increases the absorbance of 23 on- by o, ooi in 30 minutes when the enzyme is applied to a 0.2% sodium alginate solution at pH 7, 0, and 30°C. did.
より詳細に述べると、後記する実施例１に示すヨウに、
アルテロモナス・エスピー ＬＢ １０２株の生産す
るアルギン酸リアーゼをアルギン酸ソーダ１ｇ当り１０
００単位添加し、ｐＨ７，０，４０℃で反応させると、
分解反応の進行と共に２３ｏｎｍの吸光度を（Ｅ；ユ）
は上昇する。種々のＥ１ユを示す反応液を用いてカイワ
レ大根に対する生長促進作用を調べると、Ｅ１″の値が
８〜６０の範囲で一
植物生長促進作用が発現し、その作用はＥ１ユの値が３
０〜６０のとき強力であることが判明した。To describe in more detail, as shown in Example 1 below,
Alginate lyase produced by Alteromonas sp. LB 102 strain is added at 10% per gram of sodium alginate.
When 00 units are added and reacted at pH 7.0 and 40°C,
As the decomposition reaction progresses, the absorbance at 23 onm (E;
will rise. When the growth-promoting effect on daikon radish was investigated using reaction solutions showing various E1 units, the plant growth-promoting effect was expressed when the E1 value was in the range of 8 to 60;
It was found to be strong when it was between 0 and 60.
また、海藻類から直接オリゴ糖を得る場合は、例えば乾
燥昆布４０部に水を１３００部加え、ｐＨを１１とした
後、ホモゲナイザーで磨砕し、６０℃で３時間処理し、
その後ｐＨを５．５とした後、セルラーゼ（商品名：メ
イセラーゼ、明治製菓■製）を対固型分０．５％添加し
、４０℃で２０時間反応後、ｐＨを７．０とし、アルギ
ン酸リアーゼを固型分１ｇ当り１０００単位添加して４
０℃で４８時間反応させることにより反応液のＥ１％値
は！−
３０〜５０に達し、昆布より直接アルギン酸オリゴ糖を
製造することができる。In addition, when obtaining oligosaccharides directly from seaweed, for example, add 1,300 parts of water to 40 parts of dried kelp, adjust the pH to 11, grind with a homogenizer, and treat at 60°C for 3 hours.
After that, the pH was adjusted to 5.5, and 0.5% of cellulase (trade name: Meicelase, manufactured by Meiji Seika) was added to the solid content, and after reacting at 40°C for 20 hours, the pH was adjusted to 7.0, and alginic acid 4 by adding 1000 units of lyase per 1g of solid content.
By reacting at 0°C for 48 hours, the E1% value of the reaction solution is! - 30 to 50, and alginate oligosaccharides can be produced directly from kelp.
このようにして得られたアルギン酸オリゴ糖は、その構
成糖成分はマンヌロン酸とグルロン酸が主成分であり、
重合度が２〜２０までのグルロン酸のみ、マンヌロン酸
のみ、またはグルロン酸とマンヌロン酸の組合せで構成
されるオリゴＩ！類およびグルロン酸、マンヌロン酸の
組成物である。The alginate oligosaccharide obtained in this way has mannuronic acid and guluronic acid as its main constituent sugar components,
Oligo I composed of only guluronic acid, only mannuronic acid, or a combination of guluronic acid and mannuronic acid with a degree of polymerization of 2 to 20! It is a composition of Guluronic acid, Guluronic acid, and Mannuronic acid.
さらに、アルギン酸を含有する海藻類に直接アルテロモ
ナス属に属し、アルギン酸リアーゼ産生能を有する微生
物を作用させることによってもアルギン酸オリゴ塘を製
造することができる０例えば昆布２４部に１％Ｎａ ！
Ｃ０３を含む海水４００部を添加し、９０〜１００℃
に１時間保持した後、これを磨砕し、２％ＨＣ２溶液２
００部を添加して中和し、次いでアルテロモナス・エス
ピー ＬＢ１０２株を接種し、２５℃で８〜２４時間培
養したのち、温度を４０℃に上昇させ、さらに２４〜４
８時間反応させると、反応液の２３ｏｎｍにおけるＥ：
よ値が３０〜６０に達し、目的とするアルギン酸オリゴ
糖が得られる。Furthermore, alginate oligosaccharides can also be produced by directly treating alginic acid-containing seaweed with a microorganism belonging to the genus Alteromonas and having the ability to produce alginate lyase.For example, 1% Na in 24 parts of kelp!
Add 400 parts of seawater containing C03 and heat to 90-100℃
After holding for 1 hour, it was ground and added with 2% HC2 solution 2
00 parts to neutralize, then inoculate Alteromonas sp. LB102 strain, culture at 25°C for 8 to 24 hours, raise the temperature to 40°C, and further inoculate for 24 to 4 hours.
After reacting for 8 hours, the E of the reaction solution at 23 onm:
The value reaches 30 to 60, and the desired alginate oligosaccharide can be obtained.
本発明者らは、上記の如くして得られたアルギン酸オリ
ゴ糖の活性を増強する方法について検討を加えたところ
、該アルギン酸オリゴ糖を含む反応液のｐＨを１．０〜
４．０、好ましくは２．０〜３．０とし、次いで１００
〜１２０℃の温度で１５〜１８０分間加熱処理を行うこ
とにより、該アルギン酸オリゴ糖の植物生長促進作用が
さらに増強することを見出した（後記実施例２参照）。The present inventors investigated a method for enhancing the activity of the alginate oligosaccharide obtained as described above, and found that the pH of the reaction solution containing the alginate oligosaccharide was adjusted to 1.0 to 1.0.
4.0, preferably 2.0-3.0, then 100
It was found that the plant growth promoting effect of the alginic acid oligosaccharide was further enhanced by heat treatment at a temperature of -120°C for 15 to 180 minutes (see Example 2 below).
このようにして得られたアルギン酸オリゴ糖は、種子な
どに塗布したり、０．２５〜０．０００２５％の水溶液
として土壌中に添加したり、葉面散布を行ったり、さら
にまた養液栽培用液体肥料中に添加混合するなどして植
物に施用すると、植物の根および地上部の生長を促進し
、その結果収穫量が向上する。このようなアルギン酸オ
リゴ糖の効果はほうれん草、レタス、小松菜などの葉菜
類、二十日大根、ジャガイモなどの根菜類、イネ、トウ
モロコシなどの穀類、その他花弁、芝、果樹などの有用
植物においてよく発揮される。The alginic acid oligosaccharide thus obtained can be applied to seeds, etc., added to soil as a 0.25 to 0.00025% aqueous solution, sprayed on leaves, and used for hydroponic cultivation. When applied to plants, such as by adding it to a liquid fertilizer, it promotes the growth of roots and above-ground parts of the plants, resulting in improved yields. The effects of alginic acid oligosaccharides are well exhibited in leafy vegetables such as spinach, lettuce, and Japanese mustard spinach, root vegetables such as daikon radish and potatoes, grains such as rice and corn, and other useful plants such as flower petals, grass, and fruit trees. Ru.
本発明においてアルギン酸オリゴ糖とは以下のように定
義される。アルギン酸もしくはその塩（例えばアルギン
酸ナトリウム）またはこれらアルギン酸を含有する昆布
などの藻類、微生物起源の多塘体などをアルギン酸リア
ーゼで加水分解して得られる組成物またはその主成分で
あるオリゴ糖で、植物生長促進作用を有する。また、オ
リゴ糖の構成糖成分はグルロン酸およびマンヌロン酸が
主成分である。その重合度が２〜２０までのグルロン酸
のみ、マンヌロン酸のみ、またはグルロン酸とマンヌロ
ン酸の組合せで構成されるオリゴ糖類およびグルロン酸
、マンヌロン酸から成る組成物、さらにまたこの組成物
をｐＨ１〜４，１００〜１２０℃の条件下で１５〜１８
０分加熱して得られる組成物をいう。In the present invention, alginate oligosaccharide is defined as follows. Compositions obtained by hydrolyzing alginic acid or its salts (e.g. sodium alginate), alginic acid-containing algae such as kelp, or microbial-derived polysaccharides with alginic acid lyase, or oligosaccharides that are the main components thereof, Has a growth promoting effect. Furthermore, the constituent sugar components of oligosaccharides are mainly guluronic acid and mannuronic acid. A composition consisting of an oligosaccharide composed of only guluronic acid, only mannuronic acid, or a combination of guluronic acid and mannuronic acid, and guluronic acid and mannuronic acid with a degree of polymerization of 2 to 20; 4,15-18 under conditions of 100-120℃
It refers to a composition obtained by heating for 0 minutes.
実施例 以下に実施例により本発明の詳細な説明する。Example The present invention will be explained in detail below using Examples.
実施例１
アルギン酸ナトリウム０．５％、ペプトン０．５％、酵
母エキス０．２５％、 Ｍ ｇ Ｓ Ｏａ・７Ｈ２００
，６４％、ＮａＣｊ！ ０．９６％および海水２０％
を含む培地（以下、ＬＢ培地と称する。）を準備し、Ｌ
Ｂ培地を２００ｍＪの三角フラスコに３０−１分注して
、１２０℃で１５分殺菌後、アルテｒｐｍで２０時間培
養して、これを種菌とした。Example 1 Sodium alginate 0.5%, peptone 0.5%, yeast extract 0.25%, M g S Oa・7H200
,64%,NaCj! 0.96% and seawater 20%
Prepare a medium containing L (hereinafter referred to as LB medium),
30-1 portions of B medium were dispensed into a 200 mJ Erlenmeyer flask, sterilized at 120° C. for 15 minutes, and cultured at Alter rpm for 20 hours to serve as a seed culture.
５１容ジャーファーメンタ−にＬＢ培地３１を仕込み、
１２０℃で１５分殺菌後、上記種菌２０分間の条件で遠
心分離処理を行い、菌体を除去した上清液を得た。Pour LB medium 31 into a 51-volume jar fermenter,
After sterilization at 120° C. for 15 minutes, centrifugation was performed under the above-mentioned conditions for 20 minutes to obtain a supernatant from which bacterial cells had been removed.
この上清液を分子！１万カットのホローファイバーを用
いて精製し、酵素液２５０ｍｌ１を得た。Molecule this supernatant! It was purified using 10,000-cut hollow fiber to obtain 250 ml of enzyme solution.
本酵素液中のアルギン酸リアーゼ活性は９８０ Ｕ／１
ｍｌであった。The alginate lyase activity in this enzyme solution is 980 U/1
It was ml.
３％（Ｗ／Ｖ）アルギン酸ナトリウム溶液を調製し、ｐ
）ｌを７．０に調整後、上記アルギン酸リアーゼをアル
ギン酸ナトリウム１ｇ当り１００ＯＵの割合で添加し、
６〜９６時間反応させ、経時的に反応液を抜きとり、Ｅ
：二値の異なった反応液を調製した。このようにして得
られたＥ：二値の異なるアルギン酸オリゴ糖液を用いて
植物生長促進作用をカイワレ大根を用いて調べた。すな
わち、カイワレ大根の種子３６粒を合成樹脂製のウール
マットをセットしたガラス容器に播種し、水道水７０ｍ
１を添加して２３℃で６日間栽培した（４日間は暗所で
、続（２日間は５０００ルツクスの照射条件下で栽培）
、アルギン酸オリゴ糖は対水道水当り０．０２５％の割
合で添加した。結果を表１に示す。Prepare a 3% (W/V) sodium alginate solution, p
)l to 7.0, the above alginate lyase was added at a rate of 100 OU per 1 g of sodium alginate,
React for 6 to 96 hours, remove the reaction solution over time, and
: Reaction solutions with different binary values were prepared. Using the alginate oligosaccharide solutions with different E: binary values obtained in this way, the plant growth promoting effect was investigated using daikon radish. That is, 36 seeds of Kaiware radish were sown in a glass container set with a synthetic resin wool mat, and then soaked in 70 m of tap water.
1 was added and cultivated at 23°C for 6 days (4 days in the dark, continued (2 days cultivated under irradiation conditions of 5000 lux)
, alginate oligosaccharide was added at a rate of 0.025% based on tap water. The results are shown in Table 1.
（ｎ＝３６）
表１中の数値はアルギン酸オリゴ糖を無添加の状態で栽
培されたカイワレ大根の茎葉長（ｅｔａ） 。(n=36) The values in Table 1 are the stem and leaf lengths (eta) of radish grown without the addition of alginate oligosaccharide.
根長（ａｍ）の平均値を１００とした場合の指数で示し
た（ｎ＝３６）。It is shown as an index when the average value of root length (am) is set to 100 (n=36).
表１から帽１なように、反応液のＥニジ値が３０〜６０
の範囲で、生長促進作用が強く現れた。As shown in Table 1, the E value of the reaction solution is 30 to 60.
A strong growth-promoting effect was observed within this range.
実施例２
アルギン酸ナトリウム３０ｇをｌ！の水に溶解し、ｐＨ
を７．０とした後、実施例１に記載の方法で調製された
アルギン酸リアーゼをアルギン酸ナトリウム１ｇ当り１
００ＯＵ添加して、４０℃で４８時間反応させた。この
時のＥｌｃｓ値は４２．３であった。この反応液１００
＋＋１１をとり、ｐｉ＋を１．０〜６．０に調整した後
、１２０℃で２時間加熱処理を行った後、中和し、実施
例１に記載の方法でカイワレ大根による生長促進作用を
調べ、加熱処理におけるｐｔｔ値と生長促進活性増強と
の関係を調べた。結果を表２に示す。Example 2 30g of sodium alginate! Dissolved in water at pH
7.0, then add alginate lyase prepared by the method described in Example 1 to 1 g of sodium alginate.
00OU was added and reacted at 40°C for 48 hours. The Elcs value at this time was 42.3. This reaction solution 100
++11 was taken, pi+ was adjusted to 1.0 to 6.0, heat treated at 120°C for 2 hours, neutralized, and the growth promoting effect of daikon radish was examined using the method described in Example 1. The relationship between the PTT value and the enhancement of growth-promoting activity during heat treatment was investigated. The results are shown in Table 2.
差ニー１
（ｎ＝３６）
表２中の数値は未加熱のアルギン酸オリゴ糖を添加して
栽培されたカイワレ大根の茎葉長（ａｍ）。Difference knee 1 (n=36) The values in Table 2 are the stem and leaf lengths (am) of daikon radish grown with the addition of unheated alginate oligosaccharide.
根長（ｃｍ ）の平均値に対する指数で示した。It is expressed as an index relative to the average root length (cm).
表２から明らかなように、反応液をｐＨ１，０〜４．０
の範囲で加熱処理することにより、植物生長促進活性が
増強される。As is clear from Table 2, the reaction solution was adjusted to pH 1.0 to 4.0.
Plant growth promoting activity is enhanced by heat treatment within this range.
実施例３
昆布２４０ｇを１ａ１角に細断し、１％Ｎ ａ ｚ Ｃ
Ｏｓ溶液４ｇを添加して９０℃で１時間加熱処理を行っ
た。次いで、これを磨砕し、２．２％ＨＣＩ溶液２１を
添加して中和し、ｐ）Ｉを５．５とした。この液中にセ
ルラーゼ製剤（メイセラーゼ、明治製菓■製）１．８ｇ
を添加し、４０℃で６時間反応させた。以後ｐＨを７．
０に調整し、牡蠣の磨砕物３８ｇを添加し、５０℃で４
８時間反応させた。反応液の２３０ｎｍにおけるＥＩａ
ｗ＋値は３７となり、これを実施例１に記載の方法でカ
イワレ大根による植物生長促進活性を調べたところ、無
添加の場合に比較して茎葉長で１２４％、根長で３１１
％の生長が認められた。Example 3 240g of kelp was shredded into 1a1 square pieces, and 1% N az C
4 g of Os solution was added and heat treatment was performed at 90° C. for 1 hour. It was then ground and neutralized by adding 2.2% HCI solution 21 to give a p)I of 5.5. In this liquid, 1.8 g of cellulase preparation (Meicelase, manufactured by Meiji Seika)
was added and reacted at 40°C for 6 hours. After that, the pH was set to 7.
0, add 38g of ground oysters, and heat at 50℃ for 4 hours.
The reaction was allowed to proceed for 8 hours. EIa of reaction solution at 230 nm
The w+ value was 37, and when the plant growth promoting activity of Kaiware radish was investigated using the method described in Example 1, the stem and leaf length was 124% and the root length was 311% compared to the case without additives.
% growth was observed.
ＮａｚＣＯ＝を含む海水４Ｎを添加し、１００℃で１時
間加熱処理を行ったのち磨砕し、２％ＨＣ１溶液２１を
添加してｐＨを６．８とした。これに実施例１に記載の
ＬＢ培地中で２４時間培養したアルテロモナス・エスピ
ー ＬＢ １０２株（ＦＥＲＭＰ−９２１８）の培養
液１ＯＯＩｌｌを添加し、２５℃で１５時間、２４０ｖ
ｐｍの条件で培養を行った。4N of seawater containing NazCO= was added, heat treated at 100° C. for 1 hour, and then ground, and 21% HC1 solution was added to adjust the pH to 6.8. To this was added 1OOIll of a culture solution of Alteromonas sp. LB 102 strain (FERMP-9218) cultured for 24 hours in the LB medium described in Example 1, and the mixture was incubated at 25°C for 15 hours at 240V.
Culture was performed under conditions of pm.
培養終了後、温度を４０℃に上昇し、２４時間酵素反応
を実施した０反応終了後、濾過処理を行い、アルギン酸
オリゴ糖を１．０％含む反応液５．４１を得た。After the completion of the culture, the temperature was raised to 40° C. and the enzymatic reaction was carried out for 24 hours. After the completion of the reaction, filtration treatment was performed to obtain reaction solution 5.41 containing 1.0% alginate oligosaccharide.
実施例１に記載の方法と同様な方法で、カイワレ大根に
対する植物生長促進効果を調べた結果、アルギン酸オリ
ゴ糖無添加の場合に比較して茎葉長で１２８％、根長で
２８９％の生長促進作用を示した。Using a method similar to that described in Example 1, we investigated the effect of promoting plant growth on daikon radish. As a result, growth was promoted by 128% in stem and leaf length and by 289% in root length compared to when no alginate oligosaccharide was added. The effect was shown.
本発明によれば、植物の生長促進作用を有するアルギン
酸オリゴ糖が効率よく得られる。このアルギン酸オリゴ
糖を使用することにより植物、特に農作物の増収を図る
ことができる。According to the present invention, alginic acid oligosaccharide having a plant growth promoting effect can be efficiently obtained. By using this alginic acid oligosaccharide, it is possible to increase the yield of plants, especially agricultural products.
Claims (10)
酸リアーゼを作用させてアルギン酸オリゴ糖を製造する
にあたり、反応液の２３０ｎｍの吸光度（Ｅ＾１＾％＿
１＿ｃ＿ｍ）が３０〜６０となったとき反応を停止する
ことを特徴とするアルギン酸オリゴ糖の製造法。(1) When producing alginate oligosaccharide by causing alginate lyase to act on a substance containing alginic acid or its salt, the absorbance at 230 nm of the reaction solution (E^1^%_
1_c_m) is 30 to 60, the reaction is stopped.
微生物に由来する酵素である特許請求の範囲第１項記載
の方法。(2) The method according to claim 1, wherein the alginate lyase is an enzyme derived from a microorganism belonging to the genus Alteromonas.
ス・エスピーＬＢ１０２株（ＦＥＲＭＰ−９２１８）で
ある特許請求の範囲第２項記載の方法。(3) The method according to claim 2, wherein the microorganism belonging to the genus Alteromonas is Alteromonas sp. LB102 strain (FERMP-9218).
素である特許請求の範囲第１項記載の方法。(4) The method according to claim 1, wherein the alginate lyase is an enzyme derived from the digestive tract of oysters.
酸もしくはその塩を含む海藻である特許請求の範囲第１
項記載の方法。(5) Claim 1, wherein the substance containing alginic acid or its salt is seaweed containing alginic acid or its salt.
The method described in section.
酸リアーゼを作用させてアルギン酸オリゴ糖を製造する
にあたり、反応液の２３０ｎｍの吸光度（Ｅ＾１＾％＿
１＿ｃ＿ｍ）が３０〜６０となったとき反応を停止し、
次いで反応液のｐＨを１．０〜４．０として加熱処理を
行うことを特徴とするアルギン酸オリゴ糖の製造法。(6) When producing alginate oligosaccharide by causing alginate lyase to act on a substance containing alginic acid or its salt, the absorbance at 230 nm of the reaction solution (E^1^%_
1_c_m) is 30 to 60, the reaction is stopped,
A method for producing an alginate oligosaccharide, which comprises then heating the reaction solution to a pH of 1.0 to 4.0.
微生物に由来する酵素である特許請求の範囲第６項記載
の方法。(7) The method according to claim 6, wherein the alginate lyase is an enzyme derived from a microorganism belonging to the genus Alteromonas.
ス・エスピーＬＢ１０２株（ＦＥＲＭＰ−９２１８）で
ある特許請求の範囲第７項記載の方法。(8) The method according to claim 7, wherein the microorganism belonging to the genus Alteromonas is Alteromonas sp. LB102 strain (FERMP-9218).
素である特許請求の範囲第６項記載の方法。(9) The method according to claim 6, wherein the alginate lyase is an enzyme derived from the digestive tract of oysters.
ン酸もしくはその塩を含む海藻である特許請求の範囲第
６項記載の方法。(10) The method according to claim 6, wherein the substance containing alginic acid or a salt thereof is seaweed containing alginic acid or a salt thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62048383A JPS63214192A (en) | 1987-03-03 | 1987-03-03 | Production of alginic acid oligosaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62048383A JPS63214192A (en) | 1987-03-03 | 1987-03-03 | Production of alginic acid oligosaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63214192A true JPS63214192A (en) | 1988-09-06 |
| JPH0528597B2 JPH0528597B2 (en) | 1993-04-26 |
Family
ID=12801788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62048383A Granted JPS63214192A (en) | 1987-03-03 | 1987-03-03 | Production of alginic acid oligosaccharide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63214192A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2690445A1 (en) * | 1992-04-28 | 1993-10-29 | Taiyo Fishery Co Ltd | Alginate oligosaccharide and its manufacturing process. |
| GB2266532A (en) * | 1992-04-28 | 1993-11-03 | Taiyo Fishery Co Ltd | Aliginate oligosaccharide and method for producing same |
| WO2001040315A1 (en) * | 1999-11-30 | 2001-06-07 | Dalian Yaweite Biology Engineering Co., Ltd. | The alginate having low molecular weight, methods of manufacturing it and its use |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101988045B (en) * | 2010-08-16 | 2012-08-22 | 厦门大学 | Composite culture medium for algae-killing bacterium and preparation method thereof |
-
1987
- 1987-03-03 JP JP62048383A patent/JPS63214192A/en active Granted
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2690445A1 (en) * | 1992-04-28 | 1993-10-29 | Taiyo Fishery Co Ltd | Alginate oligosaccharide and its manufacturing process. |
| GB2266532A (en) * | 1992-04-28 | 1993-11-03 | Taiyo Fishery Co Ltd | Aliginate oligosaccharide and method for producing same |
| US5460957A (en) * | 1992-04-28 | 1995-10-24 | Maruha Corporation | Calcium alginate oligosaccharide and method for producing the same from potassium or sodium alginate |
| US5516666A (en) * | 1992-04-28 | 1996-05-14 | Maruha Corporation | Alginate oligosaccharide and method for producing the same |
| GB2266532B (en) * | 1992-04-28 | 1996-09-04 | Taiyo Fishery Co Ltd | Alginate oligosaccharides methods for their production and their use in foodstuffs |
| WO2001040315A1 (en) * | 1999-11-30 | 2001-06-07 | Dalian Yaweite Biology Engineering Co., Ltd. | The alginate having low molecular weight, methods of manufacturing it and its use |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0528597B2 (en) | 1993-04-26 |
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