JPS6363378A - Cultivation of bacterium of genus pseudomonas - Google Patents
Cultivation of bacterium of genus pseudomonasInfo
- Publication number
- JPS6363378A JPS6363378A JP61206030A JP20603086A JPS6363378A JP S6363378 A JPS6363378 A JP S6363378A JP 61206030 A JP61206030 A JP 61206030A JP 20603086 A JP20603086 A JP 20603086A JP S6363378 A JPS6363378 A JP S6363378A
- Authority
- JP
- Japan
- Prior art keywords
- medium
- culture
- bacterium
- protease
- amino acid
- 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
- 241000589516 Pseudomonas Species 0.000 title claims abstract description 12
- 241000894006 Bacteria Species 0.000 title abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 21
- 244000068988 Glycine max Species 0.000 claims abstract description 10
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 10
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- 108091005804 Peptidases Proteins 0.000 claims abstract description 7
- 239000004365 Protease Substances 0.000 claims abstract description 7
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 7
- 150000003863 ammonium salts Chemical class 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 7
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 abstract 1
- -1 protease Chemical class 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 18
- 239000001963 growth medium Substances 0.000 description 6
- 241000589771 Ralstonia solanacearum Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 150000007513 acids Chemical class 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 241000918585 Pythium aphanidermatum Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000004254 Ammonium phosphate Substances 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 241000208292 Solanaceae Species 0.000 description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 2
- 235000019289 ammonium phosphates Nutrition 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical group [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
- RCJVRSBWZCNNQT-UHFFFAOYSA-N dichloridooxygen Chemical compound ClOCl RCJVRSBWZCNNQT-UHFFFAOYSA-N 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MFBBZTDYOYZJGB-HAONTEFVSA-L (2s,3s,4s,5r)-4-[(2r,3r,4r,5s,6r)-5-[(2r,3r,4r,5s,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2,3,5,6-tetrahydroxyhexanoate;iron(3+);oxyg Chemical compound O.[OH-].[O-2].[Fe+3].O[C@@H]1[C@@H](O)[C@@H](O[C@@H]([C@H](O)CO)[C@@H](O)[C@H](O)C([O-])=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@@H](CO)O1 MFBBZTDYOYZJGB-HAONTEFVSA-L 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282373 Panthera pardus Species 0.000 description 1
- 244000309560 Ralstonia solanacearum race 1 Species 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 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
- 235000021240 caseins Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229960004131 ferric carboxymaltose Drugs 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000009247 menarche Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
Landscapes
- Cultivation Of Plants (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (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］ 本発明はシュドモナス属菌の培養方法に関する。[Detailed description of the invention] [Industrial application field] The present invention relates to a method for culturing Pseudomonas bacteria.
さらに詳しくはシュドモナス属菌の培養促進および培養
収量の増加に関する。More specifically, the present invention relates to promoting the culture of Pseudomonas bacteria and increasing the culture yield.
シュドモナス属に剋する微生物はタバコ植物の立枯病お
よびナス科植物の青枯病の病原菌として知られている。Microorganisms belonging to the genus Pseudomonas are known as pathogens of damping-off of tobacco plants and bacterial wilt of plants of the Solanaceae family.
この菌を効率的に培養することは、農薬の効力検定や品
種の抵抗性検定等に利用する菌を効率的に得るという意
味で、一般的に意義あることである。Efficient cultivation of this bacterium is generally significant in the sense of efficiently obtaining bacteria for use in testing the efficacy of agricultural chemicals, testing the resistance of varieties, and the like.
しかし、これに止まらず、本発明方法によって、特に、
同国の非病原性でかつ植物に免疫性を付与する変異株を
大量に効率的に培養することは、大きな意義をもつもの
である。However, the method of the present invention does not stop there, in particular,
It is of great significance to efficiently cultivate large quantities of the country's non-pathogenic mutant strains that confer immunity to plants.
［従来の技術］
先に、本出願人は病原性のシュドモナス・ソラナシアラ
ム菌（Ｐｓｅｕｄｏｍｏｎａｓ ｓｏＬａｎａｃｅａｒ
ｕｍ）に対して交叉防御免疫（Ｃｒｏｓｓ ｐｒｏｔｅ
ｃｔｉｏｎ’）作用を有する同国の変異株（例えば、同
Ｍ４Ｓ菌株）を見出し、その菌株を用いたタバコ植物立
枯病およびナス科植物青枯病の防除方法を提供した。（
特開昭６０−１８６２３０）
このようないわゆる微生物農薬において利用する菌を大
量に効率的に培養ことば重要である。[Prior Art] Previously, the present applicant has developed a method for developing pathogenic Pseudomonas solanacearum bacteria.
cross-protective immunity against
We have discovered a mutant strain (for example, the M4S strain) of the same country that has the effect of ction'), and provided a method for controlling damping-off of tobacco plants and bacterial wilt of solanaceous plants using this strain. (
JP-A-60-186230) It is important to efficiently cultivate a large amount of bacteria used in such so-called microbial pesticides.
従来、微生物の培養に用いられる培地は、微生物の種類
により、あるいは研究の目的により数えきれないほどの
報告がある（微生物学実＠法、微生物研究法懇談会編、
錨談社刊、４２１−４４３．１９８１年）が、これまで
シュドモナス属の非病原性菌の培養、測えばシュドモナ
ス・ソラナシアラムＭ４Ｓ菌においては、カザミノ酸、
ペプトン、グルコースよりなるＣＰＧ培地が用いられて
きた（特開昭６Ｏ−１８６２３０）
［発明が解決しようとする問題点］
シュドモナス菌を特に農薬として用いるためには大量に
かつ安価に培養増殖ことが必要である。Conventionally, there have been countless reports on the culture medium used for culturing microorganisms, depending on the type of microorganism or the purpose of research (Microbiology Practical @ Method, edited by the Microbial Research Methods Council,
Anchordansha, 421-443.1981) has so far cultivated non-pathogenic bacteria of the genus Pseudomonas, and in Pseudomonas solanacearum M4S, casamino acids,
A CPG medium consisting of peptone and glucose has been used (Japanese Unexamined Patent Publication No. 6O-186230) [Problems to be solved by the invention] In order to use Pseudomonas bacteria as a pesticide, it is necessary to grow them in large quantities and at low cost. It is.
しかし、ＣＰＧ培地は高価なカザミノ酸（市価約５万円
／Ｋｇ）、ペプトン（市価的２・、５万円／Ｋｇ）を使
用するため製造原価が高くなり、実用化上難点があった
。However, since CPG medium uses expensive casamino acids (market price approximately 50,000 yen/Kg) and peptone (market price 2.50,000 yen/Kg), the manufacturing cost is high and there are difficulties in practical use.
本発明者らは安価な培地による大量培養方法を確立する
ことを目的として、窒素源にアミノ酸粉末（市価的２．
５千円／Ｋｇ）、脱脂大豆粉末（市価的１．６百円／Ｋ
ｇ）を用いた培地組成に注目し、試験検討したとこ゛ろ
、培養の増殖速度が低下し生産性が悪（なるという問題
が生じてきた。The present inventors aimed to establish a mass culture method using an inexpensive medium, and used amino acid powder as a nitrogen source (market price 2.
5,000 yen/Kg), defatted soybean powder (market price 1.6 million yen/Kg)
When we focused on the medium composition using (g) and conducted tests, we found that the growth rate of the culture decreased and the productivity deteriorated.
これらの問題を解決すべく鋭意研究を重ねた結果、本発
明を完成するに至った。As a result of intensive research to solve these problems, the present invention has been completed.
すなわち、アミノ酸粉末、脱脂大豆粉末、ブドウ糖の他
に、無機アンモニウム塩および蛋白分解酵素を加えた組
成の培地を用いるシュドモナス属菌の培養方法である。That is, this is a method for culturing Pseudomonas bacteria using a medium containing an inorganic ammonium salt and a protease in addition to amino acid powder, defatted soybean powder, and glucose.
本発明のアミノ酸粉末は、カゼインを原料として食品用
に市販されているもの（例えばアミノ酸パウダーＳＬ）
でよい。これを培地組成として１〜２ｇ／ｌで用いる。The amino acid powder of the present invention is one that is commercially available for food use and uses casein as a raw material (for example, amino acid powder SL).
That's fine. This is used as a medium composition at 1 to 2 g/l.
脱脂大豆粉末は通常に市販されているものでよく、例え
ば粗蛋白含有量的５６％（対乾物重量換算）の市販品の
場合、１０〜２０ｇ／ｌの水溶液ないし水懸濁液を用い
る。望ましくは、原料粉末の熱水抽出液がよい。The defatted soybean powder may be any commercially available product; for example, in the case of a commercially available product with a crude protein content of 56% (based on dry weight), an aqueous solution or suspension of 10 to 20 g/l is used. Preferably, a hot water extract of the raw material powder is used.
ブドウ糖は、１０〜２０ｇ／ｌの範囲でよい。Glucose may range from 10 to 20 g/l.
無機アンモニウム塩は、リン酸塩、硝酸塩、硫酸塩など
が有効であり、リン酸第−アンモニウムが最もよい。添
加量は０．５〜１ｇ／ｌでよい。Effective inorganic ammonium salts include phosphates, nitrates, and sulfates, with tertiary ammonium phosphate being the best. The amount added may be 0.5 to 1 g/l.
蛋白分解酵素としては、良品加工用に市販されているも
のでよく（例えばプロテアーゼ）を０゜０１〜０．１ｇ
／ｌ添加すればよい。The protease may be one commercially available for processing good quality products (e.g. protease) at a concentration of 0.01 to 0.1 g.
/l may be added.
培豹は、上述の培地にシュドモナス属菌を接種し、温度
約３０°Ｃ，ＰＨ５〜７で３０〜５０時間通気攪拌培養
する。For culturing leopard, Pseudomonas bacteria are inoculated into the above-mentioned medium, and cultured with aeration and stirring at a temperature of about 30° C. and a pH of 5 to 7 for 30 to 50 hours.
［作用］
本発明の方法が従来培地の方法に比べて生産性が上がる
理由は必ずしも明確ではないが、一般的に微生物の培養
における培地は、微生物がこれを資化する際、自己の代
謝系における酵素による栄養源の分解能に対し、培養初
潮段階に補助的役割を果たすものと思われる。[Effect] The reason why the method of the present invention improves productivity compared to the conventional culture medium method is not necessarily clear, but in general, when microorganisms use the medium for culturing microorganisms, their own metabolic system It is thought that it plays a supplementary role during the menarche stage of culture, with respect to the ability of enzymes to decompose nutrient sources.
［実施例］
内容積５０１のステンレス裂通気攪拌壓培養槽を用い、
あらかじめ′：Ａａしておいた培地３０１を張り込み、
これにシュドモナス ソラナシアラムＭ４Ｓ菌（ＦＥＲ
Ｍ ＢＰ−７００）を、１００ｍ１注入して３０”Ｃ
１槽内圧カ０−５ Ｋ ｇ ／ ｃ ｍｃ、ｔｆ２拌速
度１°５０ｒｐｍ、通気Ｈｚ ’Ｌ ５ ／分、初発Ｐ
Ｈ６，４で５０時間培養した。[Example] Using a stainless steel cracked aerated stirring bottle culture tank with an internal volume of 501,
Pour in the culture medium 301 that has been adjusted to ':Aa in advance,
This is combined with Pseudomonas solanacearum M4S bacterium (FER
Inject 100ml of M BP-700) and heat to 30”C.
1 tank internal pressure 0-5 K g/cm mc, tf2 stirring speed 1°50 rpm, ventilation Hz'L5/min, initial P
Cultured in H6.4 for 50 hours.
試験に用いた培地１１当たりの組成を、以下に示す
培ｔｔｈＡ（本発明方法による培地）
アミノ酸粉末 １ｇ
説脂大豆粉末 ２０ｇ
ブドウ糖 １０ｇ
リン酸第−アンモニウム ０．３ｇ
硝酸アンモニウム ０．２ ｇパンチクーゼＮ
Ｐ２ ０．０５ｇ（ヤクルト本社、登録商ａ）
培地Ｂ（従来方法の培地）（対象培地）カザミノ酸
１ｇ
ペプトン １０ｇ
ブドウ糖 １０ｇ
培地培地培地Ｂの窒素源を代替した培地）（対象培地）
アミノ酸粉末 １ｇ
脱脂大豆粉末 ２０ｇ
ブドウ糖 Ｌｏｇ
なお、脱脂大豆粉末は１０倍量の水に懸濁さぜ、１２１
°Ｃｌ２Ｏ分で抽出した水溶液を用いた。The composition of each medium 11 used in the test is as follows: Culture medium 11 (medium according to the method of the present invention) Amino acid powder 1 g Soybean powder 20 g Glucose 10 g Tertiary ammonium phosphate 0.3 g Ammonium nitrate 0.2 g Panchikuse N
P2 0.05g (Yakult Honsha, registered trader a) Medium B (conventional method medium) (target medium) Casamino acids
1g Peptone 10g Glucose 10g Medium Medium Substituting the nitrogen source of Medium B) (Target medium) Amino acid powder 1g Defatted soybean powder 20g Glucose Log In addition, defatted soybean powder was suspended in 10 times the volume of water, 121
An aqueous solution extracted with Cl2O was used.
培養開始後４時間毎に培養槽内から１００ ｍ ｌサン
プルを抜き取り、これを１２０００Ｇ遠心分離後８０゛
Ｃで２４時間！２燥した後秤量し、換算して菌体目量を
求めた。結果を図１に吊子。Every 4 hours after the start of culture, a 100ml sample was taken from the culture tank, centrifuged at 12,000G, and then heated at 80°C for 24 hours! 2 After drying, it was weighed and converted to determine the weight of bacterial cells. The results are plotted in Figure 1.
ＣＰＧ培地のカザミノ酸、ペプトンを価格の安いアミノ
酸粉末、脱脂大豆粉末に・代えた培地では、増殖速度が
低下し、生産性が悪いが、本発明の方法によれば、ＣＰ
Ｇ培地のものより増殖速度、培養収量ともに優れている
ことが図１かられかる。In a medium in which casamino acids and peptone in CPG medium are replaced with cheap amino acid powder or defatted soybean powder, the growth rate decreases and productivity is poor, but according to the method of the present invention, CP
It can be seen from FIG. 1 that both the growth rate and the culture yield are superior to those using G medium.
また、本方法により得られたシュドモナス・ソラナシア
ラムＭ４ＳＷのナス科植物の立枯病、青枯病防踪効果を
調べたところＣＰＧ培地で培養したものと効果に差がな
かった。Furthermore, when the effect of Pseudomonas solanacearum M4SW obtained by this method on suppressing damping-off and bacterial wilt of Solanaceae plants was investigated, there was no difference in the effect from that of Pseudomonas solanacearum M4SW when cultured in a CPG medium.
［発明の効果］
本発明方法によれば、従来の培養方法に比ベシュドモナ
ス肥菌の培養速度、培養収量が高まると共に、培地のコ
ストを大幅に低減させることが可能である。[Effects of the Invention] According to the method of the present invention, the culture speed and culture yield of B. fertilizing bacteria can be increased compared to conventional culture methods, and the cost of the culture medium can be significantly reduced.
図１は本発明による培養試験の結果を示す３グラフて°
ある。
培養時間（ｈｒ）
図、−１Figure 1 shows three graphs showing the results of the culture test according to the present invention.
be. Culture time (hr) Figure, -1
Claims (1)
脱脂大豆粉末、ブドウ糖、無機アンモニウム塩および蛋
白分解酵素からなる培地を用いることを特徴とするシュ
ドモナス属菌の培養方法。In the method for culturing Pseudomonas bacteria, amino acid powder,
A method for culturing Pseudomonas bacteria, the method comprising using a medium comprising defatted soybean powder, glucose, an inorganic ammonium salt, and a protease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61206030A JPH064021B2 (en) | 1986-09-03 | 1986-09-03 | Cultivation method for genus Cichudomonas |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61206030A JPH064021B2 (en) | 1986-09-03 | 1986-09-03 | Cultivation method for genus Cichudomonas |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6363378A true JPS6363378A (en) | 1988-03-19 |
| JPH064021B2 JPH064021B2 (en) | 1994-01-19 |
Family
ID=16516732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61206030A Expired - Lifetime JPH064021B2 (en) | 1986-09-03 | 1986-09-03 | Cultivation method for genus Cichudomonas |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH064021B2 (en) |
-
1986
- 1986-09-03 JP JP61206030A patent/JPH064021B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH064021B2 (en) | 1994-01-19 |
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