JP2007008960A5

JP2007008960A5 -

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Publication number: JP2007008960A5
Application number: JP2006273010A
Authority: JP
Filing date: 2006-10-04
Publication date: 2007-03-01

Description

Antibacterial agent and method for producing the same

この発明は、トリコデルマハルジアナムＳＫ−５５（Trichoderma hurzianum ＳＫ−５５）菌の培地から得られる糸状菌及び細菌に抗菌性を有する抗菌剤及びその製造方法に関する。 The present invention relates to an antibacterial agent having antibacterial properties against filamentous fungi and bacteria obtained from a culture medium of Trichoderma hurzianum SK-55, and a method for producing the same.

トリコデルマ菌は、微生物農薬として病害防除に実用化されている糸状菌であり、Trichoderuma lignourum がタバコの白絹病の防除用農薬として登録されている（農薬登録 No.7023）。また英国、フランスでもTrichoderuma hurzianumを有効成分とする野菜の立枯病、苗腐病に有効な微生物農薬（F-ｓtop）が登録されている。 Trichoderma is a fungus that has been put to practical use as a microbial pesticide for disease control, and Trichoderuma lignourum has been registered as a pesticide for control of tobacco silkworm (Agricultural Chemical Registration No. 7023). In the UK and France, microbial pesticides (F-stop) are also registered, which are effective against vegetable wilt and seedling rot, which contain Trichoderuma hurzianum as an active ingredient.

近来減農薬・生態系保全型の病害防除の必要性が強く唱えられ、特に生物防除方法は環境負荷への影響が少ないと考えられるので、精力的に研究が進められている。 Recently, there is a strong need for reducing pesticides and ecosystem-conserving diseases. Especially, biological control methods are considered to have little impact on the environmental impact, so research is being conducted energetically.

植物の根や根圏から分離される細菌や、菌類の中には植物の生育を促進するものがあり、それぞれ植物生育促進根圏（plant growth-promoting rhizobacteria：ＰＧＰＲ)、植物生育促進菌類（plant growth-promoting fungi：ＰＧＰＦ)と呼ばれている。 Some bacteria and fungi isolated from plant roots and rhizospheres promote plant growth. Plant growth-promoting rhizobacteria (PGPR) and plant growth-promoting fungi (plant) It is called growth-promoting fungi (PGPF).

前記有用根圏微生物であるＰＧＰＲ、ＰＧＰＦは植物の生育を促進するのみならず、各種の土壌病害を抑制することも知られている。また最近では、土壌病害のみならず、地上病害も抑制する事実も明らかにされ、前記ＰＧＰＲ、ＰＧＰＦは、植物の全身抵抗性の誘導が関わっていることが見出されたと紹介されている（１９９９年６月号、「今月の農業」誌）。 The useful rhizosphere microorganisms PGPR and PGPF are known not only to promote plant growth but also to suppress various soil diseases. Recently, the fact that not only soil diseases but also ground diseases are also clarified, and it has been introduced that the PGPR and PGPF were found to be involved in the induction of systemic resistance of plants (1999). June issue, “This month's agriculture” magazine).

またコウライシバから分離選抜したPhoma，Trichoderma, Fusarium，Penicillium，Sterileなどの菌が、キュウリにおける炭そ病に対し、誘導抵抗性を示すことが報告されている（１９９９年６月号、「今月の農業」誌）。
特開平６−１９２０２８号 In addition, it has been reported that fungi such as Phoma, Trichoderma, Fusarium, Penicillium, and Sterile isolated and selected from cucumber shiba show induction resistance against anthracnose in cucumber (June 1999 issue, “Agriculture of the Month”). "magazine).
JP-A-6-192028

この発明は、植物病原菌に抗菌性を有する物質を得ることを課題として研究しており、北海道十勝地方の土壌からTrichoderuma hurzianum ＳＫ−５５(トリコデルマハルジアナムＳＫ−５５)菌を分離した。この菌は、植物病原菌の８０％を占めるとされる土壌伝染性病原糸状菌に対して広く拮抗性を示す菌であり、現在芝のブラウンバッチ、ラージバッチなどの Rhizoctonia類に微生物農薬（生菌製剤）として開発中のものである。前記トリコデルマハルジアナムＳＫ−５５菌の拮抗作用は、相手菌糸に接触し、コイリングをする事により、細胞質凝集を促し死滅させるものである。その外観的観察経過より見てペニシリアム（Penicillium sp）等が生産する抗生物質のような培地上で阻止円を示すほど活性が高いものではなく、菌糸同志の接触により発現するものと推定される。 This invention has been studied as an object to obtain a substance having antibacterial properties against plant pathogens, and Trichoderuma hurzianum SK-55 (Trichoderma harzianum SK-55) bacteria have been isolated from soil in Tokachi region, Hokkaido. This bacterium is widely antagonizing against soil-borne pathogenic fungi, which accounts for 80% of plant pathogens. Currently, Rhizoctonia such as turf brown batches and large batches have microbial pesticides (live bacteria) It is under development as a formulation. The antagonism of Trichoderma harzianum SK-55 bacteria promotes cytoplasmic aggregation and kills by contacting the other hyphae and coiling. It is presumed from the appearance observation process that the activity is not so high as to show a circle of inhibition on a medium such as an antibiotic produced by penicillium (Penicillium sp) and the like, and is expressed by contact between mycelia.

前記細胞質凝集作用が、トリコデルマハルジアナムＳＫ−５５菌の生産する何らかの物質によってもたらされると考えられ、これを究明することを第一の課題としたものである。 It is considered that the cytoplasm aggregating action is brought about by some substance produced by Trichoderma harzianum SK-55, and the first task is to investigate this.

なお、この発明で用いるトリコデルマハルジアナムＳＫ−５５菌は、日本国内工業技術院微生物工業研究所に受託番号「微工研菌寄第１３３２７号」として寄託されている（受注の日：１９９２年１２月９日、寄託者：株式会社北海道グリーン興産、代表者佐々木進）。微工研は、当該原寄託よりブダペスト条約に基づく寄託への移管請求を１９９２年１２月９日付で行ない、受託番号ＢＰ−４３４６が付与されている。 The Trichoderma harzianum SK-55 bacterium used in the present invention has been deposited at the National Institute of Advanced Industrial Science and Technology of Japan under the accession number “Saiken Laboratories No. 13327” (Order Date: December 1992) 9th, Depositary: Hokkaido Green Kosan Co., Ltd., Representative Susumu Sasaki). NIKK filed a request for transfer from the original deposit to a deposit based on the Budapest Treaty on December 9, 1992, and was given a deposit number of BP-4346.

前記従来のＰＧＰＲ、ＰＧＰＦは、特定植物（例えばキュウリ）に対し、炭そ病についての有効性を示すもので、今後研究の結果、その使用方法の改善などにより、他の植物、病原菌に対して有効なことが判明する可能性はあるとしても、未だ具体的植物、病原菌に対しては今後の課題とされている。 The conventional PGPR and PGPF are effective against anthracnose for specific plants (for example, cucumber). As a result of future research, improvement of the method of use, etc., against other plants and pathogens Even though it may prove effective, it is still considered as a future problem for specific plants and pathogens.

然し乍ら単に病原菌を殺菌するという従来の生物農薬の思考形態が、植物の根茎に亘り抗菌性を付与する方向に変りつつあることは、今後の植物栽培上重要な示唆を含むものである。 However, the fact that the conventional form of biological pesticides, which simply sterilizes pathogenic bacteria, is changing in the direction of imparting antibacterial properties to the rhizomes of plants, includes important suggestions for future plant cultivation.

この発明は、トリコデルマハルジアナムＳＫ−５５菌について、各種実験を重ねている間に、前記生物農薬の将来性と合致することに想到し、更に使用方法、対象植物等を選定、研究の結果、この発明を完成したのである。将来の植物栽培に多大の影響を与えるものとして、この発明はきわめて有望であり、将来の農業等を支える重要な手段の１つとなることに疑はない。 This invention was conceived to be consistent with the future potential of the biopesticides during various experiments on Trichoderma harzianum SK-55, and further, the method of use, target plants, etc. were selected and researched. The invention has been completed. The present invention is very promising as having a great influence on future plant cultivation, and there is no doubt that it will be one of the important means for supporting future agriculture and the like.

この発明は、トリコデルマハルジアナムＳＫ−５５菌の生産する物質により、細胞凝集作用（抗菌性）がもたらされるものと推定し、前記菌を培養、精製、同定した所、糸状菌又は細菌に抗菌性を有する物質を得たのである。 The present invention presumes that the substance produced by Trichoderma harzianum SK-55 produces cell agglutination (antibacterial activity), and when the bacterium is cultured, purified and identified, it has antibacterial activity against filamentous fungi or bacteria. The substance which has was obtained.

また他の発明は、植物栽培に際し、トリコデルマハルジアナムＳＫ−５５菌の分生胞子を覆土中に共存させて増殖させることにより、その生成物質により、各種植物の根茎活性を付与し、耐菌性を向上させることが判明し、前記従来の問題点を発展的に改善させて、実用性を確立したものである。 In addition, in the case of plant cultivation, conidial spores of Trichoderma harzianum SK-55 are coexisted in the cover soil and proliferated, thereby imparting rhizome activity of various plants by the generated substances, and providing resistance to bacteria. It has been found that the above problems have been improved, and the conventional problems have been progressively improved to establish practicality.

即ち抗菌剤の発明は、トリコデルマハルジアナムＳＫ−５５菌を固体培地で培養して得た病原微生物に抗菌性を有する物質であって、下記成分を含むことを特徴とした抗菌剤であり、トリコデルマハルジアナムＳＫ−５５菌を液体培地で培養して得た病原微生物に抗菌性を有する物質であって、下記成分を含むことを特徴とした抗菌剤である。 That is, the invention of the antibacterial agent is a substance having antibacterial properties against pathogenic microorganisms obtained by culturing Trichoderma harzianum SK-55 bacteria in a solid medium, and includes the following components. It is a substance having antibacterial properties against pathogenic microorganisms obtained by culturing SK-55 bacteria in a liquid medium, and is an antibacterial agent characterized by containing the following components.

Ａ成分：Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-...であってその分子量１９３３
Ｂ成分：Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-...であってその分子量１９４９または１９６４
Ｃ成分：

Component A: Ac-Aib-Ala-Aib -Aib-Aib-Aib-Gln-Aib-Aib -... a was the molecular weight 1933
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib -... and its molecular weight 1949 or 1964
C component:

...であってその分子量１６８（Ｃ _８Ｈ ₈ Ｏ _４）
Ｄ成分：Ac-Aib-Ala-Aib-Aib-Aib-... であってその分子量１８１０ Its molecular weight ... a a and _{_{_{168 (C 8 H 8 O 4}}} )
Component D: Ac-Aib-Ala-Aib -Aib-Aib -... a it was in its molecular weight 1810

また、病原微生物は、Rhizoctonia solani(AG-1IA), Bacillus subtilis ATCC6633, Micrococcus luters ATCC6633, Staphylococcus aureus 209Pとしたものであり、病原微生物は、Bacillus subtilis ATCC6633, Micrococcus luters ATCC6633, Staphylococcus aureus 209P, Escherichia coli NIHJとしたものである。 The pathogenic microorganisms were Rhizoctonia solani (AG-1IA), Bacillus subtilis ATCC6633, Micrococcus luters ATCC6633, Staphylococcus aureus 209P, and the pathogenic microorganisms were Bacillus subtilis ATCC6633, Micrococcus luters ATCC6633, Staphylococcus acherus 209P It is what.

更に、固体培地は、米を主材料とし、これに少量の大豆かす、滅菌水を添加するものであり、液体培地はグルコースを主材とし、これに少量のポリペプトン、Nacl、酵母エキス及び炭酸カルシウムと、適量の滅菌水を加えたものである。 Furthermore, the solid medium is mainly made of rice, and a small amount of soybean meal and sterilized water are added thereto. The liquid medium is mainly made of glucose, and a small amount of polypeptone, NaCl, yeast extract and calcium carbonate. And an appropriate amount of sterilized water.

次に製造方法の発明は、トリコデルマハルジアナムＳＫ−５５菌を固定培地に植菌し、２５℃〜３０℃で７日〜１５日間静置培養した後、請求項１記載の抗菌剤を得ることを特徴とした抗菌剤の製造方法であり、トリコデルマハルジアナムＳＫ−５５菌を液体培地に植菌し、２５℃〜３０℃で４日〜１０日間振盪培養した後、請求項２記載の抗菌剤を得ることを特徴とした抗菌剤の製造方法である。 Next, the invention of the production method is to inoculate Trichoderma harzianum SK-55 in a fixed medium, and after standing at 25 ° C. to 30 ° C. for 7 to 15 days, the antibacterial agent according to claim 1 is obtained. A method for producing an antibacterial agent characterized in that Trichoderma harzianum SK-55 is inoculated in a liquid medium and shaken and cultured at 25 ° C to 30 ° C for 4 days to 10 days, and then the antibacterial agent according to claim 2 is obtained. This is a method for producing an antibacterial agent.

この発明において、使用する固体培地として、米培地を用いた。米培地は、米に対し、大豆かす３％、滅菌水１０％よりなる固定培地であって、培養中に培地の表面が乾燥しないよう、滅菌水を添加した。 In this invention, a rice medium was used as the solid medium to be used. The rice medium was a fixed medium composed of 3% soybean meal and 10% sterilized water, and sterilized water was added so that the surface of the medium was not dried during the cultivation.

この発明において、使用する液体培地は、グルコース３．０％〜５．０％、ポリペプトン０．５％、Ｎａｃｌ０．８％、酵母エキス０．２％、炭酸カルシウム１．０％である。 In this invention, the liquid medium to be used is glucose 3.0% to 5.0%, polypeptone 0.5%, Nacl 0.8%, yeast extract 0.2%, calcium carbonate 1.0%.

前記のように固定培地と、液体培地によれば、夫々の特性により、異なる物質を生成することが明らかとなった。このような結果についてのメカニズムは明らかでないが、トリコデルマハルジアナムＳＫ−５５菌に対する刺激その他の作用の相違、かつ培地構成物質に関する特性により、異なる物質を生成するものであって、前記米培地及び液体培地以外の成分の培地であっても同様の物質を生成することは十分考えられ、かつ生成効率向上等今後の研究課題は多大である。 As described above, it has been clarified that the fixed medium and the liquid medium generate different substances depending on the respective characteristics. Although the mechanism for such a result is not clear, it produces different substances depending on differences in stimulation and other actions on Trichoderma harzianum SK-55 bacteria and on the characteristics of the medium constituents, and the rice medium and liquid medium Even if it is a culture medium of components other than these, it is thought enough to produce | generate the same substance, and future research subjects, such as improvement of production | generation efficiency, are great.

前記におけるトリコデルマハルジアナムＳＫ−５５菌の分生胞子を培養し、その単離精製物を検討した所、ＵＶ吸収スペクトラム、質量分析及びＮＭＲ測定結果より、Aib（α-Aminoisobutyric acid）を含むPolypeptideの、Peptibols系と判断した。Peptibols系抗生物質はアミノ酸配列の中に α-Aminoisobutyric acid（Aib）を含んでおり、Ｎ末端がAcetyl基で、Ｃ末端が Amino alcohol結合であることが特徴である（多くは Phenylalaniol基でおわる）。なお今回の精製物、Ａ成分、Ｂ成分、Ｄ成分は質量分析より一部のアミノ酸配列を下記のように推定し、その部分構造を既存のPeptibols のアミノ酸配列と検索した結果、一致する物が無く、新規 Peptibols系であると推定した。Ｅはアミノ酸配列の推定が困難であるため判断できなかった。 The conidia of Trichoderma harzianum SK-55 in the above were cultured, and the isolated and purified product was examined. From the results of UV absorption spectrum, mass spectrometry and NMR measurement, Polypeptide containing Aib (α-Aminoisobutyric acid) Peptibols system was judged. Peptibols antibiotics contain α-Aminoisobutyric acid (Aib) in the amino acid sequence, and are characterized by an N-terminal Acetyl group and a C-terminal Amino alcohol bond (mostly Phenylalaniol groups). ). As for the purified product, A component, B component, and D component, the partial amino acid sequence was estimated by mass spectrometry as follows, and the partial structure was searched with the existing amino acid sequence of Peptibols. There was no new Peptibols system. E could not be determined because it was difficult to estimate the amino acid sequence.

ＵＶ吸収波長：４成分とも末端吸収
Ａ成分の分子量＝１９３３
Ｂ成分の分子量＝１９４９または１９６４
Ｄ成分の分子量＝１８１０
Ｅ成分の分子量＝１８２９
推定構造：Ａ成分：Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-......
Ｂ成分：Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-......
Ｄ成分：Ac-Aib-Ala-Aib-Aib-Aib-..... UV absorption wavelength: molecular weight of terminal absorption A component for all four components = 1933
Molecular weight of component B = 1949 or 1964
Molecular weight of component D = 1810
E component molecular weight = 1829
Estimated structure: Component A: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib -......
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib -......
D component: Ac-Aib-Ala-Aib-Aib-Aib -.....

前記発明においては、トリコデルマハルジアナムＳＫ−５５を固体培地又は液体培地で培養し、その生成物質中Ａ、Ｂ、Ｃ、Ｄ、Ｅ成分について単離精製したが、活性物質は、新規アミノ酸配列をもつ新規物質で、その分子量は、Ａ成分の分子量＝１９３３，Ｂ成分の分子量＝１９４９または１９６４，Ｄ成分の分子量＝１８１０，Ｅ成分の分子量＝１８２９であって、当該アミノ酸又はその周辺物が植物の活性化に有効であろうと推定される。 In the above invention, Trichoderma harzianum SK-55 is cultured in a solid medium or liquid medium, and the components A, B, C, D, and E in the product are isolated and purified, but the active substance has a novel amino acid sequence. in the new material, its molecular weight is a molecular weight = 1829 molecular weight = 1810, E component having a molecular weight = 1949, or 1964, D component having a molecular weight = 1933, B component of the a component, the amino acid or peripheral thereof is a plant It is presumed that it will be effective for activation.

そこで前記トリコデルマハルジアナムＳＫ−５５菌を従来法によって多量培養し、その生成物質を抽出精製して植物の活性付与剤を得ることができる。前記生成物質は、前記Ａ成分、Ｂ成分、Ｃ成分、Ｄ成分及びＥ成分が判明している。 Therefore, the Trichoderma harzianum SK-55 bacterium can be cultured in a large amount by a conventional method, and the product can be extracted and purified to obtain a plant activity-imparting agent. The product A, B component, C component, D component, and E component are known.

この発明によれば、トリコデルマハルジアナムＳＫ−５５菌を固形培地に培養した場合には、表２のように、糸状菌について抗菌性を示すことが認められた。 According to this invention, when Trichoderma harzianum SK-55 was cultured in a solid medium, as shown in Table 2, it was confirmed that the fungus exhibited antibacterial properties.

またこの発明の製造方法によれば、糸状菌及び細菌に有効な抗菌剤を容易に製造することができる。 According to the production method of the present invention, an antibacterial agent effective against filamentous fungi and bacteria can be easily produced.

この発明は、トリコデルマハルジアナムＳＫ−５５菌を固定培地又は液体培地に植菌し、２５℃〜３０℃で４日〜１５日間静置培養又は振盪培養した後、抗菌性を有する下記Ａ成分、Ｂ成分、Ｃ成分及びＤ成分を含む物質を分離してこの発明の抗菌剤を得た。 In the present invention, Trichoderma harzianum SK-55 is inoculated into a fixed medium or a liquid medium, followed by static culture or shaking culture at 25 ° C. to 30 ° C. for 4 to 15 days. The substance containing the component, the C component and the D component was separated to obtain the antibacterial agent of the present invention.

...であってその分子量１６８（Ｃ _８Ｈ _３Ｏ _４）
Ｄ成分：Ac-Aib-Ala-Aib-Aib-Aib-... であってその分子量１８１０ Its molecular weight ... a a and _{_{_{168 (C 8 H 3 O 4}}} )
Component D: Ac-Aib-Ala-Aib -Aib-Aib -... a it was in its molecular weight 1810

前記において、固定培地による培養には、７日〜１５日を要し、一液体培地による培養には４日〜１０日を要した。 In the above, the culture with the fixed medium required 7 to 15 days, and the culture with the one liquid medium required 4 to 10 days.

（供試菌名称）
Trichoderuma hurzianum ＳＫ−５５（トリコデルマハルジアナムＳＫ−５５）
供試菌は97年1月北海道グリーン興産より寒天培地のスラント1本を受領した。供試菌は下記の斜面培地に植菌し、２８℃で５日間培養後、冷蔵にて保存した。 (Test bacteria name)
Trichoderuma hurzianum SK-55 (Trichoderuma hurzianum SK-55)
The test bacteria received a slant of agar medium from Hokkaido Green Kosan in January 1997. The test bacteria were inoculated in the following slant culture medium, cultured at 28 ° C. for 5 days, and stored in a refrigerator.

寒天斜面培地組成
オートミール５．０％
シュクロース５．０％
寒天１．０％
２８℃、５日間培養後冷蔵保存 Agar slope medium composition oatmeal 5.0%
Sucrose 5.0%
Agar 1.0%
Refrigerated after incubation at 28 ° C for 5 days

（培養）
培養は５００ｍｌ三角フラスコを用い、米培地（Ａ培地）と液体培地（Ｂ培地Ｉ）の２種類で行った。Ａ培地の条件は、２８℃で静置培養（培養途中に滅菌水１０ｍｌを添加）とし、Ｂ培地Ｉの培養は、２８℃で270rpmの振盪培養をした。 (culture)
Cultivation was performed in two types of rice medium (A medium) and liquid medium (B medium I) using a 500 ml Erlenmeyer flask. The condition of the medium A was stationary culture at 28 ° C. (10 ml of sterilized water was added during the culture), and the culture of the medium B was shake culture at 28 ° C. at 270 rpm.

培地組成：Ａ培地
米１００％
大豆かす３％
滅菌水１０％
培地組成：Ｂ培地Ｉ
グルコース５．０％
ポリペプトン０．５％
ＮａＣｌ０．８％
酵母エキス０．２％
炭酸カルシウム１．０％ Medium composition: A medium 100% rice
Soybean ground 3%
Sterile water 10%
Medium composition: B medium I
Glucose 5.0%
Polypeptone 0.5%
NaCl 0.8%
Yeast extract 0.2%
Calcium carbonate 1.0%

（検定法）
検定は、すべてペーパーディスク平板法によるin vitroで行い、供試菌には下記の菌を用いた。 (Test method)
All tests were performed in vitro by the paper disk plate method, and the following bacteria were used as test bacteria.

（１）Rhizoctonia solani（AG-1IA）
検定用培地にはＰＤＡ（Nissui）1.3%、Chloramphenicol 0.002%の組成からなる培地を用いた。Rhizoctonia の菌糸の先端をコルクボーラーで抜いたものを平板培地の中央にのせ、ブロスを染み込ませたペーパーディスクを置き菌糸の伸長の様子を観察した。 (1) Rhizoctonia solani (AG-1IA)
A medium having a composition of PDA (Nissui) 1.3% and Chloramphenicol 0.002% was used as an assay medium. The tip of Rhizoctonia hyphae was removed with a cork borer and placed in the center of the plate medium, and a paper disk soaked with broth was placed to observe the growth of the hyphae.

（２）Botrytis cinerea検定用培地にはPotato-extract 20.0%、Sucrose 2.0%、寒天1.5%の組成からなる培地を用いた。検定はペーパーディスクに試料をのせ風乾後シャーレにのせた。シャーレをインキュベーターに入れて培養し、ペーパーディスクの周りに阻止円が形成されているか否かを観察した。 (2) A medium having a composition of Potato-extract 20.0%, Sucrose 2.0%, and agar 1.5% was used as the Botrytis cinerea assay medium. The test was carried out by placing the sample on a paper disc, air-drying and then placing on a petri dish. The petri dish was placed in an incubator and cultured, and it was observed whether or not a blocking circle was formed around the paper disk.

（３）抗菌スペクトラム検定菌
抗菌スペクトラムを下記の検定菌を用いて測定した。 (3) Antibacterial spectrum test The antibacterial spectrum was measured using the following test bacteria.

Bacillus subtilis ATCC6633
Micrococcus luters ATCC6633
Staphylococcus aureus 209P
Escherichia coli NIHJ
Saccharomyces cerevisiae SHY3
Candida albicans M9001
Candida pseudotropicalis M9035
Cryptococcus neoformans M9010
Debaryomyces hansenii M9011
Trigonopsis variabilis M9031
Schizosaccharomyces pombe M9025
Hansenula schneggi IAM4269 Bacillus subtilis ATCC6633
Micrococcus luters ATCC6633
Staphylococcus aureus 209P
Escherichia coli NIHJ
Saccharomyces cerevisiae SHY3
Candida albicans M9001
Candida pseudotropicalis M9035
Cryptococcus neoformans M9010
Debaryomyces hansenii M9011
Trigonopsis variabilis M9031
Schizosaccharomyces pombe M9025
Hansenula schneggi IAM4269

（培養法）
（１）Ａ培地培養法
Rhizoctonia solani（AG-1IA）活性成物
糸状菌Rhizoctonia solani（AG-1IA）に対するＡ培地の培養抽出液活性物の単離精製。 (Culture method)
(1) A medium culture method
Rhizoctonia solani (AG-1IA) active product Isolation and purification of the culture extract actives of medium A against the filamentous fungus Rhizoctonia solani (AG-1IA).

Ａ培地（米培地１kg）５００ｍｌの三角フラスコ１本に米１００ｇと大豆かす３ｇを加え、同じ条件のもの１０本分の培地を調製した。滅菌後エーゼで植菌して、２８℃１０日間（菌がよく増殖するように途中何度かフラスコを振る。また培地の表面が乾いてきたら滅菌水を添加する。）静置培養後、５０％アセトン水を２リットル加え抽出をした。ここで培養物５０％アセトン抽出液２リットルを得た。 100 g of rice and 3 g of soybean meal were added to a 500 ml Erlenmeyer flask of A medium (1 kg of rice medium) to prepare 10 mediums under the same conditions. After sterilization, inoculate with ase and shake at 28 ° C for 10 days (shake the flask several times in the middle so that the bacteria can grow well. Add sterilized water when the surface of the medium is dry). Extraction was performed by adding 2 liters of% acetone water. Here, 2 liters of 50% acetone extract of the culture was obtained.

（２）Ｂ培地培養法
Staphylococcus aureus 209P 活性成物
細菌Staphylococcus aureus 209Pに対するＢ培地培養抽出液中活性物の単離精製。

(2) B medium culture method
Staphylococcus aureus 209P active product Isolation and purification of active product in B medium culture extract against bacterium Staphylococcus aureus 209P.

液体培地であるＢ培地Ｉの抗細菌活性成分をより多く生産する目的で培地組成の窒素源と炭素源の割合が異なるＩ〜ＩＶの４種を比較検討した。各培地５００ｍｌ三角フラスコ２本を用い、培養５日間後培養液を５倍濃縮に調製し、検定に用いた。 For the purpose of producing more antibacterial active ingredients of B medium I, which is a liquid medium, four types I to IV having different ratios of nitrogen source and carbon source in the medium composition were compared. Two 500 ml Erlenmeyer flasks were used for each medium, and after 5 days of culture, the culture solution was concentrated to 5 times and used for the assay.

（３）Ｂ培地Ｉ培養法
培地検討の結果よりＢ培地Ｉで精製用培養を行った。 (3) B medium I culture method Purified culture was performed on B medium I based on the results of medium examination.

Ｂ培地（グルコース５．０％、ポリペプトン０．５％、ＮａＣｌ０．８％、酵母エキス０．２％、炭酸カルシウム１．０％）５００ｍｌ三角フラスコに、１００ｍｌの培地を調製した。滅菌後植菌し、２８℃、５日間、振盪培養を行った。計１．５リットルの培養液と等量のアセトンを加え、５０％アセトン抽出液３リットルを得た。 B medium (glucose 5.0%, polypeptone 0.5%, NaCl 0.8%, yeast extract 0.2%, calcium carbonate 1.0%) A 100 ml medium was prepared in a 500 ml Erlenmeyer flask. After sterilization, the cells were inoculated and cultured at 28 ° C. for 5 days with shaking. A total of 1.5 liters of the culture solution and an equal amount of acetone were added to obtain 3 liters of 50% acetone extract.

（精製法）
（１）Ａ培地培養抽出物精製法
Ａ培地培養５０％アセトン抽出液２リットルのアセトン留去後、１リットルをＨＰ−２０−Sephadex カラム（60.0cm×4.5cm φ）に全量を通過吸着させた。等量の水で洗浄後、等量の５０％アセトン、次いで等量の１００％アセトンで溶離させた。それぞれの分画を検定菌Rhizoctonia solani（AG-1IA）で検定後、活性物質が１００％アセトン溶離部に溶離されていることを確認した。続いて１００％アセトン溶離部１リットルを濃縮し、1098mgの粗精製物を得て、その中の１００ｍｇを展開溶媒メタノール系のＬＨ−２０カラムにかけた（100.0cm×2.0cmφ）。分画をアッセイし、活性分画を確認した。これら活性分画を回収後シリカゲルＴＬＣ（酢酸エチル：酢酸：水＝５：１：１）で展開し、モリブデン硫酸呈色反応をしたところ、Ｒｆ＝０．３付近にスポットが検出された。同時に不活性分画の展開部にはこれらのスポットは検出されなかった。このことから活性物はＲｆ＝０．３付近に検出されるスポットと推定し、さらに精製を進めた。シリカゲルＰＴＬＣ展開（酢酸エチル：酢酸：水＝５：１：１）、かき取りメタノール抽出後、ＨＰＬＣを用いて単離精製をした。単離精製物は器機分析を実施し同定をした（図３１）。 (Purification method)
(1) A medium culture extract purification method A medium culture 50% acetone extract 2 liters of acetone was distilled off, and 1 liter was adsorbed through HP-20-Sephadex column (60.0 cm × 4.5 cm φ). . After washing with an equal volume of water, it was eluted with an equal volume of 50% acetone and then with an equal volume of 100% acetone. After each fraction was assayed with the assay bacterium Rhizoctonia solani (AG-1IA), it was confirmed that the active substance was eluted in the 100% acetone eluate. Subsequently, 1 liter of eluate with 100% acetone was concentrated to obtain 1098 mg of a crude product, and 100 mg of the crude product was applied to a developing solvent methanol-based LH-20 column (100.0 cm × 2.0 cmφ). Fractions were assayed to confirm active fractions. These active fractions were collected and then developed on silica gel TLC (ethyl acetate: acetic acid: water = 5: 1: 1) and subjected to a color reaction of molybdenum sulfate. As a result, a spot was detected around Rf = 0.3. At the same time, these spots were not detected in the developed part of the inactive fraction. From this, it was estimated that the active substance was a spot detected in the vicinity of Rf = 0.3, and further purification was advanced. After silica gel PTLC development (ethyl acetate: acetic acid: water = 5: 1: 1), scraping and methanol extraction, the product was isolated and purified using HPLC. The isolated and purified product was identified by performing instrumental analysis (FIG. 31).

（２）Ｂ培地Ｉ培養抽出物精製法
Ｂ培地培養５０％アセトン抽出液３リットルのアセトン留去後（１．５リットル）、活性炭吸着カラム（60.0cm×2.0cm φ）に全量１．５リットルを通過、吸着させた。等量の水で洗浄後、等量の５０％アセトン、続いて等量の１００％アセトンで溶離させた。各分画を検定菌Staphylococcus aureus 209Pを用いて検定した結果、１００％アセトン溶離部に活性物質が溶離されていることが確認された。１００％アセトン溶離部１．５リットルを濃縮し、酢酸エチルで分配抽出を行い、次いでメタノールを展開溶媒とするＬＨ−２０を実施し、ＨＰＬＣで単離精製を行った。単離精製物は機器分析を実施し同定をした（図３２）。 (2) B medium I culture extract purification method B medium culture 50% acetone extract 3 liters of acetone distilled off (1.5 liters), then activated carbon adsorption column (60.0 cm × 2.0 cm φ) 1.5 liters in total Passed through and adsorbed. After washing with an equal volume of water, it was eluted with an equal volume of 50% acetone followed by an equal volume of 100% acetone. As a result of assaying each fraction using the test bacterium Staphylococcus aureus 209P, it was confirmed that the active substance was eluted in the 100% acetone elution part. The eluate (1.5 liters) of 100% acetone was concentrated, partitioned and extracted with ethyl acetate, followed by LH-20 using methanol as a developing solvent, and isolated and purified by HPLC. The isolated and purified product was identified by performing instrumental analysis (FIG. 32).

（単離精製物生物活性法）
（１）Ａ培地単離精製物評価法
４成分の生物評価法
検定用培地にはＰＤＡ（Nissui） 1.3%、Choloramphenicol 0.002%の組成からなる培地を用いた。Rhizoctonia solani（AG-1IA）の菌糸の先端をコルクボーラーで抜いたものを平板培地の中央に乗せ、サイドに調製した単離物質を染み込ませたペーパーディスクを置き菌糸の伸長の様子を観察した。 (Isolated and purified biological activity method)
(1) A medium isolated and purified product evaluation method 4-component biological evaluation method A medium comprising PDA (Nissui) 1.3% and Choloramphenicol 0.002% was used as the assay medium. The hyphae of Rhizoctonia solani (AG-1IA), which had been extracted with a cork borer, was placed on the center of the plate medium, and a paper disk impregnated with the isolated substance was placed on the side to observe the state of hyphal elongation.

（２）Ｂ培地Ｉ単離精製物評価物
Ｃ成分の生物評価（ＭＷ１６８）
単離精製したＣ成分（MW:168）は新規物であり、きわめて単純な構造を保持している。母核としての興味が持たれた為生物評価を拡大して実施した。 (2) B medium I isolated and purified product evaluation biological evaluation of component C (MW168)
The isolated and purified component C (MW: 168) is novel and retains a very simple structure. The biological evaluation was expanded and carried out because of interest as a mother nucleus.

（３）バクテリア属の評価
Bioassay
試験菌を加えた寒天培地をシャーレに入れ固める。その上に試料を含むペーパーディスクをのせ、37℃で18時間培養した後、発育阻止円の形成を確認する。 (3) Evaluation of bacteria
Bioassay
Put the agar medium with the test bacteria in a petri dish and harden. A paper disk containing the sample is placed on it and cultured at 37 ° C. for 18 hours, and then the formation of a growth inhibition circle is confirmed.

（ａ）使用菌株
Staphylococcus aureus 209P、Pseudomonas syringal（タバコ野火病菌）、Xanthomonoas Campestris pv.citri（カンキツかいよう病）、Erwinia sp（ウメかいよう病）の４菌株を用いた。 (A) Strain used
Four strains were used: Staphylococcus aureus 209P, Pseudomonas syringal (tobacco wildfire fungus), Xanthomonoas Campestris pv.citri (citrus canker), Erwinia sp (plum canker).

（ｂ）使用培地
前培養にはブイヨン培地（DIFCO）を用い、一晩培養後、×10²に希釈し、これを上層の培地０．５％に混ぜ、菌測定培地には MYCIN AGAR （ミクニ化学）を用いた。 (B) Medium used Bouillon medium (DIFCO) was used for preculture, and after overnight culture, diluted to 10 ² , mixed with 0.5% of the upper medium, and MYCIN AGAR (Mikuni Chemistry) was used.

上層 − MYCIN AGAR 1.5%（ミクニ化学）＋ブイヨン２％培地（DIFCO）にて一晩培養した培養液×10²に希釈したものを０．５％加える。 Upper layer—Add 0.5% of MYCIN AGAR 1.5% (Mikuni Chemical) + Broth 2% culture medium (DIFCO) cultured overnight × 10 ² diluted.

下層 − MYCIN AGAR 2.0%（ミクニ化学） Lower layer-MYCIN AGAR 2.0% (Mikuni Chemical)

（ｃ）抗菌測定
ペーパーディスクに1000ppm,500ppm,250ppmに調製した単離精製物を染み込ませ、風乾後シャーレに乗せ、37℃で18時間培養し、発育阻止円の形成の有無を観察した。 (C) Antibacterial measurement The isolated and purified products prepared at 1000 ppm, 500 ppm, and 250 ppm were soaked in paper discs, air-dried, placed on a petri dish, cultured at 37 ° C. for 18 hours, and the presence or absence of growth-inhibiting circles was observed.

（４）黄色ブドウ球菌に対する抗菌力（ＭＩＣ）の測定
（ａ）使用菌株
S.aureusu 209P JC−1，第１Ｇ保存の臨床分離黄色ブドウ球菌２０株（ＭＳＳＡ，ＭＲＳＡ各１０株）及び基準菌株である E.coli NIHJ JC−2の計２２株を用いた。 (4) Measurement of antibacterial activity against Staphylococcus aureus (MIC) (a) Strains Used
A total of 22 strains of S. aureusu 209P JC-1, 20 clinical isolates of Staphylococcus aureus (MSSA, MRSA each 10 strains) preserved in the 1G and the reference strain E. coli NIHJ JC-2 were used.

（ｂ）使用抗菌薬
MW:168、methicllin（DMPPC，注射用スタフシリン、Lot.No.FSB 19,900μg/mg、萬有製薬）、vancomycin （VCM,Lot.No.41H0457,10750SIGMA）
（ｃ）使用培地
抗菌力測定にはMueller-Hinton agar （MIA:Difco）を用い前培養には、Mueller Hinton broth（MHB:Difco）を用いた。 (B) Antibacterial drugs used
MW: 168, methicllin (DMPPC, Stafcillin for injection, Lot.No.FSB 19,900μg / mg, Ariyu Pharmaceutical), vancomycin (VCM, Lot.No.41H0457,10750SIGMA)
(C) Medium used Mueller-Hinton agar (MIA: Difco) was used for antibacterial activity measurement, and Mueller Hinton broth (MHB: Difco) was used for preculture.

（ｄ）抗菌力測定
使用菌株に対する各薬剤の最小発育阻止濃度（ＭＩＣ）は日本化学療法学会標準法に準拠し、寒天平板希釈法にて測定した。菌株はＭＨＡに塗抹し、３７℃で一晩培養して生育したコロニーをＭＨＢにて３７℃で一晩培養し、その菌液を１００倍希釈（E.Coliのみ１０００倍希釈）したものを接種菌液とした。 (D) Antibacterial activity measurement The minimum inhibitory concentration (MIC) of each drug against the strain used was measured by the agar plate dilution method in accordance with the standard method of the Japanese Society of Chemotherapy. The strain is smeared on MHA, and the colony grown by overnight culture at 37 ° C is cultured overnight at 37 ° C in MHB, and the bacterial solution is diluted 100 times (only E. Coli is diluted 1000 times). Bacterial fluid was used.

（結果）
（１）検定菌の結果
Ａ培地とＢ培地Ｉの培養液活性は表２の通りである。
抗菌スペクトラムを下記の検定菌を用いて測定した。

(result)
(1) Results of Test Bacteria The culture medium activities of A medium and B medium I are as shown in Table 2.
The antibacterial spectrum was measured using the following test bacteria.

Ａ培地の培養抽出液中の検定の結果、Rhizoctonia solani（AG-1IA）の菌糸がＡ培地培養抽出物のペーパーディスクを避けている様子が観察された。本菌が生産するＡ培地培養液中の活性物が、菌糸の成長を妨げる物質を生産していると推察した。このためＡ培地培養液中より、Rhizoctonia solani（AG-1IA）に阻害活性を示す物質を指標に単離精製を試みた。 As a result of the assay in the culture medium A extract, it was observed that the mycelium of Rhizoctonia solani (AG-1IA) avoided the paper disk of the A medium culture extract. It was inferred that the active substance in the medium A culture medium produced by this bacterium produced a substance that hinders mycelial growth. For this reason, isolation and purification were attempted from the medium A culture medium using as an index a substance exhibiting inhibitory activity against Rhizoctonia solani (AG-1IA).

またＢ培地Ｉの培養抽出液中の検定の結果、Botrytis cinerea、Rhizoctoniasolaniに活性は確認されず、バクテリアにのみ発育阻止円の形成が確認された。このことは、本菌は拮抗作用以外の抗菌力があると見られ、抗生物質を生産している可能性が考えられた。特にStaphylococcus aureus 209Pに形成された発育阻止円はクリアーであった為、Ｂ培地ＩではバクテリアStaphylococcus aureus 209Pに対する活性物質を指標に単離精製を実施することにした。Ａ培地培養物とＢ培地Ｉ培養物の抗菌スペクトラムは異なっており、それぞれ系統の異なるものを生産していると思われた。それぞれの活性物を単離精製することとした。 Further, as a result of the assay in the culture extract of B medium I, the activity was not confirmed in Botrytis cinerea and Rhizoctoniasolani, and the formation of growth inhibition circles was confirmed only in bacteria. This indicates that this bacterium has antibacterial activity other than antagonism and may produce antibiotics. In particular, since the growth inhibition circle formed in Staphylococcus aureus 209P was clear, it was decided to carry out isolation and purification in B medium I using the active substance for bacteria Staphylococcus aureus 209P as an index. The antibacterial spectrum of the A medium culture and the B medium I culture were different, and it seemed that they produced different strains. Each active product was isolated and purified.

（２）培養の結果
（ａ）Ａ培地培養結果
Ａ培地（米培地１kg）５００ｍｌの三角フラスコ１０本分を２８℃で１０日間、静置培養し、培養物５０％アセトン抽出液２リットルを得た。 (2) Culture results (a) A medium culture results A medium (1 kg of rice medium) Ten 500 ml Erlenmeyer flasks were statically cultured at 28 ° C. for 10 days to obtain 2 liters of 50% acetone extract of the culture. It was.

（ｂ）Ｂ培地検討と培養結果
表１に表したＢ培地ＩとＢ培地ＩＩに活性が認められ、Ｂ培地Ｉにより強い活性が認められた本菌は、窒素源が少ない条件で活性物を生産することが確認された。この結果よりＢ培地Ｉを用いて２８℃で５日間、振盪培養をした。 (B) B medium examination and culture results The B medium I and B medium II shown in Table 1 showed activity, and the B cell I showed strong activity in B medium I. Production was confirmed. From this result, shaking culture was performed using B medium I at 28 ° C. for 5 days.

（３）精製結果
（ａ）Ａ培地培養精製結果
シリカゲルＰＴＬＣ処理後の活性分画を粗精製物として、ＨＰＬＣ（0.05%TFA含 MeOH/H₂O）で純度の確認を行った。分析では０分から３０分にかけてメタノール０％から１００％のグラジエントをかけ、さらに３０分から１００％メタノールで流速１ml/minで分析したところ、ＲＴ（リテションタイム）32.5分（Fr65）で検出された（図１）。一見、単一物質のピークが検出されたかのように思われたが、拡大することにより複数ピークの存在が明らかとなった（図２）。ＲＴの速い方からＡ，Ｂ，Ｃ，Ｄ，Ｅと称し（図２）、そのうちの４成分を単離精製した。単離精製物の成分と量は、Ａ成分 7.2mg，Ｂ成分12.1mg，Ｄ成分 3.5mg，Ｅ成分 4.5mgであった。ピークＣは量が少量であり、精製は断念した。 (3) Purification result (a) A culture medium purification result The purity was confirmed by HPLC (0.05% TFA-containing MeOH / H ₂ O) using the active fraction after silica gel PTLC treatment as a crude product. In the analysis, a gradient from 0% to 100% methanol was applied from 0 to 30 minutes, and an analysis was performed at a flow rate of 1 ml / min from 30 minutes to 100% methanol. RT (retention time) was detected at 32.5 minutes (Fr65) ( FIG. 1). At first glance, it seemed as if a single substance peak was detected, but the presence of multiple peaks became apparent by enlargement (FIG. 2). From the fastest RT, they were called A, B, C, D, and E (FIG. 2), and four of them were isolated and purified. The components and amount of the isolated and purified product were A component 7.2 mg, B component 12.1 mg, D component 3.5 mg, and E component 4.5 mg. Peak C was a small amount and purification was abandoned.

（ｂ）Ｂ培地Ｉ培養精製結果
それぞれの分画のアッセイより活性Fr41〜54に活性があることを確認し回収した。ＨＰＬＣ（0.05%TFA含 MeOH/H₂O）分取、分析を行い検出されたピークのＲＴの速い方からＡ，Ｂ，Ｃ，Ｄ，Ｅと数え、５成分の存在を確認した。このうちＣ成分 8.1mg単離精製することが出来た。 (B) Culture medium I culture purification result It was confirmed that the active Fr41 to 54 had activity from the assay of each fraction and recovered. HPLC (0.05% TFA-containing MeOH / H ₂ O) was collected and analyzed, and the detected peaks were counted as A, B, C, D, E from the fastest RT, and the presence of 5 components was confirmed. Of these, 8.1 mg of component C was isolated and purified.

（４）機器分析の結果
（ａ）Ａ培地単離精製物
ＵＶ吸収スペクトラム、質量分析及びＮＭＲ測定結果（図３〜１４）より、Aib（α-Aminoisobutyric acid）を含むPolypeptideの、Peptibols系と判断した。Peptibols系抗生物質はアミノ酸配列の中に α-Aminoisobutyric acid（Aib）を含んでおり、Ｎ末端がAcetyl基で、Ｃ末端が Amino alcohol結合であることが特徴である（多くは Phenylalaniol基でおわる）。なお今回の精製物、Ａ成分、Ｂ成分、Ｄ成分は質量分析より一部のアミノ酸配列を下記のように推定し、その部分構造を既存のPeptibols のアミノ酸配列と検索した結果、一致する物が無く、新規 Peptibols系であると推定した。Ｅはアミノ酸配列の推定が困難であるため判断できなかった。 (4) Results of instrumental analysis (a) A medium isolated and purified product Based on UV absorption spectrum, mass spectrometry and NMR measurement results (FIGS. 3 to 14), Polypeptide containing Aib (α-Aminoisobutyric acid) is judged as a Peptibols system did. Peptibols antibiotics contain α-Aminoisobutyric acid (Aib) in the amino acid sequence, and are characterized by an N-terminal Acetyl group and a C-terminal Amino alcohol bond (mostly Phenylalaniol groups) . As for the purified product, A component, B component, and D component, the partial amino acid sequence was estimated by mass spectrometry as follows, and the partial structure was searched with the existing amino acid sequence of Peptibols. There was no new Peptibols system. E could not be determined because it was difficult to estimate the amino acid sequence.

ＵＶ吸収波長：４成分とも末端吸収（図３、図６、図９、図１２）
Ａ成分＝１９３３（図５）
Ｂ成分＝１９４９または１９６４（図８）
Ｄ成分＝１８１０（図１１）
Ｅ成分＝１８２９（図１４）
推定構造
Ａ成分：Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib-...
Ｂ成分：Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib-...
Ｄ成分：Ac-Aib-Ala-Aib-Aib-Aib-... UV absorption wavelength: terminal absorption of all four components (FIGS. 3, 6, 9, and 12)
A component = 1933 (FIG. 5)
B component = 1949 or 1964 (FIG. 8)
D component = 1810 (FIG. 11)
E component = 1829 (Fig. 14)
Presumed structure A component: Ac-Aib-Ala-Aib-Aib-Aib-Aib-Gln-Aib-Aib -...
B component: Ac-Aib-Ala-Aib-Aib-Val-Aib-Gln-Aib-Aib -...
D component: Ac-Aib-Ala-Aib-Aib-Aib -...

（ｂ）Ｂ培地Ｉ（液体培地）活性物
機器分析の結果Ｃ成分は、分子量１６８（C₈H₈O₄）の新規物質であることが推定された。他の成分について単離精製は非常に困難であり、不可能と判断したが、LC/MS を実施した。Ａ成分のＵＶ吸収波長は末端であった（図１５）。ＨＰＬＣの条件はカラムCapcel pac C18 UG120 （2×150mm）を用い、１％酢酸：アセトニトリル＝９８：２、流速0.2ml/min、温室の条件で分析した。Ａ成分は、ＲＴ７．４８分に検出された（図１６）。Ａ成分が検出された分画の質量分析の結果、分子量は１９２であることを推定した（図１７）。同じくＢ成分についても同様の分析をしたところＢ成分もＵＶ吸収波長が末端（図１８）で、ＨＰＬＣのＲＴ１２、４５分に検出された（図１９）。質量分析の結果、分子量は２０６であることを推定した（図２０）。Ｃ成分は単離精製が可能であった分画である。Ｃ成分のＵＶ吸収波長は２７０ｎｍ（図２１）で、ＨＰＬＣのＲＴは１５．９２分に検出された（図２２）。質量分析の結果、分子量は１６８であることを推定した（図２３、２４）。また溶媒CD₃ODで溶解し、ＮＭＲを測定（図２５）、解析したところ五員環を含み、共役結合をもつ新規物質であることが推定された。推定構造を図２６に示した。Ｄ成分はＵＶ吸収波長が２３０ｎｍ付近にあり（図２７）、ＨＰＬＣのＲＴは３０．２８分に検出された（図２８）。しかし再度ＨＰＬＣを実施し、ＵＶスペクトラム検出器の波長を末端と２３０ｎｍで分析した結果、ＲＴ０．６分の差で２成分（Ｄ１，Ｄ２）存在していることが確認された（図２９）。それぞれの質量分析の結果、ＲＴの速いＵＶ吸収が２３０ｎｍのものは分子量１５４であった（Ｄ１）。たまＲＴが０．６分遅れて検出される成分の分子量は２２０であることを推定した（Ｄ２）（図３０）。これらの分子量の明らかとなった成分を分子量の少ない順にならべると１５４（Ｄ１），１６８（Ｃ），１９２（Ａ），２０６（Ｂ），２２０（Ｄ２）であることが確認された。 (B) B medium I (liquid medium) active substance As a result of instrumental analysis, it was estimated that component C is a novel substance having a molecular weight of 168 (C ₈ H ₈ O ₄ ). Although it was judged that isolation and purification of other components were very difficult and impossible, LC / MS was performed. The UV absorption wavelength of component A was terminal (FIG. 15). The HPLC was performed using a column Capcel pac C18 UG120 (2 × 150 mm), 1% acetic acid: acetonitrile = 98: 2, flow rate 0.2 ml / min, and greenhouse conditions. A component was detected at RT 7.48 minutes (FIG. 16). As a result of mass spectrometry of the fraction in which the A component was detected, it was estimated that the molecular weight was 192 (FIG. 17). Similarly, the same analysis was performed on the B component, and the B component was detected at RT 12, 45 minutes of HPLC (FIG. 19) with the UV absorption wavelength at the end (FIG. 18). As a result of mass spectrometry, it was estimated that the molecular weight was 206 (FIG. 20). Component C is a fraction that could be isolated and purified. The UV absorption wavelength of component C was 270 nm (FIG. 21), and HPLC RT was detected at 15.92 minutes (FIG. 22). As a result of mass spectrometry, it was estimated that the molecular weight was 168 (FIGS. 23 and 24). The dissolved in a solvent CD ₃ OD, measured NMR (FIG. 25), includes a five-membered ring was analyzed, it was estimated that a novel substance having a conjugated bond. The estimated structure is shown in FIG. The D component had a UV absorption wavelength around 230 nm (FIG. 27), and HPLC RT was detected at 30.28 minutes (FIG. 28). However, HPLC was performed again, and the wavelength of the UV spectrum detector was analyzed at 230 nm from the end. As a result, it was confirmed that two components (D1, D2) were present with a difference of RT 0.6 minutes (FIG. 29). As a result of the respective mass spectrometry, those having a fast UV absorption at RT of 230 nm had a molecular weight of 154 (D1). It was estimated that the molecular weight of the component detected with a delay of RT 0.6 minutes was 220 (D2) (FIG. 30). It was confirmed that these components with the molecular weights revealed were 154 (D1), 168 (C), 192 (A), 206 (B), and 220 (D2) when arranged in descending order of molecular weight.

（５）生物評価結果
（ａ）Ａ培地培養単離精製物生物評価結果
４成分の生物評価結果
Ａ，Ｂ，Ｃ，Ｄ成分について5000ppmから希釈し、5000ppm，2500ppm，1250ppm，625ppmで検定した。結果Ａ成分、Ｅ成分は1250ppmまで Rhizoctonia solaniの菌糸の伸長を阻害しおり、Ｂ成分、Ｄ成分は625ppmまでRhizoctonia solaniの菌糸の伸長を阻害しているのが確認された。 (5) Biological evaluation result (a) A culture medium isolated and purified product Biological evaluation result Biological evaluation result of 4 components The components A, B, C, and D were diluted from 5000 ppm and tested at 5000 ppm, 2500 ppm, 1250 ppm, and 625 ppm. As a result, it was confirmed that the A component and the E component inhibited the mycelial elongation of Rhizoctonia solani up to 1250 ppm, and the B component and the D component inhibited the mycelial elongation of Rhizoctonia solani up to 625 ppm.

（ｂ）Ｂ培地Ｉ培養単離精製物生物評価結果
（イ）バクテリア属の評価結果
Ｂ培養物（MW168）のバクテリアに対する生物活性は発育阻止円が形成されていなかったことから抗菌力は無いか、弱いものと考えられた。 (B) B culture medium I culture isolated and purified product biological evaluation results (b) Bacterial genus evaluation results Is the B culture (MW168) biological activity against bacteria free of anti-bacterial activity because no growth inhibition circle was formed? It was considered weak.

（ロ）黄色ブドウ球菌に対する抗菌力（ＭＩＣ）の測定結果は表３の通りである。 (B) measurement of the antibacterial against Staphylococcus aureus (MIC) are shown in Table 3.

表３に示したようにＢ培養物（MW168）は今回測定した濃度において抗菌力を示さず、黄色ブドウ球菌に対する抗菌力は極めて弱いものと考えられた。

B cultures as indicated in Table 3 (MW168) showed no antibacterial activity at the concentrations currently measured, antibacterial against Staphylococcus aureus was considered weak Te order pole.

考察Ａ培地培養物より単離精製した成分からは、Ａｉｂ（α-Aminoisobutyric acid）を含むPeptibols系のもの４成分を単離することが出来た。Trichodermaが生産する Peptibols系のものは幾つかすでに報告されているが、今回単離精製したものは、それらのものとは一致しなかった。また、それぞれの成分のアミノ酸配列の結果、３成分が新規の配列であることが推定された（Ｅ成分のみ配列を測定するのが不可能だった）。さらにin vitro試験ではRhizoctonia solani（AG-1IA）の菌糸を阻害している様子が観察された。この結果から本菌の米培養抽出物中にRhizoctonia solani（AG-1IA）に対する菌糸伸長阻害物質が生産されていることが明らかとなった。本菌の拮抗作用である相手菌糸との接触により発現する細胞質凝集を促し死滅させる物質そのものであるという断言はまだ出来ないが、本菌の米培養抽出物中に菌糸成長伸長抑制物が生産されていることは明らかである。 Discussion From the components isolated and purified from the culture medium A, 4 components of Peptibols containing Aib (α-Aminoisobutyric acid) could be isolated. Several Peptibols strains produced by Trichoderma have already been reported, but those isolated and purified this time did not match those. Moreover, as a result of the amino acid sequence of each component, it was estimated that 3 components were novel sequences (it was impossible to measure the sequence of only the E component). Furthermore, in vitro studies showed that the mycelium of Rhizoctonia solani (AG-1IA) was inhibited. From these results, it was revealed that a mycelial elongation inhibitory substance against Rhizoctonia solani (AG-1IA) was produced in the rice culture extract of this bacterium. Although it is not yet possible to assert that the substance itself is a substance that promotes and kills cytoplasmic aggregation that is expressed by contact with the partner mycelium, which is an antagonistic action of this fungus, a mycelium growth elongation inhibitor is produced in the rice culture extract of this fungus. It is clear that

またＢ培地Ｉ培養より単離精製した成分はMW:168の新規物（3(3-hydroxy-cyclopropene 5-one-2yl)2-propenoic asid）であり、Bioassay を幾つか実施したが活性は無いか、きわめて弱いものと判断した。おそらく活性本体はMW:168周辺化合物であり、MW:168は活性物の副成物であると推測している。またMW:168周辺化合物の分子量はMW:154、MW:192、MW:206、MW:220であり、分子量の差がそれぞれ１４であることがわかる。これはメチル基一個分と同量であり、メチル基の増減による活性の違いも考えられる。またMW:168は新規な構造を保持しているが、構造検索より類似していた化合物が幾つか報告されていた。その中でMW:168に一番近い構造であったもの（図３３）は、鳥取大学のＰＧＲ活性スクリーニングで見出されたものであり、糸状菌Penicillium valiabile SOPPより代謝され単離精製されたと報告され、Ｃ₈Ｈ₈Ｏ₅（MW:198）の組成式で表されている新規物質であるが、活性は弱いものと報告されている。 In addition, the component isolated and purified from B medium I culture is a new product of MW: 168 (3 (3-hydroxy-cyclopropene 5-one-2yl) 2-propenoic asid). It was judged that it was extremely weak. Presumably, the active body is a peripheral compound of MW: 168, and MW: 168 is a by-product of the active substance. The molecular weights of the MW: 168 peripheral compounds are MW: 154, MW: 192, MW: 206, and MW: 220, and the difference in molecular weight is 14 respectively. This is the same amount as that of one methyl group, and a difference in activity due to increase or decrease of the methyl group is also considered. In addition, although MW: 168 has a new structure, several similar compounds have been reported by structure search. Among them, the structure closest to MW: 168 (Fig. 33) was found by PGR activity screening at Tottori University, and was reported to be isolated and purified from the filamentous fungus Penicillium valiabile SOPP. Although it is a novel substance represented by the composition formula of C ₈ H ₈ O ₅ (MW: 198), its activity is reported to be weak.

この発明の菌のＡ培地培養におけるＨＰＬＣ分析グラフ。The HPLC analysis graph in A culture medium culture | cultivation of the microbe of this invention. 同じく図１の拡大グラフ。The enlarged graph of FIG. 同じくＡ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of A component. 同じくＡ成分の¹Ｈ−ＮＭＲ（ＣＤ₃ＯＤ）を示すグラフ。Similarly graph showing the ^{_{1 H-NMR (CD 3 OD}} ) of the A component. （ａ）、（ｂ）同じくＡ成分の質量分析測定のグラフ。(A), (b) The graph of the mass spectrometry measurement of A component similarly. 同じくＢ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of B component. 同じくＢ成分の¹Ｈ−ＮＭＲを示すグラフ。The graph which similarly shows ¹ H-NMR of B component. （ａ）、（ｂ）同じくＢ成分の質量分析測定のグラフ。(A), (b) The graph of the mass spectrometry measurement of B component similarly. 同じくＤ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of D component. 同じくＤ成分の¹Ｈ−ＮＭＲを示すグラフ。Similarly graph showing the ¹ H-NMR of the D component. （ａ）、（ｂ）同じくＤ成分の質量分析測定のグラフ。(A), (b) The graph of the mass spectrometry measurement of D component similarly. 同じくＥ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of E component. 同じくＥ成分の¹Ｈ−ＮＭＲを示すグラフ。Similarly graph showing the ¹ H-NMR of E component. （ａ）、（ｂ）同じくＥ成分の質量分析測定のグラフ。(A), (b) The graph of the mass spectrometry measurement of E component similarly. 同じくＡ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of A component. 同じくＡ成分のＨＰＬＣを示すグラフ。The graph which similarly shows HPLC of A component. 同じく図１６の検出されたＡピークの質量分析のグラフ。The graph of the mass spectrometry of the detected A peak of FIG. 同じくＢ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of B component. 同じくＢ成分のＨＰＬＣを示すグラフ。The graph which similarly shows HPLC of B component. 同じく図１９の検出されたＡピークの質量分析のグラフ。20 is a graph of mass spectrometry of the detected A peak in FIG. 19. 同じくＣ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of C component. 同じくＣ成分のＨＰＬＣを示すグラフ。The graph which similarly shows HPLC of C component. 同じく図２２の検出されたＡピークの質量分析のグラフ。22 is a graph of mass spectrometry of the detected A peak in FIG. 同じくＣ成分の質量分析グラフ。Similarly, mass analysis graph of C component. 同じくＣ成分のピークの¹Ｈ−ＮＭＲ（ＣＤ₃ＯＤ）を示すグラフ。Similarly graph showing the ¹ H-NMR peaks of the component C (CD ₃ OD). 同じくＣ成分の構造図。The structural drawing of C component similarly. 同じくＤ成分のＵＶ吸収波長を示すグラフ。The graph which similarly shows the UV absorption wavelength of D component. 同じくＤ成分のＨＰＬＣを示すグラフ。The graph which similarly shows HPLC of D component. （ａ）同じくＵＶλ２３０ｎｍで検出されたＤ１成分を示すグラフ、（ｂ）同じくＵＶが末端吸収でＤ１成分とＲＴ０．６差で検出されたＤ２成分を示すグラフ。(A) The graph which shows D1 component similarly detected by UV (lambda) 230nm, (b) The graph which shows D2 component similarly detected by RT0.6 difference with D1 component by UV terminal absorption. （ａ）同じく図２９で検出されたＤ１成分の質量分析を示すグラフ、（ｂ）同じく図２９で検出されたＤ２成分の質量分析を示すグラフ。(A) The graph which similarly shows the mass spectrometry of D1 component detected in FIG. 29, (b) The graph which shows the mass spectrometry of D2 component similarly detected in FIG. 同じく米培地の培養抽出物精製経過の系統図。The systematic diagram of the culture extract refinement | purification process of a rice culture medium. 同じくＢ培地Ｉの培養抽出物精製経過の系統図。The systematic diagram of the culture extract refinement | purification process of B culture medium I similarly. 同じく新規物質（ＭＷ：１９８）１、２、３の誘導体の構造図。The structure figure of the derivative | guide_body of the novel substance (MW: 198) 1,2,3 similarly.