JP5511112B2 - 菌体破砕物及びその配合物 - Google Patents
菌体破砕物及びその配合物 Download PDFInfo
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- JP5511112B2 JP5511112B2 JP2013520572A JP2013520572A JP5511112B2 JP 5511112 B2 JP5511112 B2 JP 5511112B2 JP 2013520572 A JP2013520572 A JP 2013520572A JP 2013520572 A JP2013520572 A JP 2013520572A JP 5511112 B2 JP5511112 B2 JP 5511112B2
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Classifications
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- A—HUMAN NECESSITIES
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- A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Description
・生菌分散液にホルマリンを0.5%になるように添加し、室温で1時間保存して殺菌した後、遠心分離器で沈殿を回収し、ホルマリンを除去して、PBSで再懸濁し、非加熱死菌を調製した(非加熱死菌体)。
・生菌分散液をオートクレーブで100℃、10分加熱した後に、そのままの加熱菌体を30尾分間、超音波処理し、菌体を破砕した(超音波破砕菌体)。
・生菌分散液を100℃、10分加熱した後、遠心分離器で沈殿を回収し、PBSで再懸濁し、加熱死菌を調製した(従来型加熱死菌体)。
・生菌分散液にホルマリンを0.5%になるように添加し、室温で1時間保存して殺菌した後、遠心分離器で沈殿を回収し、ホルマリンを除去して、PBSで再懸濁し、非加熱死菌を調製した(非加熱死菌体)。
・生菌分散液をオートクレーブで100℃、10分加熱した後に、遠心分離器で沈殿を回収し、その後、沈殿をPBSで分散し、ホモジネート(ポリトロンホモジナイザー)処理した(従来型前処理ホモジネート処理菌体)。
・生菌分散液をオートクレーブで100℃、10分加熱した後に、そのままの加熱菌体をホモジネート(ポリトロンホモジナイザー)処理により菌体を破砕した(ホモジネート破砕菌体)。
LPSの精製は従来法に基づいて抽出精製を行った(1992CPB-I)。
・生菌分散液を100℃、10分加熱した後、遠心分離器で沈殿を回収し、PBSで再懸濁し、加熱死菌を調製した(従来型加熱死菌体)。
各処理した菌体は寒天培地を用いて増殖菌がないこと(死んでいること)を確認した。
菌体は農大改変寒天培地を用いて増殖菌がないこと(死んでいること)を確認した。
大腸菌およびパントエア菌体の各種処理によるマクロファージ活性化能を一酸化窒素産生能で評価した。マクロファージはリポ多糖あるいはリポタイコ酸あるいはリポアラビノマンナン、ペプチドグリカン、フラジェニン、リポプロテイン、ムラミルジペプチド、プロテオグリカン、非メチル化シトシン・グアニン配列を含む遺伝子などで刺激を受けると、マクロファージ内で一酸化窒素合成酵素が誘導され、その結果、一酸化窒素を産生する。一酸化窒素は活性化ガスの一種であり、細菌やウイスル、がん細胞などに対して障害活性を示すため、活性化マクロファージの異物排除の実行分子として働く。その一酸化窒素は不安定であって亜硝酸に変異するため、その亜硝酸を測定した。
各処理大腸菌サンプルの各濃度の産生亜硝酸濃度を表1に示した。未刺激のRAW246.7細胞の培養上清は亜硝酸検出限界2.8〜3.1μMであった。各処理大腸菌体の各濃度の亜硝酸濃度から10μMの亜硝酸濃度を誘導する菌体濃度を片対数グラフにプロットして求めると、従来法による大腸菌死菌(従来型加熱死菌体で500ng/mlとなり、2unitであったが、ホモジネート破砕菌体は16ng/ml (62.5unit)、超音波破砕菌体は58ng/ml
(17.2unit)であり、高圧破砕菌体は200ng/ml (5unit)となった。すなわち、この結果から、同じ大腸菌の菌体由来であっても、処理によりマクロファージ活性化能が2.5倍〜31倍も効果が高くなることが明らかである。
未刺激のRAW246.7細胞の培養上清の亜硝酸を亜硝酸産生量0μMとし、各処理パントエア菌サンプルの各濃度の産生亜硝酸濃度を表2に示した。各サンプルが10μMの亜硝酸を誘導する菌体濃度を片対数グラフにプロットして各サンプルのユニット数を求めて表2に示した。従来型加熱死菌体で400ng/mlとなり、2.5unitであったが、従来型前処理高圧破砕菌体、従来型前処理ホモジネート破砕菌体は300ng/ml (3.3unit)、非加熱死菌体は185ng/ml (5.4unit)であり、ホモジネート破砕菌体は44ng/ml (23unit)、高圧破砕菌体は12ng/ml (83unit)となった。この結果から、従来型加熱死菌体を基準にして、相対比を求めることが出来るので、表2にまとめた。従来型の処理でも、1.3倍程度の能力向上(1.3分の1で同等の効果が出る)が認められた。一方、従来型の処理をせずに菌体をホモジネート破砕または高圧破砕すると9.2〜33.2倍の著しい能力向上がみられた。
大腸菌体を用いたマウス静脈内投与によるTNF産生におけるプライミング作用の誘導及びその用量依存性についての実験方法は我々が確立した方法を用いた(非特許文献11)。 陰性対照は生理食塩水を投与した。従来法として、従来型加熱死菌体、従来型前処理高圧破砕菌体を用いた。試験品としては、高圧破砕菌体、超音波処理菌体、ホモジネート破砕菌体を用いた。試験検体投与量群は3匹のC3H/Heマウスを試験に用いた。
インターフェロン−γとOK−432で誘導される血清中のTNF量を表6に示した。OK−432の1KEを単独投与した場合の血清中のTNF量は125pg/mlであった。インターフェロン−γの0.1μgをマウスに1μg静脈投与した後に、OK−432を投与した場合のTNF濃度は950pg/mlであった。従って、インターフェロン−γの0.1μgのTNFの増幅倍率は950÷125=7.6倍であった。この1/2の3.6倍のTNF増幅倍率となる最小濃度を与える各処理菌体サンプルの濃度を調べた。各処理菌体サンプルの投与量とOK−432により誘導される血清中のTNF量の結果を表7に示した。また、従来型加熱死菌体に比べて、処理方法がどの程度活性化能を増強するか示すために従来型加熱死菌体のTNF誘導能(unit数)を1としたときの各サンプルで相対値も表7に示した。従来型加熱死菌体では34ng(2.9unit)、従来型前処理高圧破砕菌体は27ng(3.7unit)、高圧破砕菌体は0.80ng(125unit)、超音波処理菌体は2.1ng(48unit)であり、ホモジネート破砕菌体は6.5ng(15unit)となった。すなわち、この結果から、同じ大腸菌の菌体由来であっても、従来処理法では1.3倍で差が無かったが、本開発処理法によりマクロファージ活性化能が5倍〜43倍も効果が高くなることが明らかである。
本明細書で引用したすべての刊行物、特許及び特許出願は、そのまま参考として、ここにとり入れるものとする。
Claims (6)
- グラム陰性菌を培養し、該グラム陰性菌の菌体を加熱後に物理的に破砕することにより得られ、該菌体の免疫活性化成分を含む前記加熱以後のすべての成分を含み、分子量20000以下のLPSを有効成分として含むことを特徴とする菌体破砕物。
- 前記グラム陰性菌が大腸菌、セラチア菌、エロモナス菌、ラーネラ菌、エンテロバクター菌、キサントモナス菌、ザイモモナス菌、パントエア菌、又は酢酸菌であることを特徴とする請求項1記載の菌体破砕物。
- 請求項1又は2記載の菌体破砕物が配合されている医薬品、医薬部外品、化粧品、食品、機能性食品、浴用剤、飼料、ペット用食品又は動物用医薬品であることを特徴とする菌体破砕物の配合物。
- 請求項1又は2記載の菌体破砕物が配合されている肥料、堆肥又は植物用医薬品であることを特徴とする菌体破砕物の配合物。
- 成長促進、免疫活性化、生活習慣病、がん若しくはアレルギー性疾患の予防、感染防除、又はストレス抵抗性を目的としたものであることを特徴とする請求項3記載の配合物。
- 成長促進、免疫活性化、感染防除、又はストレス抵抗性を目的としたものであることを特徴とする請求項4記載の配合物。
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CN104446942A (zh) * | 2014-11-21 | 2015-03-25 | 苏州市吴中区光福香雪苗圃 | 一种新型菌肥 |
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KR102174525B1 (ko) * | 2019-09-17 | 2020-11-05 | 코스맥스 주식회사 | 신규한 판토에아 왈리시 루미테리아 균주 및 그 균주 배양액을 포함하는 피부 미용 개선용 조성물 |
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EP2722386A1 (en) | 2014-04-23 |
WO2012173163A1 (ja) | 2012-12-20 |
EP2722386B1 (en) | 2017-09-27 |
EP2722386A4 (en) | 2014-04-23 |
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US20140194287A1 (en) | 2014-07-10 |
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