JPH04166080A - Novel bacillus subtilis subspecies and fungicidal substance krf-001 complex produced therefrom - Google Patents

Novel bacillus subtilis subspecies and fungicidal substance krf-001 complex produced therefrom

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Publication number
JPH04166080A
JPH04166080A JP2336898A JP33689890A JPH04166080A JP H04166080 A JPH04166080 A JP H04166080A JP 2336898 A JP2336898 A JP 2336898A JP 33689890 A JP33689890 A JP 33689890A JP H04166080 A JPH04166080 A JP H04166080A
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JP
Japan
Prior art keywords
krf
component
bacillus subtilis
2cooh
antifungal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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JP2336898A
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Japanese (ja)
Inventor
Song-Hae Bok
卜　成海
Song-Uk Kim
金　成郁
Kwang-Hui Son
孫　光▲き▼
Song-Ki Kim
金　成淇
Young-Kuk Kim
永國 金
Hang W Lee
李　項雨
Jee W Lee
李　知雨
Hye-Kyong Kwon
権　恵敬
Tae Suk Jeong
鄭　泰淑
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Korea Research Institute of Chemical Technology KRICT
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Korea Research Institute of Chemical Technology KRICT
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Publication of JPH04166080A publication Critical patent/JPH04166080A/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Storage Of Fruits Or Vegetables (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

Abstract

NEW MATERIAL: Bacillus subtilis subsp. Krictiensis ATCC 55079 capable of producing peptide antifungal complex.
USE: Production of low-toxic/non-pollutant new antifungal complex KRF-001 useful as e.g. a therapeutic agent for fungal infectious diseases.
PREPARATION: This new microorganism is isolated by means of any known method from the soil in Korea.
COPYRIGHT: (C)1992,JPO

Description

【発明の詳細な説明】 〔産業上の利用分野〕 本発明は新規なバシラス属微生物及びこれから生産され
る抗生物質に関するものである。更に、具体的に、本発
明は土壌試料から採取分離の新規なバシラス・サブチリ
ス亜種（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ　５ｕｂｓ
ｐｅｃｉｅｓ　）とその変異株およびそれらを培養また
は醗酵させて生産した新規な抗真菌物質およびこの真菌
感染症治療剤、皮膚保湿剤、保存剤および殺真菌剤等と
しての用途に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel microorganism of the genus Bacillus and an antibiotic produced therefrom. Furthermore, specifically, the present invention provides a novel Bacillus subtilis subspecies collected and isolated from soil samples.
pecies) and its mutants, novel antifungal substances produced by culturing or fermenting them, and their uses as therapeutic agents for fungal infections, skin moisturizers, preservatives, fungicides, etc.

〔従来の技術〕[Conventional technology]

最近、数十年間有機合成の生物学的活性物質等か医薬と
農薬等多方面に広く用いられるに伴い、人体と環境とに
深刻な弊害を与えているため、あらゆる研究者等により
、その生物学的活性を保ちなからも、低毒性乃至は無公
害性の天然化合物を探索開発しようとする試みもあった
。その結果、自然界特に微生物資源から医薬や農薬等の
応用の可能な、種々の天然物質が発見され、そのまま或
いは半合成の変形された形態て力価か高いなからも、そ
の特性や環境的な汚染の少ない物質が実用化されるのが
可能となり、今日薬害問題や農作物への残留毒性問題、
環境汚染問題を解決し得る突破口となって来た。Recently, over the past few decades, organically synthesized biologically active substances have been widely used in various fields such as medicine and agrochemicals, causing serious harm to the human body and the environment. There have also been attempts to explore and develop natural compounds that have low toxicity or are non-polluting while still maintaining scientific activity. As a result, various natural substances have been discovered from nature, especially from microbial resources, that can be used as medicines and agricultural chemicals. It has become possible to put substances with less contamination into practical use, and today problems such as phytotoxicity, residual toxicity in agricultural crops, etc.
This has become a breakthrough in solving environmental pollution problems.

今まで、抗真菌物質は既に相当な数か微生物から発見さ
れ農薬と医薬に広く利用されている。例を挙げれば、グ
リセオフウルビン（ｇｒｉｓｅｏｆｕｌｖｉｎ）か１９
３９９３９年オフスフオードｆｏｒｄ、　Ａ、　Ｅ）等
によって、ペニシリュウム・グリセオフウルブオム（Ｐ
ｅｎｉｃｉｌｌｉｕｍ　ｇｒｉｓｅｏｆｕｌｖｕｍ）の
培養液から分離され（Ｏｘｆｏｒｄ、Ａ、Ｅ、、　Ｒａ
１ｓｔｒｉｃｋ、Ｈ，、Ｓｉｍｏｎａｒｔ、Ｐ。To date, a considerable number of antifungal substances have been discovered from microorganisms and are widely used in pesticides and medicines. For example, griseofulvin (19)
39939 Penicillium griseophorubum (P. Ford, A., E.) et al.
enicillium griseofulvum) (Oxford, A.E., Ra.
1strick, H., Simonart, P.

（１９３９）　、Ｂｉｏｃｈｅｍ、Ｊ、　　３３　：　
２４０参照〕始めには農薬として開発されたが、その後
皮膚糸状菌症に効果的な経口剤として開発され、現在も
広く利用されているのは、その代表的な例であり、この
他にも１９５９年ハセン（Ｈａｚｅｎ、　Ｅ、　［、）
とブラウン（Ｂｒｏｗｎ、　Ｒ，）によってニスタチン
（ｎｙｓｔａｔｉｎ）（Ｈａｚｅｎ、Ｅ、Ｌ、ａｎｄ　
Ｂｒｏｗｎ、Ｒ，（１９５０）　、５ｃｉｅｎｃｅ。(1939), Biochem, J, 33:
[Refer to 240] A typical example of this is that it was first developed as a pesticide, but was later developed as an oral agent effective against dermatophytosis, and is still widely used today. 1959 Hazen (Hazen, E, [,)
Nystatin (Hazen, E, L, and
Brown, R. (1950), 5science.

１１２：４２３参照〕か、１９５５年ゴールド（Ｇｏｌ
ｄ、　Ｗ）等によってエムボテリシンＢ（ａｍｐｈｏｔ
ｅｒ　１ｃｉｎ　Ｂ）　［Ｇｏｌｄ、　Ｗ、　、　５ｔ
ｏｕｔ、　Ｈ，Ａ、　、　Ｐａｇａｎｏ。112:423] or the 1955 Gol
Embotericin B (amphot
er 1cin B) [Gold, W, , 5t
out, H, A, , Pagano.

Ｊ、Ｐ、ａｎｄ　Ｄｏｎｏｖｉｃｋ、Ｒ，（１９５５）
　、　Ａｎｔｉｂｉｏｔ、Ａｎｎ、。J. P. and Donovick, R. (1955).
, Antibiot, Ann.

１９５６：５７９参照］が各々放線菌等から発見され真
菌症に対する化学療法が確率されるようになった。1956:579] were discovered in actinomycetes, etc., and chemotherapy became available for mycoses.

しかし、細菌感染の化学療法が相当多く発展をして来た
ことに比すれば、真菌症の化学療法はその発展速度が緩
慢な実情であり、長期的に副作用か少なくケンシタ（Ｃ
ａｎｄｉｄａ）やアスパージラス（Ａｓｐｅｒｇｉｌｌ
ｕｓ）等いろいろな真菌類に対して効果的な広範囲抗真
菌剤の開発か要求されている。However, compared to the considerable development of chemotherapy for bacterial infections, the development of chemotherapy for fungal diseases has been slow, and in the long term it has fewer side effects and fewer side effects.
andida) and Aspergill
There is a need for the development of a broad-spectrum antifungal agent that is effective against various fungi such as U.S.

一方、農業用抗真菌物質の開発は有機合成剤農薬か主流
を成していた１９６０年代初頭から日本を中心として成
され、その間稲熱病に対するプラスチシジン５（ｂｌａ
ｓｔｉｃｉｄｉｎ　Ｓ）　［Ｔａｋｅｕｃｂｉ、　Ｓ、
　。On the other hand, the development of agricultural antifungal substances began in the early 1960s, when organic synthetic pesticides were the mainstream.
sticidin S) [Takeucbi, S,
.

Ｈｉｒａｙａｍａ、　Ｋ、　、　Ｕｅｄａ、　Ｋ、　、
　５ａｋａｉ、　Ｋ、　ａｎｄ　Ｙｏｎｅｈａｒａ、　
Ｈ。Hirayama, K., Ueda, K.,
5akai, K. and Yonehara,
H.

（１９５８）　、Ｊ、Ａｎｔｉｂｉｏｔｉｃｓ、　１１
Ａ　：　１−５参照］とカスがマイシン（Ｋａｓｕｇａ
ｍｙｃｉｎ）　［Ｕｍｅｚａｗａ、Ｈ，。(1958), J. Antibiotics, 11
A: 1-5] and Kasugamycin (see A: 1-5)
mycin) [Umezawa, H.

Ｏｋａｍｉ、　Ｙ、　、　Ｈｉｓｈｉｍｏｔｏ、　Ｔ、
　５ｕｈａｒａ、　Ｙ、　、　Ｈａｍａｄａ、　Ｍ、　
ａｎｄＴａｋｅｕｃｈｉ、Ｔ、　　（１９０５）　、　
Ｊ、Ａｎｔｉｂｉｏｔｉｃｓ、　１８・１０１−１０８
参照１．紋枯病に対するポリオツクシン（ｐｏｌｙｏｘ
ｉｎ）　［［５ｏｎｏ、　Ｋ、　、　Ｎａｇａｔｓｕ、
　Ｊ、　。Okami, Y., Hishimoto, T.
5uhara, Y., Hamada, M.
and Takeuchi, T. (1905),
J. Antibiotics, 18, 101-108.
Reference 1. Polyoxin against sheath blight
in) [[5ono, K, , Nagatsu,
J.

Ｋａｗａｓｈｉｍａ、Ｙ、ａｎｄ　５ｕｚｕｋｉ、Ｓ、
　　（１９６５）　。Kawashima, Y., and 5uzuki, S.
(1965).

Ａｇｒｉｃ、Ｂｉｏｌ、Ｃｈｅｍ、、　　２９　：　３
４８−８５４参照１やバリダマイシン（ｖａｌ　ｉｄａ
ｍｙｃｉｎ）　［［ｗａｓａ、　Ｔ、　。Agric, Biol, Chem, 29:3
48-854 reference 1 and validamycin (valida
mycin) [[wasa, T, .

Ｙａｍａｍｏｔｏ、Ｈ，ａｎｄ　５ｈｉｂａｔａ、Ｍ、
　　（１９７０）　。Yamamoto, H, and 5hibata, M.
(1970).

Ｊ、Ａｎｔｉｂｉｏｔｉｃｓ、　２３−５９５−６０２
参照〕、麦類の白粉病に対するミルジオマイシン （ｍｉｌｄｉｏｍｙｃｉｎ）［Ｈａｒａｄａ、Ｓ、ａｎ
ｄ　Ｋ１５ｈｉ、Ｊ、　　（１９７８）　、Ｊ、Ａｎｔ
ｉｂｉｏｔｉｃｓ、　　３１　：　５１９−５２４　］
等の抗生物質か開発された毒性の強い一部有機合成剤抗
生物質か開発され毒性の強い一部有機合成剤農薬を切替
えた。J. Antibiotics, 23-595-602
Reference], mildiomycin against white powder disease of wheat [Harada, S, an
d K15hi, J. (1978), J. Ant.
Ibiotics, 31: 519-524]
Antibiotics such as antibiotics or some highly toxic organic synthetic agents that have been developed have been replaced with antibiotics or some highly toxic organic synthetic agents that have been developed and pesticides.

〔発明か解決しようとする課題〕[Invention or problem to be solved]

この発明の目的は、このような見地から、土壌試料から
微生物資源を探索して新規な微生物を分離し、それから
新規の有用な抗生物質を提供するにある。From this perspective, the purpose of the present invention is to search for microbial resources from soil samples, isolate new microorganisms, and provide new and useful antibiotics.

更に、本発明の目的は上記抗生物質の医薬・農薬および
その他いろいろな用途に提供するのである。A further object of the present invention is to provide the above-mentioned antibiotics for use in medicine, agrochemicals, and various other uses.

このような一連の抗真菌物質開発努力の次元において、
本発明の発明者等は各種真菌類に対して広範囲な抗真菌
力を現し、毒性の少ない抗真菌抗生物質を探索しようと
国内外の各種土壌から新規な微生物を分離し、それから
生産される抗生物質を分離精製して、その生物学的およ
び理化学的性質を究明した結果、有用なる新規物質たる
ことを確認して本発明を完成するに至った。In this series of efforts to develop antifungal substances,
The inventors of the present invention have isolated new microorganisms from various soils in Japan and abroad in an effort to search for antifungal antibiotics that exhibit a wide range of antifungal activity against various types of fungi and are less toxic. As a result of separating and purifying the substance and investigating its biological and physicochemical properties, it was confirmed that the substance is a useful new substance, and the present invention was completed.

〔課題を解決するための手段〕[Means to solve the problem]

本発明の発明者等は５２００余種に達する土壌微生物探
索を通じて、その内の約３４％の微生物等か抗真菌作用
を顕著に現すことを確認のうえ、生体内試験（ｉｎ　ｖ
ｉｖｏ　ｔｅｓｔ）によるスクリーニングを通じて、本
発明の新規且つ有用なる微生物を得るのに成功した。Through a search for more than 5,200 species of soil microorganisms, the inventors of the present invention confirmed that approximately 34% of them exhibit significant antifungal activity, and conducted an in vitro test (in vitro test).
We succeeded in obtaining the novel and useful microorganism of the present invention through screening using ivo test).

試験菌株としては、主にピリキュラリア・オライゼ（Ｐ
ｙｒｉｃｕｌａｒｉａ　　ｏｒｙｚａｅ）とトリコフィ
トン・メンタグロフィテス（Ｔｒｉｃｈｏｐｈｙｔｏｎ
ＡＴＣＣ１０２３１）等を利用しており、土壌から菌株
を分離するための分離用培地としては澱粉−硝酸塩寒天
培地（ｓｔａｒｃｈ−ｎｉｔｒａｔｅ　ａｇａｒ）［Ｋ
ｕｓｔｅｒ、Ｅ、ａｎｄ　Ｗｉｌｌｉａｍｓ、Ｓ、Ｔ、
　　（１９６４）Ｎａｔｕｒｅ、　　２０２　：　９２
８−９２９］　とＨＶ寒天培地（ＨＶ　ａｇａｒ）　［
Ｈａｙａｋａｗａ、　Ｍ、　ａｎｄ　Ｎｏｎｏｍｕｒａ
、　Ｈ。The test strains were mainly Piricularia oryzae (P.
yricularia oryzae) and Trichophyton mentagrophytes
ATCC 10231), etc., and the isolation medium for isolating bacterial strains from soil is starch-nitrate agar [K
Uster, E. and Williams, S.T.
(1964) Nature, 202: 92
8-929] and HV agar medium (HV agar) [
Hayakawa, M. and Nonomura
, H.

（１９８７）　、Ｊ、Ｆｅｒｍｅｎｔ、ＴｅｃｈｎｏＬ
、　　６５　：　５０１−５０９］等を基本培地として
用いた。(1987), Ferment, J., TechnoL.
, 65: 501-509] was used as the basic medium.

本発明に基づく新規の枯草菌中野生菌株は大韓民国忠清
南道鶏龍山甲寺附近において、採集した土壌試料から分
離され、変異菌株は実施例に詳細に説明された通常の紫
外線に因る方法によって、作製された。これら微生物の
分類学的同定は文献［Ｂｅｒｇｅｙ’ｓ　Ｍａｎｕａｌ
　ｏｆ　Ｓｙｓｔｅｍａｔｉｃ　Ｂａｃｔｅｒｉｏｌｏ
ｇｙ。The novel Bacillus subtilis strain based on the present invention was isolated from a soil sample collected near Gyeryongsan Gaksa Temple, Chungcheongnam-do, Republic of Korea, and the mutant strain was isolated by the conventional method using ultraviolet light as detailed in the Examples. , was created. The taxonomic identification of these microorganisms can be found in the literature [Bergey's Manual
of Systematic Bacteriolo
gy.

Ｖｏｌ、２．　Ｗｉｌｌｉａｍｓ　＆Ｗｉｌｋｉｎｓ　
Ｃｏ、　（１９８６）　］に記述の方法に基づいて行っ
た。Vol.2. Williams & Wilkins
Co, (1986)].

本発明の新規微生物等は標準菌株として用いられたバシ
ラス・サブチリス（Ｂａｃｉｌｌｕｓ　５ｕｂｔｉｌｉ
ｓＡＴＣＣ６６３３）と殆ど同等の微生物として同定さ
れたか、胞子の位置やオギシターゼ反応に陰性であり、
塩分許容度か僅かに低く、ラクトース利用が可能であり
、ｏ、ｏｏｉ％のライソザイムにより感受性のあること
等は、特記する程のものであった（下記表２参照）。The novel microorganisms of the present invention are Bacillus subtilis, which was used as a standard strain.
sATCC6633), or it was negative for spore location and ogistase reaction,
It should be noted that the salt tolerance was slightly low, lactose utilization was possible, and sensitivity to o, ooi% lysozyme was noted (see Table 2 below).

このような理由で、本発明の新規微生物はバシラス・サ
ブチリス亜種（Ｂａｃｉｌｌｕｓ　　５ｕｂｔｉｌｉｓ
ｓｕｂｓｐ、　）に同定され、野性菌株はバシラス・サ
ブチリス亜種クリックチェンシス（Ｂａｃｉｌｌｕｓｓ
ｕｂｔｉｌｉｓ　５ｕｂｓｐ、　Ｋｒ１ｃｔｉｅｎｓｉ
ｓ　）と、変異菌株はバシラス・サブチリス亜種クリツ
クチェンシスＭ１８−９１）と各々命名された。For this reason, the novel microorganism of the present invention is Bacillus subtilis subsp.
subsp, ), and the wild strain is Bacillus subtilis subsp.
ubtilis 5ubsp, Kr1ctiensi
s) and the mutant strain was named Bacillus subtilis subsp. kritschensis M18-91), respectively.

これら新規な微生物等は１９９０年７月２６日アメリカ
合衆国菌株保存協会（Ａｍｅｒｉｃａｎ　ＴｙｐｅＣｕ
ｌｔｕｒｅ　Ｃｏ１１ｅｃｔｉｏｎ：　ＡＴＣＣ）に、
各々寄託番号ＡＴＣＣ５５０７９，およびＡＴＣＣ５５
０７８として寄託されている。These new microorganisms were published on July 26, 1990 by the American Type Cuisine Society.
lture Co11ection: ATCC),
Deposit numbers ATCC55079 and ATCC55, respectively.
It has been deposited as 078.

下記表１・２・３は本発明の微生物の特性を記述したも
のである。Tables 1, 2, and 3 below describe the characteristics of the microorganisms of the present invention.

表１１本発明の新規微生物（Ｂａｃｉｌｌｕｓ　　５ｕ
ｂｔｉｌｉｓｓｕｂｓｐ、Ｋｒ１ｃｔｉｅｎｓｉｓ　）
の形態学的および培養上の特徴 表２．標準菌株と本発明の新規な微生物の生理的特性の
比較 表３８標準菌株と本発明の新規微生物との糖料用性の比
較 本発明において、新規な微生物等から生産された新規な
抗真菌物質ＫＲＦ−００１は、上記新規の微生物を培養
するなり醗酵させることによって生産される一連のペプ
チド系化合物等の複合体を意味する。Table 11 New microorganism of the present invention (Bacillus 5u
btilissubsp, Kr1ctiensis)
Table 2. Morphological and cultural characteristics of Table 38 Comparison of physiological properties of standard strain and novel microorganism of the present invention Comparison of sugar usability between standard strain and novel microorganism of the present invention Novel antifungal substance produced from novel microorganism, etc. of the present invention KRF-001 refers to a complex of a series of peptide compounds produced by culturing or fermenting the above novel microorganism.

即ち、ＫＲＦ−００１は、各々成分Ａ、Ｂ、Ｃ。That is, KRF-001 has components A, B, and C, respectively.

Ｄ、ＥおよびＦと命名の一連のペプチド系化合物の複合
体てあって、本発明の新規微生物を培養したり醗酵させ
た後、生産された抗真菌物質を精製して高圧液体クロマ
トグラフィー（ＨＰＬＣ）て分析した時、上記６種類の
ペプチド成分に分離された。分離された成分等をＦＡＢ
−ＭＳ　（ＪＢＯＬ社製品、Ｍｏｄｅｌ　ＤＸ　　３０
３．　ｐｏｓｉｔｉｖｅｉｏｎｉｚａｔｉｏｎ、ａｒｇ
ｏｎ　ｇａｓ、ｇｕｎ　ｖｏｌｔａｇｅ３ｋｖ。The novel microorganism of the present invention is a complex of a series of peptide compounds named D, E, and F. After culturing or fermenting the antifungal substance, the produced antifungal substance is purified and subjected to high-pressure liquid chromatography (HPLC). ), it was separated into the above six types of peptide components. FAB the separated components etc.
-MS (JBOL product, Model DX 30
3. positionionization, arg
on gas, gun voltage 3kv.

ｅｍｉｓｓｉｏｎ　　３０　ｍＡ）で分析した結果、分
子量が各各１０４２．１０５６．１０５６．１０７０．
１０７０および１０８４と確認された。The molecular weight was 1042.1056.1056.1070.
Confirmed as 1070 and 1084.

ＫＲＦ−００１の薄層クロマトグラフィー（ＴＬＣ）上
におけるＲｆ値は展開溶媒としてメタノールだけを用い
た場合０．７５、メタノ−ル：アセトニトリル（ｌ・１
）を用いた場合は０．５３として現われ、水やメタノー
ルに良く溶解され、クロロホルムやヘキサンには良く溶
解されないことから見て、親水性の物質であることを知
り得る。更に、ＫＲＦ−００１はｐＨ１，０乃至１１．
０で比較的安定てあり、高温処理（１００−１２１°Ｃ
）した時相当期間（１００°Ｃて５時間、１２１　’Ｃ
で１５分）安定な物質として明らかにされた。　ＫＲＦ
−００１はニンヒドリン反応に対して陰性を現したか、
６Ｎの塩酸で加水分解した後分解産物をＧ（、−ＭＳで
分析した結果、アスパラキン、グルタミン、プロリン、
セリン、チロシンか各成分に共通的に存在することを確
認した。更に、元素分析結果に伴うと、上記Ｋ　ＲＦ　
−００１からの各ペプチド成分中ＡはＣｌ４Ｈ２゜ＮＯ
３，ＢとＣとはＣ，５Ｈ３，ＮＯ□、ＤとＥとはＣｌ６
Ｈ３３ＮＯ２およびＦはＣ１□Ｈ３ａ　Ｏ２の特異アミ
ノ酸を含有するものと各々確認された。The Rf value of KRF-001 on thin layer chromatography (TLC) is 0.75 when only methanol is used as the developing solvent;
), it appears as 0.53, and it can be seen that it is a hydrophilic substance because it dissolves well in water and methanol but does not dissolve well in chloroform and hexane. Furthermore, KRF-001 has a pH of 1.0 to 11.
It is relatively stable at 0 and can be treated at high temperature (100-121°C).
) for an equivalent period of time (5 hours at 100°C, 121'C
(15 minutes) was found to be a stable substance. KRF
-001 showed negative results for ninhydrin reaction;
After hydrolysis with 6N hydrochloric acid, the decomposition products were analyzed by G(,-MS), and the results showed that asparaquine, glutamine, proline,
It was confirmed that serine and tyrosine commonly exist in each component. Furthermore, according to the elemental analysis results, the above K RF
A in each peptide component from -001 is Cl4H2゜NO
3, B and C are C, 5H3, NO□, D and E are Cl6
H33NO2 and F were each confirmed to contain the unique amino acid of C1□H3a O2.

これらを各々６Ｎ　　ＨＣＩに溶解させ１１６°Ｃて６
時間反応させアミノ酸自動分析器（ａｍｉｎ。Dissolve each of these in 6N HCI and heat at 116°C for 6 hours.
Time-reacted amino acid automatic analyzer (amin).

ａｃｉｄ　ａｕｔｏａｎａｌｙｚｅｒ、Ｗａｔｅｒｓ　
Ｃｏ製品）で分析した結果、アスパラキン、グルタミン
、セリン、プロリン、チロシン等の存在か確認され、そ
のモル比は３：１：１：１：１と明らかになった。した
かって、ＫＲＦ−００１はアスパラギン３モル、グルタ
ミン１モル、セリン１モル、プロリン１モル、チロノン
１モルおよび特にアミノ酸１モルのオリゴペプチド化合
物複合体として見ることかでき、ギモトリップシン（ｃ
ｈｙｍｏｔｒｙｐｓｉｎ）蛋白質分解酵素によって、加
水分解されないため、このようなオリゴペプチドは、下
記の一般式のような環状構造（ｃｉｒｃｕｌａｒ　５ｔ
ｒｕｃｔｕｒｅ）なることを判ることかできた。acid autoanalyzer, Waters
As a result of analysis using Co product), the presence of asparaquine, glutamine, serine, proline, tyrosine, etc. was confirmed, and the molar ratio was revealed to be 3:1:1:1:1. Therefore, KRF-001 can be seen as an oligopeptide compound complex of 3 moles of asparagine, 1 mole of glutamine, 1 mole of serine, 1 mole of proline, 1 mole of thyronone and, in particular, 1 mole of amino acids, with gimotlipsin (c
hymotrypsin), such oligopeptides have a circular structure (circular 5t) as shown in the general formula below.
I was able to see that the structure was the same.

上記構造不明の特異アミノ酸の構造を高分解Ｈ’−ＮＭ
Ｒに解釈した結果、この構造にはメチレン鎖に基つくδ
１２５における積分値と３番炭素（Ｃ−３）の陽性子か
外の４個の陽性子とカップリングしていることを現す吸
収か６４．２５で現れたため、」１記アミノ酸の構造か
β−アミノβ　　　ＣＯＯＨ の構造を持っていることを判ることができた。High-resolution H'-NM structure of the above-mentioned unique amino acid with unknown structure
As a result of interpretation in R, this structure has a δ based on the methylene chain.
Since the integral value at 125 and the absorption indicating coupling with the proton at carbon number 3 (C-3) or the other four protons appeared at 64.25, the structure of amino acid 1 is β It was found that it had the structure -aminoβCOOH.

更に、各成分Ａ、Ｂ、Ｃ，Ｄ、Ｅ及びＦのペプチド化合
物の６０９に現れるメチル基のシグナルを通して確認さ
れるβ−アミノ酸の末端構造は、ペプチドＡは正常タイ
プ（δ０．　９．　３Ｈ，ｔ）、ペプチドＢはａｎｔｅ
ｉｓｏタイプ（δ０．９，６Ｈ。Furthermore, the terminal structure of the β-amino acid confirmed through the signal of the methyl group appearing at 609 in the peptide compounds of each component A, B, C, D, E, and F shows that peptide A is of the normal type (δ0.9.3H, t), peptide B is ante
iso type (δ0.9, 6H.

ｍ）、ペプチドＣはｉｓｏタイプ（δ０．　９．　６Ｈ
。m), peptide C is iso type (δ0.9.6H
.

ｄ）、ペプチドＤはｉｓｏタイプ（δ０．　９．　６Ｈ
。d), peptide D is iso type (δ0.9.6H
.

○）、ペプチドＥは正常タイプ（δ０．　９．　３Ｈ。○), peptide E is normal type (δ0.9.3H).

ｔ）、そしてペプチドＦはａｎｔｅｉｓｏタイプ（δ０
．９，６Ｈ，ｍ）　、であることを判ることかできる。t), and peptide F is of the anteiso type (δ0
．． 9,6H,m).

したかって、ＮＭＲ分析によるＫＲＦ−００１複合体を
構成する各ペプチドのβ−アミノ酸構造は下記一般式の
ようである。Therefore, the β-amino acid structure of each peptide constituting the KRF-001 complex as determined by NMR analysis is as shown in the following general formula.

成分Ａ：ＣＨｈ　　（ＣＩ□）ｔｏ　　ＣＨ（ＮＩ−１
２）ＣＨＩ　Ｃ０ＯＨ成分Ｂ　：ＣＨ３ＣＨ２ＣＨ（Ｃ
ＨＩ）（ＣＨ２）ｌ　Ｃ１−Ｔ　（ＮＩ２）ＣＨ２ＣＯ
ＯＨ 成分Ｃ：ＣＨｓ　ＣＨ（ＣＩ−１５）（ＣＩ２）９　Ｃ
Ｈ（ＮＩ２）ＣＨ２ＯＯＨ 成分Ｄ・ＣＨＩ　ＣＨ（ＣＨＩ）（ＣＨ２）Ｉ。ＣＨ（
Ｎｌ−１２）ＣＨ２ＯＯＨ 成分Ｅ　：ＣＨＩ　　（ＣＨ２）、２ＣＨ（ＮＨＩ）Ｃ
ＨＩ　Ｃ０ＯＨ成分Ｆ・ＣＨＩ　ＣＨＩ　ＣＨ（ＣＨＩ
）（ＣＨ２）ｌ。ＣＨ（ＮＨＪＣＨ２Ｃ００Ｈ ＫＲＦ−００１の生物学的活性 ＫＲＦ−００１は実験室条件の下に非常に広範囲な抗真
菌作用があることか実施例２から確認された。特に、既
存の市販されていた抗真菌物質と類似する活性を現して
おり、各種植物の疾病の原因となる真菌類に対して広範
囲なる真菌作用を現すものと明らかにされた。（下記実
施例２２表５参照）。Component A: CHh (CI□) to CH(NI-1
2) CHI C0OH component B: CH3CH2CH (C
HI) (CH2)l C1-T (NI2)CH2CO
OH Component C: CHs CH (CI-15) (CI2) 9 C
H(NI2)CH2OOH Component D CHI CH(CHI)(CH2)I. CH(
Nl-12) CH2OOH Component E: CHI (CH2), 2CH(NHI)C
HI C0OH component F・CHI CHI CH (CHI
)(CH2)l. CH(NHJCH2C00H) Biological activity of KRF-001 It was confirmed from Example 2 that KRF-001 has a very wide range of antifungal activity under laboratory conditions. It has been revealed that it exhibits activity similar to that of the compound, and that it exhibits a wide range of fungal effects against fungi that cause various plant diseases (see Table 5 of Example 22 below).

一方、ＫＲＦ−００１複合体の各種細菌等に対する最小
抑制濃度（Ｍｉｎｉｍｕｍ　ＩｎｈｉｂｉｔｉｏｎＣｏ
ｎｃｅｎｔｒａｔｉｏｎ：Ｍ　Ｉ　Ｃ）の測定方法およ
びその結果は下記の実施例２に詳しく記述されており、
ＫＲＦ−００１の細菌に対する抗生作用は非常に弱いも
のと確認された（実施例２１表６参照）ＫＲＦ−００１
の毒性実験 ＫＲＦ−００１の鼠を対象とした場合経口急性試験結果
ＬＤ５ｏ値か２５００乃至５０００ｍｇ／ｋｇと現れて
おり、兎を対象とした経皮急性毒性試験において゛も、
皮膚刺激やアレルギー等副作用か現れなかった。したが
って、ＫＲＦ−００１は低毒性抗真菌物質たることか判
明された。On the other hand, the minimum inhibitory concentration of KRF-001 complex against various bacteria, etc.
The method for measuring M I C) and its results are detailed in Example 2 below.
It was confirmed that the antibiotic effect of KRF-001 against bacteria was very weak (see Example 21 Table 6) KRF-001
Toxicity experiments of KRF-001 in rats showed an LD5o value of 2500 to 5000 mg/kg, and in acute dermal toxicity tests in rabbits,
There were no side effects such as skin irritation or allergies. Therefore, KRF-001 was found to be a low toxicity antifungal substance.

ＫＲＦ−００１の用途 先ず農産物の疾病予防の側面で、ＫＲＦ−００１を水田
に１００乃至２００　ｐｐｍで処理した場合稲の稲熱病
防除効果かあり、各種灰色の黴病予防にも２００乃至１
１０００ｐｐで有効なることが観察された。更に、発芽
する種、野菜および果物の保存にも効果かあって、畑で
収穫した苺をＫＲＦ一００１醗酵液に１０分間漬けてか
ら取出して置いた時、黴による腐敗が防止された。Uses of KRF-001 First, in terms of disease prevention in agricultural products, when KRF-001 is applied to rice fields at 100 to 200 ppm, it is effective in controlling rice fever disease, and it is also effective in preventing various gray mildew diseases.
It was observed that 1000 pp was effective. Furthermore, it is effective in preserving germinated seeds, vegetables, and fruits, and when strawberries harvested in the field were soaked in KRF-1001 fermentation solution for 10 minutes and then taken out and put aside, rot due to mold was prevented.

一方、ＫＲＦ−００１を水に溶解させた後、人体の皮膚
に塗ったり、クリームや軟膏を造って皮膚に塗る際各種
の皮膚関連の黴の病が治癒されるのを観察した。On the other hand, when KRF-001 was dissolved in water and applied to human skin, or when a cream or ointment was made and applied to the skin, various skin-related fungal diseases were observed to be cured.

ＫＲＦ−００１は水、メタノール、エタノール等に良く
溶けるし、熱に強く、１２１’Ｃて１５分間処理しても
抗真菌活性を失わず、特にＫ　ＲＦ　−ｏｏｉは吸湿性
が強＜ＫＲＦ−００１粉末を室温に露出する時、大気中
から湿気を吸収してゲル状態に変わるのか観察された。KRF-001 is highly soluble in water, methanol, ethanol, etc., is resistant to heat, and does not lose its antifungal activity even after being treated at 121'C for 15 minutes. In particular, KRF-ooi is highly hygroscopic. When the powder was exposed to room temperature, it was observed whether it absorbed moisture from the atmosphere and turned into a gel state.

従って、Ｋ　ＲＦ−００１は各種の化粧品、化学製品お
よび食品の保湿剤として使用することができる。Therefore, KRF-001 can be used as a humectant in various cosmetics, chemical products, and foods.

一方、ＫＲＦ−００１成分を動物飼料や、その他外の工
業製品例えば洗浄剤や消毒剤中に添加した時、天然的な
抗真菌および防腐作用を見せた。On the other hand, when the KRF-001 component was added to animal feed and other industrial products such as cleaning agents and disinfectants, it exhibited natural antifungal and antiseptic properties.

新規微生物変異株作製方法、ＫＲＦ−００１の生産精製
および分析方法、そして、詳細なるＫＲＦ−００１の用
途に関しては、後述の実施例において詳しく説明される
筈である。The method for producing a novel microbial mutant strain, the production and purification and analysis method for KRF-001, and the detailed use of KRF-001 will be explained in detail in the Examples below.

〔実施例〕〔Example〕

実施例　１ 変異菌株バシラス・ザブチリス亜種りリッチェンシス　
Ｍ−１８，７９１（ＡＴＣＣ５５０７８）の作製 本発明の新規物質ＫＲＦ”−００１’の生産収率を高め
るため、母菌株のバシラス・サブチリス亜種クリッチエ
ンシス（Ｂａｃｉｌｌｕｓ　　５ｕｂｔｉｌｉｓ　５ｕ
ｂｓｐ。Example 1 Mutant strain Bacillus subtilis subsp. richensis
Preparation of M-18,791 (ATCC55078) In order to increase the production yield of the novel substance KRF''-001' of the present invention, the mother strain Bacillus subtilis subsp.
bsp.

Ｋｒ１ｃｔｉｅｎｓｉｓ　　：　ＡＴＣＣ５５０７９）
に紫外線を照射して変異さた後生産能の高い菌株を選別
する方法を模索した。Kr1ctiensis: ATCC55079)
We sought a method to select strains with high productivity after mutating them by irradiating them with ultraviolet light.

母菌株をＬＢＧ培地（１−リップトン１％、酵母抽出物
０．５％、ＮａＣ１１％、グルコス　１％）に接種した
後、代数増殖期後期に集め遠心分離した後、濁度か５５
０ｎｍで１．０となるように調節し同量の１０μＭカベ
イン（ｃａｆｆｅｉｎ）溶液を添加した後１分間紫外線
を照射した。After inoculating the mother strain into LBG medium (1-Lipton 1%, yeast extract 0.5%, NaC 11%, glucose 1%), it was collected at the late algebraic growth phase, centrifuged, and the turbidity reached 55.
After adjusting the wavelength to 1.0 at 0 nm and adding the same amount of 10 μM caffeine solution, ultraviolet rays were irradiated for 1 minute.

その後ＰＤＡ培地に塗末して現れた集落を試験菌株とし
て利用した（６０４５株）。これら菌株等をＬＢＧ培地
において４０時間培養して高密度（２×１０５胞子／ｍ
１）Ｐ、　　肛Ｕ些平盤において母菌株より生育阻止環
が大きく現れる菌株を２１２株選別した。これら２１２
株の菌株を醗酵用培地が５ｍｌ入っている試験管に接種
した後、４０時間培養し更に母菌株とＰ、　ｏｒｙｚａ
ｅ平盤において阻止環の大きさを比較して１６菌株を選
別し、３次に５００ｍ１三角フラスコに醗酵用培地１０
０ｍ１を入れて１６菌株を接種した後同じ方法てＭ　１
８−９１、Ｍ　　２６−８７、Ｍ　　２７−８、Ｍ　　
２８−９、Ｍ　　３１−１３９の５菌株を選定した。Thereafter, the colonies that appeared after being applied to a PDA medium were used as a test strain (strain 6045). These strains were cultured in LBG medium for 40 hours to achieve high density (2 x 105 spores/m2).
1) P. We selected 212 strains that showed a larger growth inhibition ring than the mother strain in the anal plate. These 212
After inoculating the strain into a test tube containing 5 ml of fermentation medium, it was cultured for 40 hours and further incubated with the mother strain and P. oryza.
16 strains were selected by comparing the size of the inhibition ring on a flat plate, and then 10 strains of fermentation medium were placed in a 500 m Erlenmeyer flask.
After inoculating 16 strains of M1 with 0ml in the same manner.
8-91, M 26-87, M 27-8, M
Five strains, 28-9 and M31-139, were selected.

このうちＭ　　１８−９１において生産されたＫＲＦ−
００１の物質をＸＡＤ−７樹脂を利用し予備精製を行い
母菌株と生産量を比較すればリットル当たり生産量が２
倍以上増加したものと現れ。Of these, KRF- produced in M 18-91
If we pre-purify the substance 001 using XAD-7 resin and compare the production with the mother strain, the production per liter is 2.
Appears to have increased more than double.

た。（表４参照） 表４．変異菌株と母菌株とのＫＲＦ−００１生産量比較 変異株Ｍｌｌｌｌ−９１の母菌株との異なる点は、一番
目に、Ｍ１１３−９１の場合は醗酵条件中ｐＨを調節し
なかった時、更に、多い量のＫＲＦ−００１物質を収得
することができ、二番目に、Ｍ２Ｓ−９１菌株か細胞成
長速度が尚更早く細胞濃度か高かったし、三番目に、Ｋ
ＲＦ−００１の生産速度かＭ　　１８−９１の場合、母
菌株に比して３０時間以上短縮された。Ta. (See Table 4) Table 4. Comparison of KRF-001 production between the mutant strain and the mother strain. A large amount of KRF-001 substance could be obtained, secondly, the cell growth rate of the M2S-91 strain was even faster and the cell concentration was high, and thirdly, the KRF-001 substance was
In the case of RF-001 or M18-91, the production rate was reduced by more than 30 hours compared to the mother strain.

以上のように、母菌株のバシラス・サブチリス５ｕｂｓ
ｐ、Ｋｒ１ｃｔｉｅｎｉｓ、　ＡＴＣＣ５５０７９）か
ら得られた変異菌株はバシラス・サブチリシス亜種クリ
ッチェンシス　Ｍ　１８−９１　　（Ｂａｃｉｌｌｕｓ
ｓｕｂｔｉｌｉｓ　５ｕｂｓｐ、　Ｋｒ１ｃｔｉｅｎｉ
ｓ　　Ｍ　１８−９１゜ＡＴＣＣ５５０７８）と命名さ
れており、母菌株と共に１９９０年７月２６日付に米合
衆国菌株保存協会（ＡＴＣＣ）に寄託された。As mentioned above, the mother strain Bacillus subtilis 5ubs
The mutant strain obtained from Bacillus subtilis subsp. kritchensis M 18-91
subtilis 5ubsp, Kr1ctieni
s M 18-91° ATCC 55078) and was deposited with the mother strain at the American Strain Conservancy (ATCC) on July 26, 1990.

実施例　２ 最小抑制濃度の測定 ■、真閑に対する測定 各種真菌（ｆｕｎｇｉ）類等をＰＤＡ、ザブロウ寒天培
地（Ｓａｂｏｕｒａｕｄ’　ｓ　ａｇａｒ）、　ＹＭ寒
天培地、Ｖ−８ｊｕｉｃｅ寒天培地等を利用して２４乃
至３０°Ｃて１乃至１４日間斜面培養後５ｍｌの蒸留水
を添加し胞子を懸濁させて接種菌液として使用した。試
験しようとする試料溶液の２倍稀釈系列を作った後、各
種真菌類等の培地と混合して最終濃度か１００乃至０．
１９５μｇ／ｍｌとなるように５ｃｍ直径の平盤に株分
けして水平に固めた。培地か固形化すれば、上記接種菌
液を各平盤毎に１白金匙宛接種し２４乃至３０℃で１日
乃至４日間培養した後、肉眼で観察して菌の生育か抑制
された濃度を最小の抑制濃度として定め、結果は表５に
示したとおりである。Example 2 Measurement of Minimum Inhibitory Concentration ■, Measurement for Mangan Various fungi, etc. were measured using PDA, Sabouraud's agar, YM agar, V-8 juice agar, etc. After slant culture at 30°C for 1 to 14 days, 5 ml of distilled water was added to suspend the spores and used as an inoculum. After making a 2-fold dilution series of the sample solution to be tested, it is mixed with a culture medium for various fungi, etc. to give a final concentration of 100 to 0.
The cells were divided into flat plates with a diameter of 5 cm to give a concentration of 195 μg/ml and solidified horizontally. Once the medium has solidified, inoculate each plate with one platinum spoonful of the above-mentioned inoculated bacterial solution, incubate at 24 to 30°C for 1 to 4 days, and then visually observe to see if the concentration of bacteria growth is suppressed. was determined as the minimum inhibitory concentration, and the results are shown in Table 5.

上記表に示したように、ＫＲＦ−００１は既存の市販さ
れている、抗真菌物質等と比較した時、エムボテリシン
　Ｂ　（ａｍｐｈｏｔｅｒｉｃｉｎ　Ｂ）やニスタチン
（ｎｙｓｔａｔｉｎ）と類似する活性を示し、各種植物
疾病および人体疾病の原因になる真菌類に対して、広範
囲な真菌作用を示す特性か兼ねて確認された。As shown in the table above, when compared with existing commercially available antifungal substances, KRF-001 exhibits similar activity to amphotericin B and nystatin, and is effective against various plant diseases and It was also confirmed that it has a wide range of fungal effects against fungi that cause human diseases.

２、細菌に対する測定 Ｆｌｅｉｓｈ　ｅｘｔｒａｃｔｂｒｏｔｈ　　（１％　
肉汁抽出物、１％ペプトン、０．３％　ＮａＣ１，０，
２％ＮａＨＰＯ４・１２Ｈ２０，２％寒天、ｐＨ７，２
〜７．５）や血清を添加したｆｌｅｉｓｈ　ｅｘｔｒａ
ｃｔｂｒｏｔｈにおいて１８時間程度培養した試験菌を
１００倍位稀釈して接種菌液として使用した。2. Measurement against bacteria Fleish extractbroth (1%
Meat juice extract, 1% peptone, 0.3% NaC1,0,
2% NaHPO4・12H20, 2% agar, pH 7.2
~7.5) or flesh extra with added serum
The test bacteria cultured in ctbroth for about 18 hours was diluted about 100 times and used as an inoculum solution.

試験しようとする試料溶液の２倍稀釈系列を造った後、
直径９ｃｍ平盤に滅菌したミュラーヒントン（Ｍｕｅｌ
ｌｅｒ　Ｈｊｎｔｏｎ）寒天培地と稀釈系列の試料を４
＝１の比率で株分けして最終試料溶液の濃度が各々１０
０乃至０．０２μｇ／ｍｌとなるように１７個の平盤を
製造した。製造した平盤上に各々の試験菌を自動接種機
（ａｕｔｏｍａｔｉｃ　１ｎｏｃｕｌａｔｏｒ。After making a 2-fold dilution series of the sample solution to be tested,
Sterilized Mueller-Hinton (Muel) into a 9 cm diameter flat plate.
ler Hjnton) agar medium and dilution series of 4 samples.
Divide the stocks at a ratio of = 1 so that the concentration of the final sample solution is 10 for each
Seventeen flat plates were manufactured so that the concentration ranged from 0 to 0.02 μg/ml. Each test bacterium was inoculated onto the prepared flat plate using an automatic inoculator.

米国Ｄｙｎａｔｅｃｈ社製品）を利用して、１０’ＣＦ
Ｌｌ／５ｐｏｔ（ＣＰＵ：Ｃｏ１ｏｎｙ　Ｆｏｒｍｉｎ
ｇ　Ｕｎｉｔ）接種し３７°Ｃて１８時間培養した後、
肉眼で観察して生育か抑制された試料の濃度を最小の抑
制濃度として定め結果は下記表６に示したとおりである
。10'CF using Dynatech product)
Ll/5pot (CPU: Co1ony Formin
g Unit) After inoculation and culturing at 37°C for 18 hours,
The concentration of the sample whose growth was inhibited by visual observation was determined as the minimum inhibitory concentration, and the results are shown in Table 6 below.

上記表に示したように、ＫＲＦ−００１の細菌に対する
抗生作用は非常に弱い。As shown in the table above, the antibiotic effect of KRF-001 against bacteria is very weak.

実施例　３ ＫＲＦ−００１の精製および構造分析 −８０°Ｃ冷凍機内に保存されているバシラス・ザブチ
リス亜種（Ｂａｃｉｌｌｕｓ　５ｕｂｔｉｌｊｓ　５ｕ
ｂｓｐ、）バアイアル（１ｍ１．）１個を溶かして１０
０ｍ１のクルコスＬＢ培地に接種した後３０°Ｃ，ｐＨ
７，０で振盪培養した。グルコスＬＢ培地はグルコス１
０ｇ、バクト−トリップトン１０ｇ、バクト−イースト
抽出物５ｇ、塩１０および蒸留水１０１００Ｏで製造し
、ｐＨは７０に調整した。上記種菌の成長かＡ３５０　
＝０．　１乃至０．　５に至ったとき、全体の培養液を
８リットルの生産用培地が入っている１４リツ１〜ル入
すの醗酵タンクに無菌的に移した。Example 3 Purification and structural analysis of KRF-001 - Bacillus 5ubtiljs 5u stored in an 80°C refrigerator
bsp,) Melt 1 piece of Baial (1ml.) and make 10
After inoculating into 0ml of Kurkos LB medium, 30°C, pH
Shaking culture was carried out at 7.0. Glucos LB medium is Glucos 1
0g, Bacto-tripton 10g, Bacto-yeast extract 5g, salt 10 and distilled water 10100O, and the pH was adjusted to 70. Growth of the above inoculum or A350
=0. 1 to 0. 5, the entire culture was aseptically transferred to a 14 liter fermentation tank containing 8 liters of production medium.

醗酵タンク内における、温度、空気疎通速度および攪拌
速度は各々３０°Ｃ１０，５ＶＶ、Ｍおよび４００ＲＰ
Ｍである。２日後に醗酵液を遠心分離して微生物細胞を
取除いた後上澄液をＸＡＤ−７カラムに通過させた後吸
着されたＫＲＦ−００１をメタノールて溶出した時、黄
褐色の相Ｋ　ＲＦ−００１（純度２５％）１２ｇを得た
。生産用培地はシュクロス３０ｇ、ソイトン１０ｇ、酵
母抽出物５ｇ、に２　ＨＰＯ４０，５ｇ、Ｍｇ５Ｏ＋　
　・７Ｈ２００，５ｇ、　ＭｎＣ１２４ｍｇ１ＣａＣ１
２５ｎ＋ｇおよびＦｅＳＯ４−７Ｈ２Ｏ２５ｍｇ、を蒸
留水１０１００Ｏに入れて溶解させｐＨ７に調整した後
１２１°Ｃて４０分間滅菌して準備した。The temperature, air communication speed and stirring speed in the fermentation tank were 30°C, 10.5VV, M and 400RP, respectively.
It is M. After 2 days, the fermentation solution was centrifuged to remove microbial cells, and the supernatant was passed through an XAD-7 column. When the adsorbed KRF-001 was eluted with methanol, a yellow-brown phase KRF- 12 g of 001 (purity 25%) was obtained. The production medium is 30g of sucrose, 10g of soyton, 5g of yeast extract, 40.5g of Ni2 HPO, Mg5O+
・7H200,5g, MnC124mg1CaC1
25n+g and 25mg of FeSO4-7H2O were dissolved in 10,100O distilled water, adjusted to pH 7, and then sterilized at 121°C for 40 minutes to prepare.

上記のように得た醗酵液１６リツトルを利用してＫＲＦ
−００１の精製を試みた。はじめに、約４リットルの醗
酵液を４リットルのＡｍｂｅｒｌｉｔｅＸＡＤ−７カラ
ム（米国Ｒｏｈｍ　＆　Ｈａｓｓ社製品、９Ｘ　９０　
ｃｍ）に吸着させた後毎分４０ｍ１の流速てべ１ぐの２
倍分の蒸留水（８リツ１〜ル）で不純物を洗出しだ後、
ベトの２倍分のメタノール（８リットル）でＫＲＦ−０
０１を溶出した。溶出した分画等に対してピリキュラリ
ア・オライセ 結乾燥して黄褐色の活性物質を得た。上の方法を４回繰
返し１６リツトルの醗酵液から約１５ｇの黄褐色の粗Ｋ
ＲＦ−００１を得た。次段階の精製はシリカゲル（ｓｉ
ｌｉｃａ　ｇｅｌ）クロマトグラフィーによって行った
。３００ｇのシリカゲル（米国Ｍ　ｅ　ｒ　ｃ　ｋ社製
品　７７３４）をカラム（４Ｘ５０ｃｍ）にクロロホル
ムを用いて満たした後、上記粗ＫＲＦ−００１を最少量
のメタノール−クロロホルム（３゜７）溶液で溶かして
吸着させた。Using 16 liters of fermentation liquid obtained as above, KRF
An attempt was made to purify -001. First, about 4 liters of fermentation liquid was transferred to a 4 liter Amberlite
cm) after adsorption at a flow rate of 40 m1 per minute.
After washing out impurities with double the amount of distilled water (8 liters, 1 to 1 liters),
KRF-0 with twice as much methanol (8 liters) as Beto.
01 was eluted. The eluted fractions were condensed and dried with Pyricularia oleicea to obtain a yellow-brown active substance. Repeat the above method 4 times to obtain about 15 g of yellow-brown crude K from 16 liters of fermentation liquid.
RF-001 was obtained. The next step of purification is silica gel (si)
lica gel) chromatography. After filling a column (4 x 50 cm) with 300 g of silica gel (7734, manufactured by Merck, USA) using chloroform, the above crude KRF-001 was dissolved in the minimum amount of methanol-chloroform (3°7) solution and adsorbed. I let it happen.

ＫＲＦ−００１の溶出はメタノール−クロロホルム（３
・７）７５０ｍｌおよびメタノール−クロロホルム（１
：　１）２１００ｍｌを流して行った。活性成分か入っ
ている部分を凍結乾燥して約７ｇの粗質のＫＲＦ−００
１を得た。予備ＴＬＣ盤を利用して次段階の精製を試み
た。展開溶媒としてはブタノール−エチルアセティ）・
−水（６１：］）を用いてＲｆＯ，３６の物質を回収し
た結果５ｇの活性物質か回収された。次の段階には上記
活性物質５ｇを２次に亘って５ｅｐｈａｄｅｘ　　Ｌ　
Ｈ−２０カラム（０，８Ｘ９０ｃｍ）を用いて精製した
。KRF-001 was eluted using methanol-chloroform (3
・7) 750ml and methanol-chloroform (1
: 1) 2100 ml was poured. Freeze-dry the part containing the active ingredient to produce approximately 7g of crude KRF-00.
I got 1. The next step of purification was attempted using a preliminary TLC panel. The developing solvent is butanol-ethyl acetate).
- Recovery of RfO, 36 material using water (61:]) resulted in recovery of 5 g of active material. The next step is to apply 5 g of the active substance to 5 ephadex L over two times.
Purification was performed using a H-20 column (0.8 x 90 cm).

試料を少量のメタノールに溶かしてカラムに吸着させ、
メタノールを溶出剤として利用して毎分０．２ｍｌの流
速で流した。得られた分画中の活性物質を凍結乾燥した
結果１ｇの白色の活性物質か得られた。Dissolve the sample in a small amount of methanol and adsorb it onto the column.
Methanol was used as the eluent at a flow rate of 0.2 ml/min. As a result of freeze-drying the active substance in the obtained fraction, 1 g of white active substance was obtained.

最終的な精製段階として、ＫＲＦ−００１の各成分等を
ＨＰ　ＣＬ　（Ｐｅｒｋｉｎ−Ｅｌｍｅｒ　Ｃｏ、モデ
ル）を用いて分離した。ＨＰＬＣカラムとしてはＡｌｔ
ｅＣｋ社のＲ５１ｌ　Ｃ（１０ｕ、　　１０Ｘ２５０ｍ
ｍ）を利用した。１ｇの試料を３５％アセトニトリルて
１０ｍｇ／ｍｌの濃度で注入した。試料の溶出は初めの
３０分間は３５％のアセトニトリル後５０分まて５０％
アセ１〜ニトリルかてきるまて、溶媒勾配て行い流速は
毎分４ｍｌてあり、ＵＶ２２５ｎｍにおける吸収でモニ
タリングした。吸収ピーク等に対する生物学性検索結果
Ｒｔ２２．８’　。As a final purification step, each component of KRF-001 was separated using HP CL (Perkin-Elmer Co, model). Alt as HPLC column
eCk R51l C (10u, 10X250m
m) was used. 1 g of sample was injected at a concentration of 10 mg/ml in 35% acetonitrile. Sample elution was performed using 35% acetonitrile for the first 30 minutes, then 50% acetonitrile for 50 minutes.
A solvent gradient ranging from acetic acid to nitrile was used at a flow rate of 4 ml per minute, and the absorption was monitored at UV 225 nm. Biological search results for absorption peaks, etc. Rt22.8'.

３３、８“、３８　７″，４５．５’。33, 8", 38 7", 45.5'.

４６、５’　、４９．８’　のピークか活性を現してお
り、これらを各々ＫＲＦ−００１の成分Ａ，Ｂ、Ｃ，Ｄ
，ＥおよびＦと命名した。最終的に得られた純粋なＫＲ
Ｆ−００１の各成分は成分Ａ２１０　Ｔ＋１ｇ、成分８
３０ｍｇ、成分Ｃ９０ｍｇ、成分Ｄ　　３０ｍｇ、成分
Ｅ３０ｍｇおよび成分Ｆ１０ｍｇてあった。したかって
、ＫＲＦ−００１は最小限６個以上の抗真菌性物質成分
等の複合体なることを知ることかできる。収得したＫＲ
Ｆ−００１の各種機器分析の結果は次のようである。The peaks at 46, 5', and 49.8' show activity, and these are compared to components A, B, C, and D of KRF-001, respectively.
, E and F. The final pure KR
Each component of F-001 is component A210 T+1g, component 8
30 mg, component C 90 mg, component D 30 mg, component E 30 mg, and component F 10 mg. Therefore, it can be seen that KRF-001 is a complex of at least six or more antifungal components. KR earned
The results of various instrumental analyzes of F-001 are as follows.

１、元素分析 ＨＰＬＣによって最終分離精製のＫＲＦ−００１試料を
元素分析機（Ｐｅｒｋｉｎ−Ｂ１ｍｅｒ社製品、Ｍｏｄ
ｅｌ　２４０　Ｃ）を利用して炭素、水素、窒素の構成
比率を決定した。ＫＲＦ−００１は炭素５２．６２％、
水素６．８５％および窒素１４．５９％で成されたもの
と現れた。1. Elemental analysis HPLC final separation and purification of the KRF-001 sample was performed using an elemental analyzer (Perkin-B1mer product, Mod
The composition ratios of carbon, hydrogen, and nitrogen were determined using el 240 C). KRF-001 is 52.62% carbon,
It appeared to be made of 6.85% hydrogen and 14.59% nitrogen.

２、アミノ酸分析 ＫＲＦ−００１試料１０ｍｇを６Ｎ　　ＨＣＩに溶かし
て１１０°Ｃて６時間反応させた後、アミノ酸自動分析
機（ａｍｉｎｏ　ａｃｉｄ　ａｕｔｏａｎａｌｙｚｅｒ
、ＷａｔｅｒｓＣｏ、製品）を利用してアミノ酸を分析
した。ＫＲＦ−００１にはアスパラギン、グルタミン、
セリン、プロリン、チロシンとロイシン等か含有されて
いるということが判った。2. Amino acid analysis 10 mg of KRF-001 sample was dissolved in 6N HCI and reacted at 110°C for 6 hours.
Amino acids were analyzed using a commercially available product, WatersCo, Inc. KRF-001 contains asparagine, glutamine,
It was found that it contains serine, proline, tyrosine, and leucine.

３、赤外線（ＩＲ）スペクトル ＫＲＦ−００１試料２ｍｇをＩ（Ｂｒ２００ｍｇと混せ
てディスクに作った後、比率記録赤外線分光機（Ｐｅｒ
ｋｉｎ−Ｂ１ｍｅｒ社製品、モデル１４２０）で分析し
た。ＫＲＦ−００１はＮＨグループの存在を現す３３０
０−３５００ｃｍ−’とＣＨグループの存在を現す２７
００−３０００ｃＦ’およびカーボニルグループの存在
を現す１６６０ｃｍ付近で吸収を現した。3. Infrared (IR) Spectrum After mixing 2 mg of KRF-001 sample with 200 mg of I (Br) to make a disk, it was analyzed using a ratio recording infrared spectrometer (Per
Kin-B1mer product, model 1420). KRF-001 indicates the existence of NH group 330
0-3500cm-' and 27 indicating the presence of CH group.
Absorption appeared at around 00-3000 cF' and 1660 cm indicating the presence of carbonyl groups.

４、質量分析 ＫＲＦ−００１試料をＪＥＯＬ　　ＤＸ−３０３質量分
析機を利用してＦ　Ａ　Ｂ　（ｐｏｓｉｔｉｖｅｉｏｎ
ｉｚａｔｉｏｎ）方法で分子量を測定した。１０６５．
１０７９．１０９３および１１０７において〔Ｍ＋Ｎａ
）“イオン分子ピークか現れ、各々分子量か１０４２．
１０５６．１０７０および１１８４として推定された。4. Mass spectrometry The KRF-001 sample was subjected to F A B (positional analysis) using a JEOL DX-303 mass spectrometer.
Molecular weight was measured by the following method. 1065.
1079.1093 and 1107 [M+Na
) "Ion molecular peaks appear, each with a molecular weight of 1042.
Estimated as 1056.1070 and 1184.

言い換えれば、ＫＲＦ−００１は分子量か異なる幾つか
の類似物質の集合体として確認された。In other words, KRF-001 was confirmed as a collection of several similar substances with different molecular weights.

５、核磁気共鳴（ＮＭＲ）実験 Ｉ（ＲＦ−００１をＣＤ３０Ｄに溶かした後５ｍｍのチ
ューブに入れて（Ｂｒｕｋｅｒ　ＡＭ　−３００とＢｒ
ｕｋｅｒ　　ＡＭ　−５００）　ＮＭＲ分析を行った。5. Nuclear magnetic resonance (NMR) experiment I (dissolve RF-001 in CD30D and put it in a 5 mm tube (Bruker AM-300 and Br
uker AM-500) NMR analysis was performed.

（内部標準試薬としてＴＭＳを用いた）。(TMS was used as an internal standard reagent).

’ＨＮＭＲは３００．１３ＭＨｚ、５００．１３ＭＨｚ
ＮＭＲで、”ＣＮＭＲは７５．４７３八ｌＨｚ。'HNMR is 300.13MHz, 500.13MHz
NMR: CNMR is 75.4738 lHz.

１２５　、　７５ＭＨｚ　ＮＭＲて、１５Ｎ　　ＮＭＲ
は５０、　６７ＭＨｚ　ＮＭＲで測定した。ＫＲＦ−０
０１の’ＨＮＭＲスペクトル、　ＩＨｌ）（ＮＭＲスペ
クトル、２Ｄ　’Ｈ−’Ｈコシスペクトル、２Ｄ　’Ｈ
−１３Ｃ相関−スベクトル、２Ｄ’Ｈ−１５Ｎ逆相関ス
ペクトル等を解釈した結果、ＫＲＦ−００１内にチロシ
ン、アスパラギン、セリン、プロリン等のアミノ酸か存
在することを判ることができた。125, 75MHz NMR, 15N NMR
was measured by 50 and 67 MHz NMR. KRF-0
'H NMR spectrum of 01, IHl) (NMR spectrum, 2D 'H-'H Koshi spectrum, 2D 'H
As a result of interpreting the -13C correlation spectrum, 2D'H-15N anticorrelation spectrum, etc., it was found that amino acids such as tyrosine, asparagine, serine, and proline were present in KRF-001.

実施例　４ ＫＲＦ−００１の皮膚関連抗真菌治療効果抗真菌性ペプ
チド物質ＫＲＦ−００１を水、７０％エタノールまたは
グリセリンと混せて存効濃度か０．１乃至０．５％（ｖ
　／　ｖ　）となるようにした後、水虫、湿疹、頑癖等
の症状を見せる皮膚の表面に塗布したとき、病症が顕著
に減少して３乃至５日後には治療効果か顕著なることを
観察することができた。Example 4 Skin-related antifungal therapeutic effect of KRF-001 The antifungal peptide substance KRF-001 was mixed with water, 70% ethanol or glycerin to give an effective concentration of 0.1 to 0.5% (v
/v), and when applied to the surface of the skin showing symptoms such as athlete's foot, eczema, and stubbornness, it was observed that the symptoms were significantly reduced and the therapeutic effect was noticeable after 3 to 5 days. We were able to.

実施例　５ ＫＲＦ−００１の皮膚保湿効果 抗真菌ペプチド物質ＫＲＦ−００１を皮膚保護用ローシ
ョンクリームやジャンプ中に０２％（Ｖ／Ｖ）入れて皮
膚に塗った時、皮膚に和かな感を与え皮膚の乾燥を抑制
し清潔な皮膚を保つことが出来た。Example 5 Skin moisturizing effect of KRF-001 When the antifungal peptide substance KRF-001 is applied to the skin with 02% (V/V) in a skin protection lotion cream or in a jump, it gives a soothing feeling to the skin and improves the skin. It was possible to suppress dryness and maintain clean skin.

実施例　６ ＫＲＦ−００１の果物保存効果 良く熟れた苺を農場から直接採取して蒸留水で土を洗い
落とした後、一つの試験区では苺の表面に灰色の黴胞子
を直接処理し、他の一つの試験区では苺等をＫＲＦ−０
０１生産菌株の培養液中に１０分間入れてから取出した
後、苺の表面に灰色の黴胞子を処理した。１週間２５°
Ｃの培養機中で培養後観察した結果、試験区（ＫＲＦ−
００１無処理区）の苺はすへて腐敗したかＫＲＦ−００
１醗酵液で処理した二番目の試験区の苺等は未だに新鮮
度を保っていた。Example 6 Effect of KRF-001 on fruit preservation Ripe strawberries were collected directly from the farm and the soil was washed off with distilled water. In one test area, the surface of the strawberries was directly treated with gray mold spores, and in the other In one test area, strawberries etc. were grown using KRF-0.
After placing the strawberries in the culture solution of the 01 production strain for 10 minutes and removing them, the surface of the strawberries was treated with gray mold spores. 25° for 1 week
As a result of observation after culturing in the incubator C, it was found that the test group (KRF-
Are the strawberries in the 001 non-treated area completely rotten?KRF-00
The strawberries, etc. in the second test group treated with the fermentation solution 1 still maintained their freshness.

実施例　７ ＫＲＦ−００１の稲熱病予防効果 洛東稲を栽培する水田における稲熱病予防のための圃場
試験を行った。粗ＫＲＦ−００１（純度２５％）を水に
稀釈して有効濃度かｌ１４ｐｐｍ、２３１ｐｐｍおよび
４６２ｐｐｍとなるようにした後、２次に亘って稲葉の
表面に処理した。対照薬剤としてプラスナシジン−Ｓ１
カスカマイシン、トリサイクラゾル等を各々２０．２０
．３７５ｐｐｍで撒布した。約２週聞役稲熱病防除効果
を比較した結果ＫＲＦ−００１は１１４ｐｐｍにて７４
％、２３１ｐｐｍ４ごて７６％、４６２ｐｐｍにて７７
％の防除効果を見せており、既存の対照薬剤であるフ゛
ラスチシジン−８は６５％、カス力マイシンは７５％、
トリサイクラゾルは８２％の防除効果を見せた。Example 7 Effect of KRF-001 on preventing rice fever disease A field test was conducted to prevent rice fever disease in paddy fields where Rakudong rice is cultivated. Crude KRF-001 (purity 25%) was diluted in water to give effective concentrations of 14 ppm, 231 ppm, and 462 ppm, and then treated on the surface of rice leaves in two steps. Plus Nasidin-S1 as control drug
20.20 each for cascamycin, tricyclazole, etc.
．． It was applied at 375 ppm. As a result of comparing the effect of controlling rice fever after about 2 weeks of use, KRF-001 was 74% at 114ppm.
%, 231ppm4 76%, 77 at 462ppm
The existing control drugs, phylasticidin-8, have shown a control effect of 65%, kasulymicin has a control effect of 75%,
Tricycrazole showed a control effect of 82%.

実施例　８ ＫＲＦ−００１の苺灰色黴病防除効果 苺灰色黴病防除実験のための圃場実験を行った。Example 8 Strawberry gray mold control effect of KRF-001 A field experiment was conducted to control strawberry gray mold.

粗ＫＲＦ−００１（純度２５％）を稀釈して有効濃度か
５００ｐｐｍ、Ｉｏｏｏｐｐｍおよび１５００ｐｐｍな
るようにした後、熟れていく苺等のある圃場に撒布した
。対照薬剤として、ベノミル５００ｐｐｍを撒布した。Crude KRF-001 (purity 25%) was diluted to give an effective concentration of 500 ppm, 100 ppm, and 1500 ppm, and then spread on fields with ripening strawberries, etc. As a control drug, 500 ppm of Benomyl was applied.

１週間後病気の罹った苺の比率を調査した結果ＫＲＦ−
００１処理区は５００ｐｐｍにて１７％、１１０００ｐ
ｐにて４７％および１５００ｐｐｍにて２１％の発病防
除率を見せており、対照薬であるベノミルは５００ｐｐ
ｍにて４８％の防除効果を見せた。After one week, we investigated the percentage of strawberries that were infected and found that KRF-
001 treatment area is 17% at 500ppm, 11000p
It showed a disease control rate of 47% at p.
It showed a control effect of 48% in M.

ＫＲＦ−００１の局所刺激性および急性毒性試験ＫＲＦ
−００１の皮膚１次刺激性に対して次のように試験を行
った。４ケ月経った雄の兎（体重２乃至３ｋｇ）２匹を
選定して２．５Ｘ２．５ｃｍ範囲で４ケ処に擦過傷を作
り０．５ｇのＫ　ＲＦ　−００１を蒸留水０．５ｍｌに
溶解させた溶液を上記擦過および非擦過部位に２４時間
の間過用させた。Local irritation and acute toxicity test of KRF-001 KRF
-001 was tested for primary skin irritation as follows. Two male rabbits (weight 2 to 3 kg) aged 4 months were selected and abrasions were made in 4 places in an area of 2.5 x 2.5 cm, and 0.5 g of K RF-001 was dissolved in 0.5 ml of distilled water. The solution was applied to the abraded and non-abrasive areas for 24 hours.

投与した１週間後、−膜状態および適用部に特異なる刺
激性反応は観察されなかった。One week after administration, no specific irritating reactions were observed in the membrane condition or at the application site.

ＫＲＦ−００１の急性経口毒性に関しては７乃至８週経
ったＴＣＰマウス雄７匹（３５±５ｇ）と雌７匹（２５
±５ｇ）を用いて試験を行った。Regarding the acute oral toxicity of KRF-001, 7 male TCP mice (35 ± 5 g) and 7 female TCP mice (25
±5g).

２乃至３匹からなる各群に５００．１０００および５０
００ｍｇ／体重ｋｇの容量でＫＲＦ−００１をゾンデ（
ｓｏｎｄｅ）を用いて経口強制的に投与した。500.1000 and 50 for each group of 2 to 3 animals.
KRF-001 was used as a sonde with a capacity of 00 mg/kg body weight.
The drug was administered orally by force using a tube (sonde).

投与してから２週間後５　ｏ　ＯＯｍｇ／ｋｇ投与群の
雄群からたけ３匹の内１匹か投与後３日目に死亡した。Two weeks after administration, one of the three male rats in the 5 o OOmg/kg administration group died on the third day after administration.

死亡前の臨床症状は鱒り（ｃｒｏｕｃｈｉｎｇ）　、閉
眼、毛の反り立ち（ｒｕｆｆｌ、ｅｄ　ｆｕｒ）等であ
ったか生存動物からは特異な内容か観察されなかった。Clinical symptoms before death included crouching, closed eyes, and ruffled fur, but no unusual symptoms were observed in the surviving animals.

各群（５００、ｌ０００、ｓ　ｏ　ｏ　ｏ　ｍｇ／ｋｇ
投与群）すべてから特異的体重減少または抑制傾向は観
察されなかった。剖検した結果死亡動物（５０００ｍｇ
／ｋｇ群の雄１匹）から腸管内に黒褐色の内容物か観察
されたかその他の特異な所見は観察されなかった。本試
験の結果、試験物質ＫＲＦ−００１は直接的な刺激でも
比較的毒性のないものと現れた。Each group (500, 1000, s o o o mg/kg
No specific tendency toward weight loss or suppression was observed in all treatment groups. Dead animal as a result of autopsy (5000mg
Blackish brown contents were observed in the intestinal tract of one male in the /kg group), and no other peculiar findings were observed. As a result of this test, the test substance KRF-001 appeared to be relatively non-toxic even when directly stimulated.

一方、マウスの急性経口毒性は５０００　ｍｇ／ｋｇ群
における１匹が死亡してＬＤ５′ｏ値は５０００ｍｇ／
ｋｇ以上の容量として推定される。On the other hand, acute oral toxicity in mice resulted in the death of one mouse in the 5000 mg/kg group, and the LD5'o value was 5000 mg/kg.
Estimated to have a capacity of more than kg.

〔発明の効果〕〔Effect of the invention〕

本発明に基づく新規な微生物バシラス・サブチリス亜種
クリッチエンシス（Ｂａｃｉｌｌｕｓ　　５ｕｂｔｉｌ
ｉｓｓｕｂｓｐ、　　Ｋｒ１ｃｔｉｅｎｓｉｓ、　　Ａ
ＴＣＣ５５０７９）およびこの変異株のバシラス・ザブ
チリス亜種りリッチェンシス　Ｍ　　１８−９１　　（
Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ　　５ｕｂｓｐ、　
　Ｋｒ１ｃｔｉｅｎｓｉｓ、　　Ｍ　　１８−９１、Ａ
ＴＣＣ５５０７８）によって生産される新規抗真菌性物
質ＫＲＦ−００１複合体、それから分離可能な各成分ま
たはそれらの誘導体は各種の用途を持っている。ＫＲＦ
−００１複合体は真菌感染症治療剤、皮膚保湿剤、保存
剤および殺真菌剤等に用いることができ、このような生
物学的活性を保ちながらも充分に低毒性乃至は無公害性
なることを示した。A novel microorganism based on the present invention, Bacillus subtilis subsp.
issubsp, Kr1ctiensis, A
TCC55079) and this mutant strain Bacillus subspecies Lichensis M 18-91 (
Bacillus subtilis 5ubsp,
Kr1ctiensis, M 18-91, A
The novel antifungal substance KRF-001 complex produced by TCC55078), its separable components, or their derivatives have various uses. KRF
The -001 complex can be used as a fungal infection treatment, a skin moisturizer, a preservative, a fungicide, etc., and has sufficiently low toxicity or non-pollution while maintaining such biological activity. showed that.

手続補正書（自発） 平成３年１月、Ｊｏ日Procedural amendment (voluntary) January 1991, Jo day

Claims (1)

【特許請求の範囲】 （１）ペプチド抗真菌物質を生産するバシラス・サブチ
リス亜種クリッチエンシス（￥Ｂａｃｉｌｌｕｓ￥￥ｓ
ｕｂｔｉｌｉｓ￥　ｓｕｂｓｐ．￥Ｋｒｉｃｔｉｅｎｓ
ｉｓ￥．ＡＴＣＣ　５５０７９）。 （２）ペプチド系抗真菌物質を生産するバシラス・サブ
チリス亜種クリッチエンシスＭ　１８−９１（￥Ｂａｃ
ｉｌｌｕｓ￥　￥ｓｕｂｔｉｌｉｓ￥　ｓｕｂｓｐ．￥
Ｋｒｉｃｔｉｅｎｓｉｓ￥Ｍ−１８−９１、ＡＴＣＣ　
５５０７８）。 （３）第１項または第２項の新規微生物を培養または醗
酵させて生産した下記成分Ａ、Ｂ、Ｃ、Ｄ、ＥおよびＦ
からなるペプチド系抗真菌物質ＫＲＦ−００１複合体。 ▲数式、化学式、表等があります▼ 上記式において、特異β−アミノ酸は成分Ａ、Ｂ、Ｃ、
Ｄ、ＥおよびＦにおいて各々次の通りである： 成分Ａ：ＣＨ＿３（ＣＨ＿２）＿１＿０ＣＨ（ＮＨ＿２
）ＣＨ＿２ＣＯＯＨ成分Ｂ：ＣＨ＿３ＣＨ＿２ＣＨ（Ｃ
Ｈ＿３）（ＣＨ＿２）＿３ＣＨ（ＮＨ＿２）ＣＨ＿２Ｃ
ＯＯＨ 成分Ｃ：ＣＨ＿３ＣＨ（ＣＨ＿３）（ＣＨ＿２）＿３Ｃ
Ｈ（ＮＨ＿２）ＣＨ＿２ＣＯＯＨ成分Ｄ：ＣＨ＿３ＣＨ
（ＣＨ＿３）（ＣＨ＿２）＿１＿０ＣＨ（ＮＨ＿２）Ｃ
Ｈ＿２ＣＯＯＨ 成分Ｅ：ＣＨ＿３（ＣＨ＿２）＿１＿２ＣＨ（ＮＨ＿２
）ＣＨ＿２ＣＯＯＨ成分Ｆ：ＣＨ＿３ＣＨ＿２ＣＨ（Ｃ
Ｈ＿３）（ＣＨ＿２）＿１＿０ＣＨ（ＮＨ＿２）ＣＨ＿
２ＣＯＯＨ（４）ＫＲＦ−００１複合体から分離可能な
抗真菌性成分Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆおよびそれらの誘
導体。 （５）ＫＲＦ−００１の人間および動物に対する皮膚関
連真菌感染症治療剤としての用途。 （６）ＫＲＦ−００１の皮膚の保湿剤としての用途。 （７）ＫＲＦ−００１の果物、野菜および食品保存剤と
しての用途。 （８）ＫＲＦ−００１の農業用殺真菌剤としての用途。[Scope of Claims] (1) Bacillus subtilis subsp. krichensis that produces peptide antifungal substances
ubtilis￥ subsp. ￥Krictiens
is￥. ATCC 55079). (2) Bacillus subtilis subspecies crichensis M 18-91 (￥Bac
illus￥￥subtilis￥ subsp. ￥
Krictiensis￥M-18-91, ATCC
55078). (3) The following components A, B, C, D, E, and F produced by culturing or fermenting the novel microorganisms described in paragraph 1 or 2.
A peptide antifungal substance KRF-001 complex consisting of. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ In the above formula, the specific β-amino acids are components A, B, C,
In D, E and F, respectively: Component A: CH_3(CH_2)_1_0CH(NH_2
) CH_2COOH component B: CH_3CH_2CH(C
H_3)(CH_2)_3CH(NH_2)CH_2C
OOH Component C: CH_3CH (CH_3) (CH_2)_3C
H(NH_2)CH_2COOH component D: CH_3CH
(CH_3)(CH_2)_1_0CH(NH_2)C
H_2COOH Component E: CH_3(CH_2)_1_2CH(NH_2
) CH_2COOH component F: CH_3CH_2CH(C
H_3)(CH_2)_1_0CH(NH_2)CH_
2COOH(4) Antifungal components A, B, C, D, E, F and derivatives thereof separable from the KRF-001 complex. (5) Use of KRF-001 as a therapeutic agent for skin-related fungal infections in humans and animals. (6) Use of KRF-001 as a skin moisturizer. (7) Use of KRF-001 as a fruit, vegetable and food preservative. (8) Use of KRF-001 as an agricultural fungicide.
JP2336898A 1990-10-31 1990-11-30 Novel bacillus subtilis subspecies and fungicidal substance krf-001 complex produced therefrom Pending JPH04166080A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR90-17551 1990-10-31
KR1019900017551A KR930001383B1 (en) 1990-10-31 1990-10-31 New microoganism bacillus subtilis subspecies

Publications (1)

Publication Number Publication Date
JPH04166080A true JPH04166080A (en) 1992-06-11

Family

ID=19305446

Family Applications (1)

Application Number Title Priority Date Filing Date
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Country Status (2)

Country Link
JP (1) JPH04166080A (en)
KR (1) KR930001383B1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011756A1 (en) * 1997-09-03 1999-03-11 Washi Kosan Co., Ltd. Novel microorganism
WO1999028441A1 (en) * 1997-12-01 1999-06-10 Eisai Co., Ltd. Novel bacillus subtilis with antibacterial effects
JP2013522315A (en) * 2010-03-17 2013-06-13 アグラクエスト インコーポレイテッド Method of using Bacillus subtilis strains for the prevention and treatment of gastrointestinal conditions
US9247757B2 (en) 2008-09-17 2016-02-02 Bayer Cropscience Lp Method for using a Bacillus subtilis strain to enhance animal health
JP2021165282A (en) * 2017-03-27 2021-10-14 テンフォールド テクノロジーズ リミティッド ライアビリティ カンパニー Methods and Agricultural Compositions for Preventing or Controlling Plant Diseases

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101704652B1 (en) 2015-08-27 2017-02-23 한국해양과학기술원 Antimicrobial agents from a marine-derived bacterium Bacillus sp. having inhibition effects of zoospore motility of the phytopathogen Phytophthora capsici

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999011756A1 (en) * 1997-09-03 1999-03-11 Washi Kosan Co., Ltd. Novel microorganism
US6399056B1 (en) 1997-09-03 2002-06-04 Washi Kosan Co., Ltd. Microorganism
WO1999028441A1 (en) * 1997-12-01 1999-06-10 Eisai Co., Ltd. Novel bacillus subtilis with antibacterial effects
US9247757B2 (en) 2008-09-17 2016-02-02 Bayer Cropscience Lp Method for using a Bacillus subtilis strain to enhance animal health
JP2013522315A (en) * 2010-03-17 2013-06-13 アグラクエスト インコーポレイテッド Method of using Bacillus subtilis strains for the prevention and treatment of gastrointestinal conditions
US9457054B2 (en) 2010-03-17 2016-10-04 Bayer Cropscience Lp Method for using a Bacillus subtilis strain for prophylaxis and treatment of gastro-intestinal conditions
JP2021165282A (en) * 2017-03-27 2021-10-14 テンフォールド テクノロジーズ リミティッド ライアビリティ カンパニー Methods and Agricultural Compositions for Preventing or Controlling Plant Diseases

Also Published As

Publication number Publication date
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KR920008183A (en) 1992-05-27

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