JP3577485B2 - Plant growth fertilizer derived from soybean meal using a novel Bacillus circulans fungus - Google Patents

Description

【００１６】
［菌株の同定］
分離選別した大豆粕分解菌の菌学的性質を調べるために、グラム染色を始めとした種々の確認試験をそれぞれ下記の手法により行なった。
【００１７】
１．グラム染色
Ｈｕｃｋｅｒの変法に従って、スライドガラス上に塗抹し、乾燥後火炎固定した菌液について、Ｈｕｃｋｅｒの液（１．５％クリスタルバイオレット−エタノール溶液）で３０秒間染色した。染色後ただちに水洗いし、Ｌｕｇｏｌ 液で１分間媒染した。媒染終了後水洗し、さらにアルコールで３０秒間脱色し、ただちに水洗した。水をよく切った後、サフラニンで３０秒間染色を行なった。染色した標本を顕微鏡で観察し、紫色に染色された菌をグラム陽性とし、赤色に染色された菌をグラム陰性とした。また、検鏡により菌の形態を観察した。
【００１８】
２．生育温度
下記組成の液状チオグリコレート培地を１２１℃で１５分間滅菌し、試験管に５ml分注した。この培地にコロニーから採取した菌を植え、３７℃，２５℃のそれぞれの中温域温度で静置培養して、１２時間または２４時間後に培地が混濁したものを生成したと判断した。
＜液状チオグリコレート培地の組成＞
Ｌ−シスチン ０．７５ｇ
ＮａＣｌ ２．５ｇ
ブドウ糖 ５．０ｇ
酵母エキス ５．０ｇ
寒天 ０．７５ｇ
カゼイン消化物 １５．０ｇ
チオグリコール酸ナトリウム ０．５ｇ
０．１％レザズリン １．０ml
蒸留水 １０００ml
ｐＨ ７．２
【００１９】
３．オキシダーゼ
テルモ（株）製バクトラボ オキシダーゼ・テストを用いて試験した。即ち、二塩化Ｎ，Ｎ，Ｎ’，Ｎ’−テトラメチル−ｐ−フェニレンジアミンを含んだ綿棒に菌体を付け、オキシダーゼ活性により生成する青紫色を生じた場合に、オキシダーゼ陽性と判断した。
【００２０】
４．カタラーゼ
毛細管の一端に菌を付けた後、毛細管の菌を付けた一端から３％過酸化水素水を吸い上げて菌体と過酸化水素水とを毛細管内で混合させた。毛細管内で過酸化水素分解にともない酸素の気泡が発生した場合をカタラーゼ陽性とした。
【００２１】
５．硝酸塩還元能
０．１％硝酸ナトリウム加ブイヨン培地に菌を接種し、最適生育温度で５日間培養した。得られた培養液に、α− ナフチルアミン液（α− ナフチルアミン０．１ｇを３０％酢酸水溶液２００ｍｌに溶解したもの）１．０ｍｌとスルファニル酸液（スルファニル酸０．５ｇを３０％酢酸水溶液１５０ｍｌに溶解したもの）１．０ｍｌを加えてよく混和させた後、３０分以内に硝酸還元により生成した亜硝酸による桃赤色を呈した場合を陽性とした。
【００２２】
６．クリグラー培地
下記組成のクリグラー培地を試験管に分注した後、１２１℃で１５分間高圧蒸気滅菌して高層斜面に固めた。斜面部分および高層部分に被検菌を植え、各菌の生育温度で１８〜２４時間培養した。培養後、斜面部分が黄変色した場合を斜面部陽性（乳糖を分解し酸を生成）とし、高層部分で黄変色した場合を高層部陽性（ブドウ糖を分解し酸を生成）とした。
尚、表２に示した（ＮＣ）は斜面部陰性を、（Ｙ）は高層部陽性を、（Ｒ）は高層部陰性を、それぞれ意味する。
＜クリグラー培地の組成＞
肉エキス ４．０ｇ ペプトン １５．０ｇ
乳糖 １０．０ｇ ブドウ糖 １．０ｇ
塩化ナトリウム ５．０ｇ チオ硫酸ナトリウム ０．０８ｇ
亜硫酸ナトリウム ０．４ｇ 硫酸第一鉄 ０．２ｇ
フェノール赤 ０．０２ｇ 寒天末 １５．０ｇ
精製水 １０００ｍｌ ｐＨ ７．２
【００２３】
７．ＳＣ培地
下記組成の培地を蒸留水に溶し約２ｍｌずつ試験管に分注し、１２１℃で１５分間高圧蒸気滅菌し全斜面に固めた。斜面部分に被検菌を植え、各菌の生育温度で１８〜２４時間培養した。この培地に含まれる唯一の炭素源であるクエン酸を利用し生育した場合を陽性とした。陽性の場合に、培地に含まれるブロム・チモールが緑色から深青色に変色した。
＜ＳＣ培地の組成＞
塩化ナトリウム ５．０ｇ
硫酸マグネシウム ０．２ｇ
リン酸二水素アンモニウム １．０ｇ
リン酸二水素カリウム １．０ｇ
クエン酸ナトリウム ２．０ｇ
ブロム・チモール青 ０．０８ｇ
寒天末 １５．０ｇ
精製水 １０００ｍｌ
ｐＨ ６．８
【００２４】
８．ＳＩＭ培地
下記組成の培地を１０００ｍｌの蒸留水に加温溶解して約３ｍｌずつ試験管に分注し、１２１℃で１５分間高圧蒸気滅菌した後高層に固めた。被検菌を高層部分に穿刺し、それぞれの菌の生育温度で１８〜２４時間培養した。培養後培地が黒変した場合を硫化水素発生（＋）とし、穿刺部分から広がって生育した場合を運動性（＋）とし、さらに培地上部にクロロホルム１ｍｌを重層し、その上にコバック試薬１ｍｌを重層してクロロホルム層が赤変した場合をインドール（＋）とした。
＜ＳＩＭ培地の組成＞
エールリッヒ肉エキス ３．０ｇ
プロテオーゼペプトン １０．０ｇ
ポリペプトン ２０．０ｇ
チオ硫酸ナトリウム ０．２ｇ
クエン酸鉄アンモニウム ０．２ｇ
寒天末 ３．０ｇ
精製水 １０００ｍｌ
ｐＨ ７．３
【００２５】
９．メチルレッド
下記組成のグルコース−リン酸塩−ペプトン水を試験管に３ｍｌ分注し、１００℃で３０分間の滅菌操作を３回繰返した。この培地に被検菌を植え、各菌の生育温度で３日間培養した。得られた培養液１ｍｌを別の試験管に移し、メチルレッドを滴下し、培地色調が赤変した場合を陽性とした。
＜グルコース−リン酸塩−ペプトン水の組成＞
ペプトン ７．０ｇ リン酸二水素カリウム ５．０ｇ
ブドウ糖 ５．０ｇ 蒸留水 １０００ｍｌ
【００２６】
１０．フォゲスプロスカウエル（ＶＰ）
上記グルコース−リン酸塩−ペプトン水を試験管に３ｍｌ分注し、１００℃で３０分間の滅菌操作を３回繰返した。この培地に被検菌を植え、各菌の生育温度で３日間培養した。得られた培養液１ｍｌを別の試験管に移し、これにα−ナフトール液（５％α−ナフトール−無水エタノール溶液）０．６ｍｌと４０％ＫＯＨ水溶液０．２ｍｌを加えてよく混和させた後、試験管を斜めに傾けて静置した。静置後１５分後と１時間後にそれぞれ培地色調の変化を観察し、培地色調が濃赤色となったものを陽性とした。
【００２７】
１１．ＯＦ（Ｏｘｉｄａｔｉｏｎ ｏｒ Ｆｅｒｍｅｎｔａｔｉｏｎ ）
Ｈｕｇｈ−Ｌｅｉｆｓｏｎの方法に従って、下記組成のＯＦ培地を１２１℃で１５分間高圧蒸気滅菌し、培地が６０℃程度に冷めたところで終濃度１％となるように濾過滅菌したグルコースを加え、得られた培地を２本の試験管にそれぞれ３ｍｌ分注し、高層に固めた。この培地に被検菌を穿刺し、１本の試験管には流動パラフィンを１〜２ｃｍの厚さに重層して嫌気的に培養し、他方の試験管はそのまま好気的に培養した。各菌体の生育温度で３〜４日間培養した後、酸化的糖分解を行なう菌で好気的条件で培養した培地色調が黄変したものを（Ｏ）と結果に表記し、発酵的糖分解を行なう菌で好気的及び嫌気的両条件で培地色調が黄変したものを（Ｆ）と結果に表記し、また４日間の培養後さらに次の７日間に糖分解が観察されたものを（Ｗ）と結果に表記した。
＜ＯＦ培地の組成＞
トリプトン ２．０ｇ 塩化ナトリウム ５．０ｇ
寒 天 ２．５ｇ リン酸二水素カリウム ０．３ｇ
Ｂ．Ｔ．Ｂ． ０．０３ｇ 蒸留水 １０００ｍｌ
ｐＨ ７．１
【００２８】
１２．Ｕｒｅａｓｅ
下記組成の栄研化学（株）製ウレアーゼ確認用尿素培地（滅菌済み）を用い、０．５〜１．０ｍｌを滅菌済の試験管に分注し、被検菌を接種した。この培地を各被検菌の生育温度で６〜２４時間培養し、アンモニアの生成にともなう培地の赤変を生じた場合を陽性とした。
＜尿素培地の組成＞
ペプトン ２．０ｇ 尿素 ３０．０ｇ
塩化ナトリウム ５．０ｇ リン酸１カリウム ９．０ｇ
リン酸２ナトリウム ３．０ｇ フェノールレッド ０．０１ｇ
ｐＨ ６．２
【００２９】
１３．ＮａＣｌ
１％ペプトン水に種々の濃度のＮａＣｌを加え、滅菌した培地（ｐＨ７．２）を約３ｍｌ試験管に加えて被検菌を植菌後、各菌体の生育温度で１８〜２４時間静置培養した。そして培養液が混濁したものを陽性とした。
【００３０】
１４．マンニット食塩（ＭＳ）培地
下記組成の栄研化学（株）製マンニット食塩培地「栄研」を用い、この培地１１２ｇを蒸留水１０００ｍｌに溶解して、１２１℃で１５分間高圧蒸気滅菌した。滅菌した培地を滅菌したシャーレに約２０ｍｌずつ分注し、平板に固めた。得られた平板培地に被検菌を植菌し、各菌体の生育温度で２４〜４８時間培養した。
マンニットを分解しコロニーの周囲が黄変したものをＭＳ陽性とし、黄変せず生育しコロニーを形成したものをＮａＣｌ陽性とした。
＜マンニット食塩培地「栄研」の組成＞
肉エキス「栄研」 ２．５ｇ ペプトン「栄研」 １０．０ｇ
マンニット １０．０ｇ 塩化ナトリウム ７５．０ｇ
フェノールレッド ０．０２５ｇ 寒 天 １５．０ｇ
【００３１】
１５．Ｈｅｍｏｌｙｓｉｓ
普通寒天１５ｍｌを溶解し４５℃に保ちながら、これに５％の割合で脱繊維素血液を加えた。この培地に微量の被検菌を植えてよく混和し、滅菌シャーレに注ぎ平板に固めた。各菌の生育温度で２４時間培養し、溶血の有無により判定した。溶血を起しハローを生じたものを陽性とした。
【００３２】
１６．エスクリン
１２１℃で１５分間高圧蒸気滅菌した下記組成のＢａｒｓｉｅｋｏｗ 培地が８０℃に冷えたところで濾過滅菌したエスクリン（配糖体）を０．５％の割合で加え、滅菌試験管に２ｍｌ分注した。この培地に被検菌を植菌し、各菌体の生育温度で１８〜２４時間培養し、エスクリン分解により培地色調が青色から黄変した場合を陽性とした。
＜Ｂａｒｓｉｅｋｏｗ培地の組成＞
ペプトン水 １００ｍｌ
０．２％Ｂ．Ｔ．Ｂ溶液 １．２ｍｌ
【００３３】
１７．ゼラチンの液化
普通ブイヨン１０００ｍｌに精製ゼラチン１００ｇを加え、１２１℃で１２分間高圧蒸気滅菌した培地を滅菌試験管に３ｍｌ分注し、高層に固めた。被検菌を高層部分に穿刺し、各菌体の生育温度で１週間培養した。穿刺線に沿ったゼラチン液化の有無を観察し、液化が観察されたものを陽性とした。
【００３４】
１８．オルニチンからの脱炭酸試験
Ｄｉｆｃｏ 社製 Ｓｔａｎｄａｒｄ Ｄｅｈｙｄｒａｔｅｄ Ｃｕｌｔｕｒｅ Ｍｅｄｉｕｍ １０．５ｇ及びＬ−オルニチン１０．０ｇを蒸留水１０００ｍｌに溶解させ、１２１℃で１５分間高圧蒸気滅菌し、滅菌試験管に３ｍｌ分注した。この培地に被検菌を植菌し、各菌体の生育温度で１８〜２４時間培養し、培地色調が小豆色から黄変したものを陽性とした。
【００３５】
１９．リジンからの脱炭酸試験
Ｄｉｆｃｏ 社製 Ｓｔａｎｄａｒｄ Ｄｅｈｙｄｒａｔｅｄ Ｃｕｌｔｕｒｅ Ｍｅｄｉｕｍ １０．５ｇ及びＬ−オルニチン１０．０ｇを蒸留水１０００ｍｌに溶解させ、１２１℃で１５分間高圧蒸気滅菌し、滅菌試験管に３ｍｌ分注した。この培地に被検菌を植菌し、各菌体の生育温度で１８〜２４時間培養し、培地色調が赤紫色から黄変したものを陽性とした。
【００３６】
２０．アルギニンからの脱炭酸試験
Ｄｉｆｃｏ 社製 Ｓｔａｎｄａｒｄ Ｄｅｈｙｄｒａｔｅｄ Ｃｕｌｔｕｒｅ Ｍｅｄｉｕｍ １０．５ｇ及びＬ−オルニチン１０．０ｇを蒸留水１０００ｍｌに溶解させ、１２１℃で１５分間高圧蒸気滅菌し、滅菌試験管に３ｍｌ分注した。この培地に被検菌を植菌し、各菌体の生育温度で１８〜２４時間培養し、培地色調が茶褐色から黄変したものを陽性とした。
【００３７】
２１．糖の資化能判定
前記ＯＦ培地を１２１℃で１５分間高圧蒸気滅菌し、培地温度が６０℃程度に冷めたところで濾過滅菌した糖を１％の割合で加え、よく混和した後、試験管に３ｍｌ分注し、高層に固めた。この培地に被検菌を穿刺し、各菌体の生育温度で２４時間培養し、培地色調が黄変したものを陽性とした。
【００３８】
以上の試験の結果を表２に示す。
【表２】

【００３９】
上記表２に示した種々の同定確認試験の結果から、ここで分離選別した菌株はグラム陽性の中桿菌であり、内胞子を形成し、またオキシダーゼやカタラーゼを産生する、等の性質を有することが確認された。
また、両菌株ともバチルス（Ｂａｃｉｌｌｕｓ）属に属する菌株であることが判明し、種名はＨＡ１２がサーキュランス（ｃｉｒｃｕｌａｎｓ ）、ＨＡ１９がステアロサーモフィルス（ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ）であると同定され、それぞれバチルス・サーキュランス（Ｂａｃｉｌｌｕｓ ｃｉｒｃｕｌａｎｓ）ＨＡ１２並びにバチルス・ステアロサーモフィルス（Ｂａｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ ）ＨＡ１９と命名した。（以後、これら菌株をそれぞれＨＡ１２，ＨＡ１９と称する。）
これらは、タンパク質を特異的早期に低分子化分解する未知の新規菌株である。そこで、これらの新規菌株は、１９９３年２月１２日に工業技術院微生物工業技術研究所においてＦＥＲＭ Ｐ−１３４２８及びＦＥＲＭ Ｐ−１３４２９として寄託した。
【００４０】
そこで、大豆粕に水を加えた溶液を原料とし、この溶液のタンパク質を早期に低分子化分解するバチルス・サーキュランスＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株を用いて高速且つ効率的に低分子化分解し、これを水で希釈の上、使用に給する大豆粕由来の植物成長肥料が得られることになった。
次に、これら新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）を用いた大豆粕分解物の挙動について説明する。
新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）を用いた大豆粕分解過程におけるタンパク質濃度の経時変化をＢＣＡ法を用いて測定した。
【００４１】
［大豆粕液体培地及び試薬の調整］
＜大豆粕液体培地の調整＞
大豆粕１．５ｇに蒸留水１５０ｍｌを加えて、５００ｍｌ振盪培養フラスコでオートクレーブ（１２１℃／２０ｍｉｎ ）を行ない大豆粕液体培地とした。
＜ＢＣＡ試薬の調整＞
試薬Ａ：１００溶＋試薬Ｂ：２溶
【００４２】
＜検量線用標準溶液（ウシアルブミン）の調整＞
下記の表３に従い、種々のタンパク質濃度の標準溶液を調整した。
【表３】

【００４３】
而して、調整した検量線用アルブミン標準溶液０．１ｍｌとＢＣＡ試薬２．０ｍｌとを試験管内で撹拌した後、３７℃の恒温槽内に３０分間放置することによりＢＣＡ反応を行なった。
反応終了後、各試験管を室温まで放置し、同時に測定した水０．１ｍｌとＢＣＡ試薬０．２ｍｌの溶液をブランクとして５６２ｎｍにおける吸光度を測定し、検量線を作成した。
【００４４】
次に、オートクレーブした１ｗｔ％大豆粕液体培地（１５０ｍｌ）から１ｍｌを無菌的に採取し、１万ｒｐｍ／１５ｍｉｎ 遠心して得られた上清についてＢＣＡ法によるタンパク質濃度測定を行ない、この操作により得られたタンパク質濃度を培養時間０（ゼロ）におけるタンパク質濃度とした。
同様にして、１ｗｔ％大豆粕液体培地から１ｍｌを無菌的に採取し、その培地に、予め大豆粕液体培地で前培養したＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）の菌液１．５ｍｌをそれぞれ植菌し、５０℃，１５０ｒｐｍ／１５ｍｉｎ の条件で培養を行ない、この培養液から経時的に培養液を無菌的に採取し、各培養時間におけるタンパク質濃度を測定した。新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）を用いた大豆粕分解過程におけるタンパク質濃度の経時変化を別紙図１に示す。
【００４５】
この図１から理解されるように、新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）は大豆粕タンパク質を高速で効率良く低分子化分解している。
尚、図１中の白丸はプロテインの濃度を示し、黒丸はペプチドの濃度を示す。
【００４６】
次に、大豆粕に水を加えた溶液を原料とし、この溶液のタンパク質を早期に低分子化分解するバチルス・サーキュラスＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株を用いて高速且つ効率的に低分子化分解し、これを水で希釈の上、使用に給する大豆粕由来の植物成長肥料を散布した場合における植物に対する肥料効果について説明する。
（１）小松菜を用いた例
［種子の前処理］
種子に水を吸収させ発芽を促進させるために、種子を蒸留水中に入れ１５時間放置した。
［苗の育成］
採取した土を篩にかけて草の根などを取り除き、市販の腐葉土を一割ほど加えてよく混ぜ合わせ、底に小石を敷いたプランターに約８ｃｍの厚さに入れた。
そして、前処理した種子を約５ｃｍ間隔で数粒ずつ１．５ｃｍほどの深さに植え、毎日朝に水をかけ、通常室温に置き雨を避け、天気の日は外に出し日に当てて、小松菜がある程度成長し間引きを必要とするようになってから実験に使用した。
【００４７】
［発芽促進剤（市販品）及び化学肥料の調整］
発芽促進剤（商品名：メネデール，メネデール社製）２０ｍｌを蒸留水で希釈し１リットルとした。
化学肥料は、リッチェル社製化学肥料５０号（組成：窒素９．０ ％，リン１１．０％，カリ１０．０％）を用い、１株につき５ｇを直接土中に入れて使用した。
［実験用土の前処理］
採取した土を篩にかけて草の根などを取り除き、その土を金属容器に入れアルミホイルで蓋をし、１８０℃で５時間乾熱滅菌を行なった。
【００４８】
［栽培の実施］
家庭栽培用の小型プランターを栽培容器として用い、その内に上記前処理した土を入れ、小松菜の苗を８株ずつ植えた。
そして、新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）でタンパク質の低分子化分解生成した大豆粕由来の植物成長肥料溶液を毎日２００ｍｌずつ与える。
比較例として、前記発芽促進剤を使用したものと化学肥料を使用したものを同時に栽培した。
【００４９】
栽培の結果を下記の表４に示す。
【表４】

【００５０】
上記表４の数値は、３２日間（７月６日〜８月７日）栽培後の小松菜重量をグラム単位で表し、括弧内の数値は水のみで栽培した小松菜重量を１．０ とした場合の各栽培条件における小松菜重量比を表す。
【００５１】
また、栽培の結果を下記の表５で示す。
【表５】

【００５２】
上記表５の数値は、４２日間（７月３０日〜９月９日）栽培後の小松菜重量をグラム単位で表し、括弧内の数値は水のみで栽培した小松菜重量を１．０ とした場合の各栽培条件における小松菜重量比を表す。
【００５３】
前記表４の栽培結果から、新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）由来の植物成長肥料は顕著な肥料効果を示すことが理解され、また表５の栽培結果からは、新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）由来の植物成長肥料が従来の化学肥料とほぼ同様の肥料効果を示すことが理解される。
【００５４】
（２）かいわれ大根を用いた例
［種子の前処理］
かいわれ大根の種子を８０個とり、蒸留水中に入れ３分間放置した。
［苗の育成］
各栽培容器に１８０℃で５時間乾燥させた土１００ｃｍ^３ を入れ、前処理した種子を１ｃｍ間隔で５列に並べ、２１℃インキュベータ内で栽培した。
【００５５】
［微生物肥料の調整］
新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）で分解生成した大豆粕由来の植物成長肥料溶液をそれぞれ１％，１０％，２５％，５０％，及び１００％になるように調整した。
【００５６】
［栽培の実施］
プラスチック製容器（寸法１９×１４×３ｃｍ）を栽培容器として用い、１８×１３×１．５ｃｍのロックウール（ニチアス製）を敷き、その上に前処理した種子２０粒を等間隔に蒔いた。
次に、各濃度に調整した植物成長肥料各２６０ｍｌを各栽培容器に入れ、その上から前処理した土６０ｃｍ^３ を均等の厚さにかぶせ、２５℃インキュベータ内で栽培した。尚、水分蒸発を防ぐために、実験開始２日後に水２０ｍｌをそれぞれの栽培容器に加えた。
【００５７】
栽培の結果を下記の表６に示す。
【表６】

【００５８】
上記表６の数値は、８日間（１２月１８日〜１２月２５日）にわたって栽培したかいわれ大根の各日における成長（長さ）をセンチ（ｃｍ）単位で表すと共に、最終的に収穫したかいわれ大根の重量をグラム（ｇ）単位で表したものである。なお、括弧内の数値は水のみで栽培したかいわれ大根の長さ及び重量を１００％とした場合の各栽培条件におけるかいわれ大根の各比を表す。
【００５９】
前記表６の栽培結果から、新菌株ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）由来の植物成長肥料はいずれも低濃度で長さ重量とも増加し、顕著な肥料効果を示すことが理解され、また低濃度においては早期（２〜３日早く）に水のみの場合の最終長さに達し、栽培期間を短縮し得ることが理解される。
【００６０】
従って、本発明のバチルス・サーキュランスＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株を用いた大豆粕由来の植物成長肥料は、早期（約２０時間程）にタンパク質を低分子化分解した溶液であって、濃度を約１０％に希釈して使用することが最も良好である。
【００６１】
【発明の効果】
本発明に係るバチルス属に属するバチルス・サーキュランスＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株は、従来において未知のものであったが、このＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株によって、大豆粕のタンパク質を特異的早期に低分子化分解することができるようになった。そして、このＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株が用いられて大豆粕のタンパク質を短期間に低分子化分解する大豆粕由来の根毛増殖を伴なう植物成長肥料である。この植物成長肥料は、従来の化学肥料や、有機質肥料だけでは得られない肥料効果を有する。
有機質肥料は、従来において、化学肥料では絶対に不可能とするタンパク質の生成を可能とし、しかも植物の根は有機質肥料を好むことも一応知られていることであったが、それも植物が要求するタンパク質を有機質肥料から直接吸収できるからである。しかしながら実際上、高分子タンパク質であるがために、植物が直接それらを吸収するには問題があり、低分子化分解されなければ円滑な吸収がされにくかった。現状では、植物へ吸収されるために、高分子タンパク質を低分子化分解するのに長期化し、１年以上も要するので、化学肥料のようなすぐにも吸収出来るようになっている肥料と同じであるとはいえず、まして大豆粕が有機質肥料として十分な肥料効果要件を備えていながらも、未使用状態であった。
ところで、ＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株を用いて大豆粕のタンパク質を短期間に低分子化分解し、これを水で希釈の上、使用に給する大豆粕由来の根毛増殖を伴なう植物成長肥料が産出されたことによって、植物へ低分子化分解されたタンパク質、即ちペプチドの吸収が行われ易く、且つ早くなり、これに伴って、植物の根毛に好まれるペプチドの吸収で根毛の増殖をすることになり、また、含有するＨＡ１２（ＦＥＲＭ Ｐ−１３４２８）新規菌株によって、地中の有機物が分解されて、更なる吸収増殖をすることになって、結果的には、植物の生育を旺盛にする。
まして、大豆粕は、安価で、且つ環境や人体に悪影響を及ぼす惧れがないので、植物の根毛増殖をする有機質肥料として大豆粕由来の植物成長肥料が提供できることになる。
従って、大豆粕を肥料として有効利用し得ると共に、大豆粕の利用促進を図ることが出来る。
【図面の簡単な説明】
【図１】本発明に係る新菌株ＨＡ１２による大豆粕分解過程におけるタンパク質濃度の経時変化を示す図。
【図２】本発明に係る新菌株ＨＡ１９による大豆粕分解過程におけるタンパク質濃度の経時変化を示す図。[0001]
[Industrial applications]
The present invention relates to a plant growth fertilizer derived from soybean meal accompanied by root hair growth on a plant produced using a novel strain in which protein of soybean meal is specifically degraded to a low molecular weight by producing a proteolytic enzyme. .
[0002]
[Technical background]
Until around 1919 in Japan, fertilizers such as soybean meal and vegetable oils and fish manure were widely used as fertilizers for plants. Soybean meal fertilizers are scarcely used due to the prolonged absorption, the sharp increase in cheap fertilizers of ammonium sulfate and urea, and the development of chemical fertilizers and pesticides that are more effective than ammonium sulfate. is the current situation.
By the way, the initial effect of using chemical fertilizers is very good, but the accumulation in soil will increase as the frequency of use increases, thereby reducing the type and amount of microorganisms in soil in a limited manner. In addition, it has been pointed out that the soil becomes hard and the root hairs cannot be put on, so that the growth of the plant is adversely inhibited.
Even under these conditions, soybean meal, which is a soybean oil squeezed cake, is seldom used and is rarely used. Is the current situation.
The present inventors have conducted intensive research to effectively use this soybean meal, and found a novel strain that specifically degrades and degrades the protein of soybean meal, using this new strain, in a short time. Plant growth fertilizers have been completed in which proteins are degraded to lower molecular weight to improve absorption into plants and to grow root hairs.
[0003]
[Problems to be solved by the invention]
The present invention provides a novel strain that produces a protease and degrades and degrades proteins at an early stage.The novel strain is used to degrade and degrade proteins in soybean meal in a short period of time, thereby increasing root hair growth on plants. It can be effectively used as a plant growth fertilizer that accompanies it, and enables early absorption by degrading proteins to lower molecular weight, which is impossible with conventional chemical fertilizers. In addition, it is intended to promote the use of soybean meal, because the raw material is inexpensive and has no fear of adversely affecting the environment and the human body.
[0004]
[Means for Solving the Problems]
The microorganism of the present invention which achieves the above object is a bacterium belonging to the genus Bacillus, which produces a proteolytic enzyme and degrades soybean meal into a specific molecule at an early stage. Specifically, Bacillus circulans HA12 (FERMP) -13428) and / or a new strain of Bacillus stearothermophilus HA19 (FERM P-13429).
Further, as the soybean meal-derived plant growth fertilizer according to the present invention, a solution obtained by adding water to soybean meal is used as a raw material, the protein in this solution is decomposed by a novel strain, and the molecular weight is reduced quickly and efficiently. And Bacillus circulans HA12 (FERM P-13428) as a novel strain that degrades soybean meal protein.
[0005]
【Example】
Hereinafter, examples of the present invention will be described.
[0006]
[Screening of soybean meal decomposing microorganisms]
<Preparation of soybean meal liquid medium>
100 g of distilled water was added to 1 g of soybean meal, and autoclave (121 ° C./20 min) was performed in a 500 ml shaking culture flask to obtain a soybean meal liquid medium.
[0007]
<Preparation of screening medium>
In a 300 ml Erlenmeyer flask, 1 g of peptone, 0.5 g of yeast extract, 0.5 g of sodium chloride, 0.5 g of agar, 2 g of casein and 100 ml of distilled water were added, and the lid was capped and subjected to high-pressure steam sterilization (121 ° C./20 min). After completion of the autoclave, after the medium has cooled down to 60 to 70 ° C., a thick melted agar is shaken well at the bottom of an Erlenmeyer flask, and dispensed into a dry-heat sterilized petri dish to form a plate of LB + agar + casein (medium). It was created.
[0008]
<Preparation of LB liquid medium for preculture>
Peptone (1 g), yeast extract (0.5 g), sodium chloride (0.5 g), and distilled water (100 ml) were added to prepare an LB liquid medium for preculture, sterilized in an autoclave, and then dispensed into sterilized test tubes (5 ml each).
[0009]
Thus, 1 g of a soil sample collected from the natural world was added to the soybean cake liquid medium, and cultured with shaking at 50 ° C. for 24 hours in a shaking incubator at 150 rpm. After completion of the shaking culture, 100 μl of the culture is dispensed and diluted in a test tube containing 10 ml of sterile water for dilution, and 100 μl of the diluted culture is dispensed into a test tube containing 10 ml of sterile water for dilution. Was performed twice to dilute the culture solution 106-fold.
[0010]
100 μl of the diluted culture solution was dispensed onto the LB + agar + casein plate serving as a screening medium, spread with a conrad stick, cultured at 50 ° C. for 12 hours, and the number, color, morphology, The state was observed.
[0011]
A single colony in which a halo was observed among the grown colonies was inoculated on the LB + agar + casein plate (medium for screening) with a sterilized toothpick and cultured at 50 ° C. for 12 hours.
Thereafter, one loopful of loop was collected from each single colony that had grown, added to a test tube containing an LB liquid medium for preculture, and cultured at 50 ° C. for 12 hours (preculture).
[0012]
Next, 1 ml of the pre-cultured liquid was added to the soybean cake liquid medium, and the culture was shake-cultured for 24 to 72 hours while maintaining the temperature at 50 ° C. in a shaking incubator at 150 rpm.
As a result, when the soybean meal in the shaking incubator was muddy, the bacterium was determined as plus (+), and the others were determined as minus (-). In addition, the hard part of the epidermis was excluded.
[0013]
[Determination of utilization of soybean meal and growth temperature of each strain]
<Culture conditions>
In a 300 ml Erlenmeyer flask, 1 g of peptone, 0.5 g of yeast extract, 0.5 g of sodium chloride, 2 g of agar and 100 ml of distilled water were added, sterilized in an autoclave, dispensed into a dry-heat sterilized petri dish, and LB + agar plate ( Medium).
[0014]
Bacteria judged as plus (+) in the screening of soybean meal-decomposing microorganisms were collected with a sterilized toothpick, inoculated on the above LB + agar plate (medium), and 30 ° C or lower, 37.5 ° C, 50 ° C, The cells were cultured for 12 hours in a thermostat set at 70 ° C, 75 ° C and 80 ° C.
Then, bacteria in which a halo producing protease was produced by decomposing casein were determined to be positive (+), and other bacteria were determined to be negative (-). As a result, a total of 20 strains growing on the soybean cake medium were obtained, and were named HA1 to HA20, respectively.
[0015]
The obtained soybean meal decomposing bacteria and the culture conditions (temperature) are summarized in Table 1 below.
[Table 1]

[0016]
[Strain identification]
In order to examine the bacteriological properties of the separated and selected soybean meal-decomposing bacteria, various confirmation tests including gram staining were performed by the following methods.
[0017]
1. Gram stain
According to Hucker's modified method, the bacterial solution smeared on a slide glass, dried and then flame-fixed was stained with Hucker's solution (1.5% crystal violet-ethanol solution) for 30 seconds. Immediately after staining, the cells were washed with water and mordanted with Lugol solution for 1 minute. After completion of the mordant, the resultant was washed with water, further decolorized with alcohol for 30 seconds, and immediately washed with water. After draining well, staining was performed with safranin for 30 seconds. The stained specimen was observed under a microscope, and bacteria stained purple were regarded as Gram-positive, and bacteria stained red were regarded as Gram-negative. In addition, the morphology of the bacteria was observed with a microscope.
[0018]
2. Growth temperature
A liquid thioglycolate medium having the following composition was sterilized at 121 ° C. for 15 minutes, and 5 ml was dispensed into a test tube. Inoculate the bacteria collected from the colony in this medium, 37 ℃, 25 ℃ each Medium temperature range The culture was allowed to stand still at the temperature, and it was determined that the medium became turbid after 12 hours or 24 hours.
<Composition of liquid thioglycolate medium>
0.75 g of L-cystine
2.5 g of NaCl
Glucose 5.0g
Yeast extract 5.0g
Agar 0.75g
Casein digest 15.0g
Sodium thioglycolate 0.5g
1.0 ml of 0.1% resazurin
Distilled water 1000ml
pH 7.2
[0019]
3. Oxidase
The test was performed using Bactlab Oxidase Test manufactured by Terumo Corporation. That is, when a bacterial cell was attached to a cotton swab containing N, N, N ', N'-tetramethyl-p-phenylenediamine dichloride and a blue-violet color produced by the oxidase activity was generated, it was determined to be oxidase positive.
[0020]
4. Catalase
After the bacteria were attached to one end of the capillary, 3% hydrogen peroxide solution was sucked up from one end of the capillary where the bacteria were attached, and the cells were mixed with the hydrogen peroxide solution in the capillary. Catalase positive was determined when oxygen bubbles were generated in the capillary tube due to decomposition of hydrogen peroxide.
[0021]
5. Nitrate reducing ability
The bacteria were inoculated into a broth medium containing 0.1% sodium nitrate, and cultured at an optimum growth temperature for 5 days. 1.0 ml of α-naphthylamine solution (0.1 g of α-naphthylamine dissolved in 200 ml of 30% acetic acid aqueous solution) and sulfanilic acid solution (0.5 g of sulfanilic acid dissolved in 150 ml of 30% acetic acid aqueous solution) After adding 1.0 ml and mixing well, a pink-red color due to nitrous acid generated by nitrate reduction within 30 minutes was regarded as positive.
[0022]
6. Crigler medium
After dispensing a Crigler medium having the following composition into a test tube, the mixture was subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes and solidified on a high slope. The test bacteria were planted on the slope part and the high part, and cultured at the growth temperature of each bacteria for 18 to 24 hours. After the cultivation, the slope portion was yellow when it turned yellow (the lactose was decomposed to generate an acid), and the yellow portion was turned yellow in the high portion and the high portion was positive (it decomposed the glucose to generate an acid).
In addition, (NC) shown in Table 2 means slope part negative, (Y) means high part positive, and (R) means high part negative.
<Composition of Krigler medium>
Meat extract 4.0 g Peptone 15.0 g
Lactose 10.0g Glucose 1.0g
Sodium chloride 5.0 g Sodium thiosulfate 0.08 g
Sodium sulfite 0.4g Ferrous sulfate 0.2g
Phenol red 0.02 g Agar powder 15.0 g
Purified water 1000ml pH 7.2
[0023]
7. SC medium
A culture medium having the following composition was dissolved in distilled water, dispensed in approximately 2 ml portions into test tubes, and subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes to solidify the whole slope. The test bacteria were planted on the slope, and cultured at the growth temperature of each bacteria for 18 to 24 hours. Positive ones were grown using citric acid, the only carbon source contained in this medium. When positive, brom thymol contained in the medium turned from green to deep blue.
<Composition of SC medium>
Sodium chloride 5.0g
Magnesium sulfate 0.2g
1.0 g of ammonium dihydrogen phosphate
1.0 g of potassium dihydrogen phosphate
2.0 g of sodium citrate
Brom thymol blue 0.08g
Agar powder 15.0g
Purified water 1000ml
pH 6.8
[0024]
8. SIM medium
A medium having the following composition was heated and dissolved in 1000 ml of distilled water, dispensed in approximately 3 ml portions into test tubes, and subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes, and then solidified in a high layer. The test bacteria were punctured into the high-rise part and cultured at the growth temperature of each of the bacteria for 18 to 24 hours. When the medium turned black after cultivation, it was regarded as hydrogen sulfide generation (+). When the medium spread and grew from the punctured part, it was regarded as motility (+). Further, 1 ml of chloroform was overlaid on the upper part of the medium, and 1 ml of Kovac reagent was placed thereon. The case where the chloroform layer was reddened due to the superposition was defined as indole (+).
<Composition of SIM medium>
Ehrlich meat extract 3.0g
Proteose peptone 10.0g
Polypeptone 20.0g
Sodium thiosulfate 0.2g
Ferric ammonium citrate 0.2g
Agar powder 3.0g
Purified water 1000ml
pH 7.3
[0025]
9. Methyl red
3 ml of glucose-phosphate-peptone water having the following composition was dispensed into a test tube, and a sterilization operation at 100 ° C. for 30 minutes was repeated three times. The test bacteria were inoculated on this medium and cultured at the growth temperature of each of the bacteria for 3 days. 1 ml of the obtained culture was transferred to another test tube, methyl red was added dropwise, and the case where the color tone of the medium turned red was regarded as positive.
<Composition of glucose-phosphate-peptone water>
Peptone 7.0 g Potassium dihydrogen phosphate 5.0 g
Glucose 5.0g Distilled water 1000ml
[0026]
10. Foge Sploss Cowell (VP)
3 ml of the glucose-phosphate-peptone water was dispensed into a test tube, and a sterilization operation at 100 ° C. for 30 minutes was repeated three times. The test bacteria were inoculated on this medium and cultured at the growth temperature of each of the bacteria for 3 days. Transfer 1 ml of the obtained culture solution to another test tube, add 0.6 ml of α-naphthol solution (5% α-naphthol-absolute ethanol solution) and 0.2 ml of 40% KOH aqueous solution, and mix well. The test tube was allowed to stand at an angle. Changes in the medium color tone were observed 15 minutes and 1 hour after standing, respectively, and those in which the medium color tone became dark red were regarded as positive.
[0027]
11. OF (Oxidation or Fermentation)
According to the method of Hugh-Leifson, an OF medium having the following composition was subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes, and after the medium was cooled to about 60 ° C., glucose sterilized by filtration so as to have a final concentration of 1% was obtained. 3 ml of the medium was dispensed into two test tubes, respectively, and was solidified in a high layer. A test bacterium was punctured into this medium, liquid paraffin was layered in a thickness of 1 to 2 cm on one test tube and anaerobically cultured, and the other test tube was cultured aerobically as it was. After cultivation at the growth temperature of each cell for 3 to 4 days, the culture medium which was cultured under aerobic conditions with a bacterium that performs oxidative sugar degradation and whose medium color tone turned yellow was indicated as (O) in the results, and the fermentative sugar was expressed. Bacteria that decompose and whose medium color tone turned yellow under both aerobic and anaerobic conditions are indicated as (F) in the results, and that glycolysis was observed for the next 7 days after 4 days of culture. Is shown in the results as (W).
<Composition of OF medium>
Tryptone 2.0g Sodium chloride 5.0g
Agar 2.5g Potassium dihydrogen phosphate 0.3g
B. T. B. 0.03g distilled water 1000ml
pH 7.1
[0028]
12. Urease
Using a urea medium for confirming urease (sterilized) manufactured by Eiken Chemical Co., Ltd. having the following composition, 0.5 to 1.0 ml was dispensed into a sterilized test tube, and the test bacteria were inoculated. This medium was cultured at the growth temperature of each test bacterium for 6 to 24 hours, and a case where the medium turned red accompanying the production of ammonia was defined as positive.
<Composition of urea medium>
Peptone 2.0g Urea 30.0g
Sodium chloride 5.0 g Potassium phosphate 9.0 g
Disodium phosphate 3.0 g Phenol red 0.01 g
pH 6.2
[0029]
13. NaCl
Various concentrations of NaCl were added to 1% peptone water, a sterilized medium (pH 7.2) was added to about 3 ml test tubes, and the test bacteria were inoculated, and allowed to stand at the growth temperature of each cell for 18 to 24 hours. Cultured. And the thing which the culture solution became cloudy was set as positive.
[0030]
14． Mannit salt (MS) medium
112 g of this medium was dissolved in 1000 ml of distilled water using a mannit salt medium “Eiken” manufactured by Eiken Chemical Co., Ltd. having the following composition, and subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. Approximately 20 ml of the sterilized medium was dispensed into a sterilized petri dish and solidified on a plate. Test bacteria were inoculated on the obtained plate medium, and cultured at the growth temperature of each cell for 24-48 hours.
Those which decomposed mannitol and yellowed around the colonies were regarded as MS-positive, and those which grew without yellowing and formed colonies were regarded as NaCl-positive.
<Composition of the mannit salt medium “Eiken”>
Meat extract "Eiken" 2.5g Peptone "Eiken" 10.0g
Mannit 10.0g Sodium chloride 75.0g
Phenol red 0.025 g Agar 15.0 g
[0031]
15. Hemolysis
15 ml of ordinary agar was dissolved and kept at 45 ° C., and defibrinated blood was added thereto at a rate of 5%. A small amount of the test bacterium was inoculated on this medium, mixed well, poured into a sterile petri dish, and solidified on a plate. The cells were cultured at the growth temperature of each bacterium for 24 hours, and the presence or absence of hemolysis was determined. Those which caused hemolysis and produced a halo were regarded as positive.
[0032]
16. Esculin
When Barsiekow's medium having the following composition sterilized by high-pressure steam at 121 ° C. for 15 minutes was cooled to 80 ° C., filter-sterilized esculin (glycoside) was added at a ratio of 0.5%, and 2 ml was dispensed into a sterilized test tube. A test bacterium was inoculated into this medium, cultivated at the growth temperature of each cell for 18 to 24 hours, and a case where the medium color tone changed from blue to yellow due to esculin decomposition was regarded as positive.
<Composition of Barsiekow medium>
100 ml of peptone water
0.2% B. T. B solution 1.2ml
[0033]
17． Liquefaction of gelatin
100 g of purified gelatin was added to 1000 ml of ordinary broth, and 3 ml of a medium subjected to high-pressure steam sterilization at 121 ° C. for 12 minutes was dispensed into a sterilized test tube to solidify into a high layer. The test bacterium was punctured into the high-rise portion and cultured at the growth temperature of each cell for one week. The presence or absence of gelatin liquefaction was observed along the puncture line, and the liquefaction was observed as positive.
[0034]
18. Decarboxylation test from ornithine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 ml), sterilized by high pressure steam at 121 ° C. for 15 minutes, and dispensed into sterile test tubes (3 ml). A test microorganism was inoculated into this medium and cultured at the growth temperature of each cell for 18 to 24 hours. A medium whose color tone changed from reddish to yellow was regarded as positive.
[0035]
19. Decarboxylation test from lysine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 ml), sterilized by high pressure steam at 121 ° C. for 15 minutes, and dispensed into sterile test tubes (3 ml). A test bacterium was inoculated into this medium and cultured at a growth temperature of each cell for 18 to 24 hours. A medium whose color tone changed from reddish purple to yellow was regarded as positive.
[0036]
20. Decarboxylation test from arginine
Difco Standard Dehydrated Culture Medium (10.5 g) and L-ornithine (10.0 g) were dissolved in distilled water (1000 ml), sterilized by high pressure steam at 121 ° C. for 15 minutes, and dispensed into sterile test tubes (3 ml). A test bacterium was inoculated into this medium and cultured at a growth temperature of each cell for 18 to 24 hours. A medium whose color tone changed from brown to yellow was regarded as positive.
[0037]
21. Sugar utilization assessment
The OF medium was subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. When the temperature of the medium was cooled to about 60 ° C., filter-sterilized sugar was added at a ratio of 1%, mixed well, and 3 ml was dispensed into a test tube. Hardened. A test bacterium was punctured into this medium and cultured at the growth temperature of each cell for 24 hours. A medium whose color tone turned yellow was regarded as positive.
[0038]
Table 2 shows the results of the above test.
[Table 2]

[0039]
From the results of the various identification confirmation tests shown in Table 2, the strains isolated and selected here are Gram-positive bacilli, have endospores, and have the properties of producing oxidase and catalase. Was confirmed.
In addition, both strains were found to be strains belonging to the genus Bacillus, and HA12 was identified as circulans, HA19 was identified as stearothermophilus, and HA19 was identified as B. circulcus. Lance (Bacillus circulans) HA12 and Bacillus stearothermophilus HA19. (Hereinafter, these strains are referred to as HA12 and HA19, respectively.)
These are unknown new strains that specifically degrade proteins into small molecules at an early stage. Therefore, these new strains were deposited as FERM P-13428 and FERM P-13429 at the Research Institute of Microorganisms and Technology on February 12, 1993.
[0040]
Therefore, a solution obtained by adding water to soybean meal as a raw material is used as a raw material, and the molecular weight of the solution is rapidly and efficiently reduced using a novel strain of Bacillus circulans HA12 (FERM P-13428), which degrades and degrades the protein at an early stage. The plant was decomposed, diluted with water, and a soybean meal-derived plant growth fertilizer to be supplied for use was obtained.
Next, the behavior of the soybean meal decomposition product using these new strain HA12 (FERM P-13428) will be described.
The time course of the protein concentration in the soybean meal decomposition process using the new strain HA12 (FERM P-13428) was measured using the BCA method.
[0041]
[Preparation of soybean meal liquid medium and reagent]
<Preparation of soybean meal liquid medium>
Distilled water (150 ml) was added to 1.5 g of soybean meal, and autoclave (121 ° C./20 min) was performed in a 500 ml shaking culture flask to obtain a soybean meal liquid medium.
<Adjustment of BCA reagent>
Reagent A: 100 dissolved + Reagent B: 2 dissolved
[0042]
<Preparation of calibration curve standard solution (bovine albumin)>
According to Table 3 below, standard solutions having various protein concentrations were prepared.
[Table 3]

[0043]
After stirring 0.1 ml of the adjusted standard solution for albumin and 2.0 ml of the BCA reagent in a test tube, the mixture was allowed to stand in a thermostat at 37 ° C. for 30 minutes to carry out a BCA reaction.
After completion of the reaction, each test tube was allowed to stand at room temperature, and the absorbance at 562 nm was measured using a solution of 0.1 ml of water and 0.2 ml of BCA reagent measured at the same time as a blank to prepare a calibration curve.
[0044]
Next, 1 ml of the autoclaved 1 wt% soybean meal liquid medium (150 ml) was aseptically collected, and the supernatant obtained by centrifugation at 10,000 rpm / 15 min was subjected to protein concentration measurement by the BCA method. The obtained protein concentration was defined as the protein concentration at culture time 0 (zero).
Similarly, 1 ml of a 1 wt% soybean meal liquid medium was aseptically collected, and 1.5 ml of a bacterial solution of HA12 (FERM P-13428) pre-cultured in the soybean meal liquid medium was inoculated into the medium. The culture was carried out at 50 ° C. and 150 rpm / 15 min, and the culture was aseptically collected from the culture over time, and the protein concentration at each culture time was measured. FIG. 1 shows the time course of the protein concentration in the process of decomposing soybean meal using the new strain HA12 (FERM P-13428).
[0045]
As can be understood from FIG. 1, the new strain HA12 (FERM P-13428) decomposes soybean meal protein at a high speed and efficiently into small molecules.
In FIG. 1, open circles indicate the protein concentration, and black circles indicate the peptide concentration.
[0046]
Next, a solution obtained by adding water to soybean meal as a raw material is used as a raw material, and the molecular weight of the solution is rapidly and efficiently reduced using a novel strain of Bacillus circularus HA12 (FERM P-13428), which degrades and degrades proteins at an early stage. The effect of the fertilizer on the plant when it is decomposed, diluted with water, and sprayed with soybean meal-derived plant growth fertilizer to be used will be described.
(1) Example using Komatsuna
[Seed pretreatment]
The seeds were placed in distilled water and allowed to stand for 15 hours to absorb water and promote germination.
[Growling of seedlings]
The collected soil was sieved to remove grass roots and the like, and about 10% of commercially available humus was added, mixed well, and put into a planter with a pebble on the bottom to a thickness of about 8 cm.
Then, plant the pre-treated seeds at a depth of about 1.5 cm at intervals of about 5 cm, spraying water every morning, keeping it at room temperature to avoid rain, and go outside on a sunny day Was used for experiments after Komatsuna grew to some extent and required thinning.
[0047]
[Adjustment of germination accelerator (commercially available) and chemical fertilizer]
20 ml of a germination accelerator (trade name: Menedale, manufactured by Menedale Co.) was diluted with distilled water to 1 liter.
Chemical fertilizer No. 50 (composition: nitrogen 9.0%, phosphorus 11.0%, potash 10.0%) manufactured by Richell Co., Ltd. was used, and 5 g per strain was directly put into the soil and used.
[Pretreatment of experimental soil]
The collected soil was sieved to remove grass roots and the like. The soil was placed in a metal container, covered with aluminum foil, and sterilized by dry heat at 180 ° C. for 5 hours.
[0048]
[Implementation of cultivation]
A small planter for home cultivation was used as a cultivation container, into which the pretreated soil was placed, and eight seedlings of Komatsuna were planted.
Then, 200 ml of a soybean meal-derived plant growth fertilizer solution, which is produced by degrading a protein into a low molecular weight by the new strain HA12 (FERM P-13428), is given daily.
As a comparative example, one using the germination promoter and one using a chemical fertilizer were cultivated at the same time.
[0049]
The results of the cultivation are shown in Table 4 below.
[Table 4]

[0050]
The values in Table 4 above represent the weight of Komatsuna after cultivation for 32 days (July 6 to August 7) in grams, and the values in parentheses assume the weight of Komatsuna grown only with water as 1.0. Represents the weight ratio of Komatsuna under each cultivation condition.
[0051]
Table 5 below shows the results of cultivation.
[Table 5]

[0052]
The values in Table 5 above represent the weight of Komatsuna after cultivation for 42 days (July 30 to September 9) in grams, and the values in parentheses assume the weight of Komatsuna grown only with water as 1.0. Represents the weight ratio of Komatsuna under each cultivation condition.
[0053]
From the cultivation results in Table 4 above, it is understood that the plant growth fertilizer derived from the new strain HA12 (FERM P-13428) exhibits a remarkable fertilizer effect, and the cultivation results in Table 5 indicate that the new strain HA12 (FERM P- It is understood that the plant growth fertilizer derived from 13428) exhibits almost the same fertilizer effect as conventional chemical fertilizers.
[0054]
(2) Example using radish
[Seed pretreatment]
Eighty seeds of radish were taken, placed in distilled water and left for 3 minutes.
[Growling of seedlings]
Soil 100cm dried in each cultivation container at 180 ° C for 5 hours ³ , And the pretreated seeds were arranged in five rows at 1 cm intervals and cultivated in a 21 ° C. incubator.
[0055]
[Adjustment of microbial fertilizer]
The soybean meal-derived plant growth fertilizer solutions decomposed and generated by the new strain HA12 (FERM P-13428) were adjusted to 1%, 10%, 25%, 50%, and 100%, respectively.
[0056]
[Implementation of cultivation]
Using a plastic container (dimensions 19 × 14 × 3 cm) as a cultivation container, rock wool (manufactured by Nichias) having a size of 18 × 13 × 1.5 cm was laid, and 20 pretreated seeds were sown at even intervals.
Next, 260 ml of each of the plant growth fertilizers adjusted to each concentration was placed in each cultivation container, and the pretreated soil 60 cm was placed thereon. ³ Was cultivated in a 25 ° C. incubator. In addition, in order to prevent water evaporation, 20 ml of water was added to each cultivation container two days after the start of the experiment.
[0057]
The results of the cultivation are shown in Table 6 below.
[Table 6]

[0058]
The numerical values in Table 6 above indicate that the radish was grown for eight days (December 18 to December 25) and that the growth (length) of each radish on each day was expressed in centimeters (cm) and that the radish was finally harvested. It is the weight of radish expressed in grams (g). In addition, the numerical value in a parenthesis shows each ratio of the dried radish under each cultivation condition when the length and weight of the dried radish are cultivated only with water and 100%.
[0059]
From the cultivation results shown in Table 6, it is understood that the plant growth fertilizers derived from the new strain HA12 (FERM P-13428) all increase in length and weight at a low concentration and exhibit a remarkable fertilizer effect. It is understood that can reach the final length in the case of water only early (2 to 3 days earlier) and shorten the cultivation period.
[0060]
Therefore, the plant-growing fertilizer derived from soybean meal using the novel strain of Bacillus circulans HA12 (FERM P-13428) of the present invention is a solution obtained by degrading a protein to a low molecular weight at an early stage (about 20 hours). It is best to use a dilution of about 10%.
[0061]
【The invention's effect】
The Bacillus circulans HA12 (FERM P-13428) novel strain belonging to the genus Bacillus according to the present invention was previously unknown, but the HA12 (FERM P-13428) novel strain was used to remove soybean meal protein. It is now possible to decompose and degrade into molecules at a specific early stage. The HA12 (FERM P-13428) novel strain is used as a plant growth fertilizer with soybean meal-derived root hair growth, which decomposes and degrades soybean meal protein in a short period of time. This plant growth fertilizer has a fertilizer effect that cannot be obtained with conventional chemical fertilizers or organic fertilizers alone.
Conventionally, it has been known that organic fertilizers allow the production of proteins that are absolutely impossible with chemical fertilizers, and that plant roots are known to prefer organic fertilizers. This is because the protein to be absorbed can be directly absorbed from the organic fertilizer. However, in practice, since it is a high-molecular protein, there is a problem in that plants directly absorb them. At present, it takes a long time to decompose and decompose high-molecular-weight proteins, and it takes more than one year to be absorbed by plants, so it is the same as fertilizers that can be absorbed immediately, such as chemical fertilizers. Notwithstanding, soybean meal was not used, although it had sufficient fertilizer effect requirements as an organic fertilizer.
By the way, using a new strain of HA12 (FERM P-13428), the protein of soybean meal is degraded to low molecular weight in a short period of time, diluted with water, and accompanied by root hair growth derived from soybean meal supplied for use. The production of plant growth fertilizer facilitates and speeds up the absorption of low-molecular-weight degraded proteins, that is, peptides, into the plant. In addition, the HA12 (FERM P-13428) new strain contained therein decomposes the organic matter in the ground and further absorbs and grows. As a result, the growth of plants To be active.
Furthermore, since soybean meal is inexpensive and has no fear of adversely affecting the environment and the human body, a plant-growing fertilizer derived from soybean meal can be provided as an organic fertilizer for growing root hairs of plants.
Therefore, soybean meal can be effectively used as a fertilizer, and utilization of soybean meal can be promoted.
[Brief description of the drawings]
FIG. 1 is a graph showing a change over time in protein concentration in a soybean meal decomposition process by a new strain HA12 according to the present invention.
FIG. 2 is a graph showing the time-dependent change in protein concentration in the process of decomposing soybean meal by the new strain HA19 according to the present invention.

Claims (1)

Using a solution obtained by adding water to soybean meal as a raw material, the protein in this solution produces a protease and is degraded to a low molecular weight. The growth temperature in the medium temperature range of 37 ° C and 25 ° C, oxidase, catalase, methyl red, and man knit, lactose, sucrose, Bacillus circulans, which is considered positive in various confirmation tests such as assimilating the determination of sugars salicin, etc. (Bacillus circulans) HA12 (FERM P -13428) using the novel strain 10 a fast 30 hours, and the 5.0-7.0 mg / ml made efficient depolymerized (peptide) degradation, in terms of this is diluted to a concentration of 5-25% with water, subjected to use, 30 A soybean meal-derived plant growth fertilizer with root hair growth for a plant cultivated 10 to 30 times its weight relative to cultivation of water only in a day .

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