JPS5940127B2 - Koushiyokubutsu Virus - Google Patents

Description

【発明の詳細な説明】 本発明は酵母菌体抽出液から抗植物ウィルス性画分を製
造する方法に係り、更に詳しくは、酵母菌体抽出液に一
価の銅化合物を加えて銅塩反応せしめ、次いで銅塩反応
に依り生じる銅塩沈殿を分離除去することにより抗植物
ウィルス性画分を得る方法に係る゜ものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an anti-plant virus fraction from a yeast cell extract, and more specifically, a monovalent copper compound is added to a yeast cell extract to perform a copper salt reaction. The present invention relates to a method for obtaining an anti-plant virus fraction by separating and removing the copper salt precipitate produced by the copper salt reaction.

近年、微生物工業の発達に依り有用物質の宝庫とでも言
うべき微生物菌体が、多量且つ安定的に、しかも比較的
安価に入手しうるようになつて来た。In recent years, with the development of the microbial industry, microbial cells, which can be called a treasure trove of useful substances, have become available stably in large quantities and at relatively low prices.

人間は古くから微生物を利用し、多大の恩恵を受けて来
たが、まだまμ微生物の持つ一部の能力しか利用してお
らず更に有効に利用することが望まれている。特に未利
用資源の有効利用が叫ばれている昨今、微生物工業に於
いても微生物の持つ能力を、食用、薬用、工業農業と多
岐に亘り総合的に有効利用することが切望されている。
その中に於いて、植物ウィルスに依る作物の被害が極め
て大きく、産業上大きな損失となつている。Humans have been using microorganisms for a long time and have received great benefits from them, but we are still only utilizing a portion of the capabilities of microorganisms, and it is desired to use them even more effectively. Particularly in recent years, where the effective use of unused resources is being called for, there is a strong desire for the comprehensive and effective use of the abilities of microorganisms in a wide range of fields, including food, medicine, and industrial agriculture, even in the field of microbial industry.
In this situation, damage to crops caused by plant viruses is extremely large, resulting in large industrial losses.

これに対し、安全な抗植物ウィルス剤として酵母菌体抽
出物（特公昭４９−２０４９０）や農業用抗ウィルス剤
（特開昭５０−１２２３０）など酵母を原料とした抗植
物ウィルス剤が提案されて（・るが、未だ実用には供さ
れていない。そこで本発明者らは、実用に供しうる抗植
物ウィルス剤の要件は、安全であり、ウィルスの感染を
効果的に阻止し、植物体内に於いてウィルスの増殖を抑
える所謂浸透移行性を有し、しかも、経済的に製造し得
るものであるという考えに基いて、酵母の未利用部分か
らの実用に供しうる抗植物ウィルス剤の製造方法につい
て鋭意研究した結果、従来酵母からのグルタチオン（以
下、ＧＳＨと称す）製造工程に於いて廃液として処理さ
れていた区分に、高い植物ウィルス感染阻止能力のある
ことを発見した。即ち、本発明は酵母菌体抽出液に一価
の銅化合物を加え銅塩反応を起こさしめ、次いで銅塩反
応により生じる銅塩沈殿を分離除去することを特徴とす
る、工業的に有利で、安全且つ抗ウィルス性の顕著な抗
ウィルス性画分の製造方法を提供するものである。本発
明に於いて用いられる酵母菌体抽出液は通常の方法で培
養して得られる通常の生もしくは乾燥酵母から水抽出さ
れたもので、抽出時のＰＨ、温度、圧、時間などは特に
限定されない。In response, yeast-based anti-plant virus agents such as yeast cell extract (Japanese Patent Publication No. 49-20490) and agricultural anti-virus agent (Japanese Patent Application Laid-open No. 12230-1983) have been proposed as safe anti-plant virus agents. However, it has not yet been put to practical use. Therefore, the present inventors believe that the requirements for a practically usable anti-plant virus agent are that it is safe, effectively inhibits virus infection, and that it is effective in plants. The production of a practically usable anti-plant virus agent from the unused parts of yeast is based on the idea that it has so-called systemic transfer properties that suppress the proliferation of viruses in yeast, and can be produced economically. As a result of intensive research on the method, it was discovered that a segment that was conventionally treated as waste liquid in the production process of glutathione (hereinafter referred to as GSH) from yeast has a high ability to inhibit plant virus infection.In other words, the present invention is an industrially advantageous, safe and antiviral method that is characterized by adding a monovalent copper compound to yeast cell extract to cause a copper salt reaction, and then separating and removing the copper salt precipitate produced by the copper salt reaction. This invention provides a method for producing a fraction with remarkable antiviral properties.The yeast cell extract used in the present invention is extracted with water from ordinary fresh or dried yeast obtained by culturing in a conventional manner. The pH, temperature, pressure, time, etc. during extraction are not particularly limited.

本発明は、該酵母菌体抽出液に、−価の銅化合物を添加
して銅塩反応せしめるのであるが、本発明に言う銅塩反
応とは、例えば臭化第一銅塩化第一銅、亜酸化銅、亜硫
酸銅などの一価の銅化合物を酸性下に於いて反応系に添
加し、ＧＳＨ、ペプチド、タンパクなどを銅塩として沈
殿させる反応を言う。In the present invention, a -valent copper compound is added to the yeast cell extract to cause a copper salt reaction.The copper salt reaction referred to in the present invention includes, for example, cuprous bromide, cuprous chloride, A reaction in which a monovalent copper compound such as cuprous oxide or copper sulfite is added to a reaction system under acidic conditions to precipitate GSH, peptides, proteins, etc. as copper salts.

この銅塩反応のＰＨ、温度、一価の銅化合物の添加量等
の反応条件は、原料酵母、培養条件抽出方法などに依つ
て異なるが、好ましくは酵母菌体抽出液中の全システイ
ン残基と当量ないし２当量に相当する一価の銅化合物を
ＰＨＯ．５乃至ＰＨ２の酸性下に添加する。The reaction conditions for this copper salt reaction, such as pH, temperature, and the amount of monovalent copper compound added, vary depending on the raw material yeast, culture conditions, extraction method, etc., but preferably all cysteine residues in the yeast cell extract are A monovalent copper compound equivalent to PHO. Add under acidic conditions of pH 5 to 2.

該銅塩反応に依り、ＧＳＨ、ペプチド、タンパクなどが
銅塩として沈殿するので、傾斜法、沢過法、遠心分離法
などの方法で沈殿物を除去する。該沈殿からは、後処理
してＧＳＨｌペプチド、タンパクなどが得られる。かく
して得られた沈殿除去した上澄を中和するだけで抗植物
ウイルス剤として供し得るが、相当量の塩類を含むので
脱塩するのが望ましい。勿論、更に、透析法、電気透析
法ゲル▲過法、イオン交換法、有機溶剤沈殿法あるいは
不溶塩形成法、その他の方法で精製することもできる。
本発明の方法に従うなら、従来廃棄していた酵母の未利
用区分からウイルス感染阻止能力に優れ、且つ、浸透移
行性を有する経済的で実用に供しうる抗植物ウイルス剤
を提供でき、その産業上の価値は著大である。Due to the copper salt reaction, GSH, peptides, proteins, etc. are precipitated as copper salts, so the precipitates are removed by a method such as a slanting method, a filtration method, or a centrifugation method. The precipitate can be post-treated to obtain GSHl peptides, proteins, and the like. The supernatant obtained in this way after removing the precipitate can be used as an anti-plant virus agent by simply neutralizing it, but since it contains a considerable amount of salts, it is desirable to desalt it. Of course, further purification can be carried out by dialysis, electrodialysis, gel filtration, ion exchange, organic solvent precipitation, insoluble salt formation, or other methods.
If the method of the present invention is followed, it is possible to provide an economical and practical anti-plant virus agent that has excellent virus infection inhibiting ability and systemic transferability from the unused portion of yeast that was previously discarded, and can be used industrially. is of great value.

以下、実施例、比較例、試験例を挙げて本発明を説明す
る。The present invention will be explained below with reference to Examples, Comparative Examples, and Test Examples.

実施例１〜３，比較例１ サツカロミセス・セルビシエの酵母ミルクを７５℃、５
分間加熱抽出し、▲過して酵母菌体抽出液（比較例１）
を得た。Examples 1 to 3, Comparative Example 1 Satucharomyces cerevisiae yeast milk at 75°C, 5
Extract by heating for ▲ for yeast cell extract (Comparative Example 1)
I got it.

該酵母菌体抽出液に硫酸を加えてＰＨＯ．７とした後、
亜酸化銅を２．３η／ｍｌになるように添加し、室温で
２時間銅塩反応せしめ、生成した銅塩沈殿を▲過除去し
て、黄色の上澄（実施例１）を得た。該上澄をビスキン
体※グチユーブを用いて流水に対し、２日間透析して残
留区分（実施例２）、及び終濃度７５％になるようメタ
ノールを加えて生じた沈殿区分（実施例３）をそれぞれ
得た。実施例４、５；比較例２ キヤンデイダ・ユチリスの乾燥粉末１００７に水９００
７を加え９０℃に加熱して３分間抽出し、遠心分離して
酵母菌体抽出液（比較例２）を得た。Sulfuric acid was added to the yeast cell extract and PHO. After setting it to 7,
Cuprous oxide was added at a concentration of 2.3 η/ml, and the copper salt reaction was allowed to occur at room temperature for 2 hours. The produced copper salt precipitate was removed by filtration to obtain a yellow supernatant (Example 1). The supernatant was dialyzed against running water using a Viskin body*gut tube for 2 days to obtain a residual fraction (Example 2) and a precipitate fraction (Example 3) produced by adding methanol to a final concentration of 75%. I got each. Examples 4 and 5; Comparative Example 2 100% dry powder of Candida utilis to 900% water
7 was added, heated to 90°C, extracted for 3 minutes, and centrifuged to obtain a yeast cell extract (Comparative Example 2).

該抽出液を硫酸でＰＨｌとした後、亜硫酸銅を４．０η
／ｍｌになるように添加し、室温で１時間銅塩反応せし
め、生成した銅塩沈殿を遠心除去して黄色上澄（実施例
４）を得た。該上澄にまず２５％濃度になるようメタノ
ールを加え、生じた沈殿を遠心除去後、更に士澄に終濃
度７５％になるようにメタノールを加えて生じた沈殿区
分（実施例５）を得た。試験例 検体としてニコチアナ・グルチノーザの１４乃至１５葉
期の最大葉を中心に、上位葉、中位葉、下位葉の３枚の
葉を付けた苗を用い、比較例及び実施例処理区は各薬剤
の０．５％水溶液を、また対照区は水をそれぞれ中位葉
の表裏にのみ散布し、乾燥後タバコ王ザイクウイルスを
常法のカーボランダム法に従い３葉の表に接種した接種
４日後、各中位葉上に形成された斑点数を数え、下式に
依り感染阻止率を求め薬効を判断した。After converting the extract to PHL with sulfuric acid, copper sulfite was added to 4.0η
/ml and allowed to react with the copper salt at room temperature for 1 hour, and the produced copper salt precipitate was removed by centrifugation to obtain a yellow supernatant (Example 4). First, methanol was added to the supernatant to give a concentration of 25%, and the resulting precipitate was removed by centrifugation, and then methanol was added to the supernatant to give a final concentration of 75% to obtain the resulting precipitate section (Example 5). Ta. As test specimens, seedlings of Nicotiana glutinosa with three leaves, the upper leaf, the middle leaf, and the lower leaf, centered on the largest leaf at the 14th to 15th leaf stage, were used. A 0.5% aqueous solution of the drug and water for the control plot were sprayed only on the front and back of the middle leaves, and after drying, Tobacco King Zaik virus was inoculated on the top of 3 leaves according to the conventional carborundum method. 4 days after inoculation. The number of spots formed on each medium leaf was counted, and the infection inhibition rate was calculated using the formula below to determine the medicinal efficacy.

Claims (1)

【特許請求の範囲】[Claims] １ 酵母菌体抽出液に一価の銅化合物を加えて銅塩反応
せしめ、次いで銅塩反応に依り生じる銅塩沈殿を分離除
去することを特徴とする抗植物ウィルス性画分の製造法
。1. A method for producing an anti-plant virus fraction, which comprises adding a monovalent copper compound to a yeast cell extract to cause a copper salt reaction, and then separating and removing a copper salt precipitate produced by the copper salt reaction.