JP2016530222A - 広い範囲の微生物の除去のための官能基及び表面に吸着された柑橘類抽出物で改質されたナノ粒子の二酸化チタンナノ材料 - Google Patents
広い範囲の微生物の除去のための官能基及び表面に吸着された柑橘類抽出物で改質されたナノ粒子の二酸化チタンナノ材料 Download PDFInfo
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Classifications
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Abstract
Description
植物は、多くの成分を含有し、そして抗菌特性を有する新規な生物学的に活性な分子の価値ある供給源を構成する。前記の成分は、規格化された抽出物又は純粋な化合物の供給源のいずれかとしてある種の植物から抽出され、その化学的多様性のために、微生物の増殖の制御のために無制限の機会を提供する。多くの植物抽出物は、各種の細菌、酵母及びカビに対する抗菌活性を有するが、しかし生体活性成分の品質及び量の変化は、顕著な欠点である。規格化された抽出物を産出する有効な単離方法の開発、並びに前記抗菌剤の安全性及び毒性評価は、更なる研究を必要とする(45−47)。
米国特許第6,117,814号。この特許は、酸化チタンを含有する支持体を記載し、これは、更にその構造中に結合剤としてシリカ及びアルミナの両方を組込んでいる。前記結合剤の目的は、支持体に改良された機械的特性を与えることである。支持体のサイズの範囲は、約20〜120ミクロンである。前記支持体は、ゾル−ゲル法によって製造された約50%の結合剤を含む。
抽出物の製造は、二つの工程を含む。第1に、エタノール工程で、選択された果実、前記果実は、ブドウ、タンジェリン、オレンジ、グレープフルーツ、レモン、グアバである、の種、葉、皮、及び殻が、24〜48時間、30〜50℃の温度で、100〜400RPMの一定の撹拌下で少なくとも70%のエチルアルコール溶液との接触に置かれる。アルコール部分は、抽出物から濾過によって除去される。
支持体を官能化するための方法は、抽出物を含ませるために改良される。
抽出物の組込みを実現するために、使用される酸化物は、50m2/gより大きい又はそれに等しい表面積を有していなければならない。
これらの例示的試験において、酸化チタンを支持体として使用した。
細胞培養
10%のウシ胎児血清(Invitrogen,Mexico D.F.)及び抗生物質(ペニシリン100IU/mL、ストレプトマイシン100mg/mL及びアムホテリシンB10mg/mL)(Sigma−Aldrich,Inc.,St.Louis M.USA)で補充された最小必須培地(MEM)(Gibco/BRL,NY,USA)を伴う25cm2フラスコにおいて、37℃で5%CO2を伴って培養されたMDCK細胞系が使用される。細胞は、80%集密まで増殖させなければならない。
試験管内、又はV字のウェルプレート上で、ウイルス含有試料の二重の希釈物を、赤血球の一定の懸濁液(一般的に10,000,000細胞/mLを使用)と一緒に混合し、そして次いでインキュベートした。結果を評価するために、加えた細胞の量を、分光光度計を使用して定量化し、完全な血液凝集(HA)を示す最終希釈度を、限界稀釈度と考え、そして血液凝集単位(HAU)として表示する。
3〜5日齢のヒヨコからの赤血球を使用する。ヒヨコを血液の抜き取りによって安楽死させ;血液をアルセバー液中に入れる。細胞を、1800×gで5分間の遠心によって数回洗浄し、上清が透明になった時点で、これを取出し、そして細胞をPBS中で10%に調節する。溶液を使用する時に、これをPBS中で0.5%に調節する。
それぞれのウイルスのバッチを滴定しなければならない。1:10〜1:2560の二重の希釈物を、0.05mLの体積のそれぞれの希釈物をV字底の96ウェルプレートのウェルに入れることによって作製した。対照としての赤血球のための一つのウェルを含めなければならない。
試験のために必要なウイルスの量並びに細胞に対する清浄薬の効果が標準化される。その表面上に吸着されたハーブ又は果実抽出物を伴う二酸化チタンナノ材料の溶液が、清浄薬容器からの推奨される希釈度(5リットルの水中に75mLを注ぐことによって調製)で使用される。40μlのウイルスを、異なったHAU(40、20及び4HAU)で希釈物と混合し、そして異なった時間:1、5及び15分の間インキュベートする。それぞれの相互作用時間からの混合物(ウイルス−ナノ材料)にMDCK細胞の集密単層を接種し、そして湿潤雰囲気及び5%CO2下で、37℃で2時間インキュベートする。その後、ウイルスの接種材料を除去し、ウシ胎児血清を含まない新鮮な培養培地を加え、そして単層を24時間インキュベートする。
MDCK細胞を、24ウェルプレート上で集密まで培養する。一定の感染性投与量のウイルスと混合された、異なった体積の清浄溶液が使用される。これらを10〜15分間室温でインキュベートし、そして次いで、マイクロタイタープレート上において単層の集密細胞を2時間接種する。
研究所の分析及び試料維持のために、分析される産物の代表的副次試料を無作為に選択し、そしてロット番号を記録する。
0.25Mのリン酸緩衝溶液
1000mLのメスフラスコで、34gの一塩基性リン酸カリウムを、500mLの水中に溶解し、水酸化ナトリウム溶液でpHを7.1〜7.3に調整し、水で体積を合わせ、混合し、そして100mLの部分に分割する。オートクレーブ中で394K(121℃)で15分間滅菌し、冷却し、そして冷蔵下で保存する。
1.25mLの0.25Mのリン酸緩衝溶液を、1Lのメスフラスコに入れ、そして水で体積を合わせ、混合し、そして試験管及びフラスコ中に、それぞれ9mL及び99mLで分割し、オートクレーブ中で394K(121℃)で15分間滅菌する。
40gのアゾレシチンを、280mLのポリソルベート80mL及び1.25mLのリン酸緩衝溶液と混合し、水で希釈して1Lを得る;水酸化ナトリウム滴定液又は塩酸滴定液でpHを7.2に調整し、次いで100mlの部分に分配する。オートクレーブ中で394K(121℃)で20分間滅菌する。
100mlの濃縮された中和用溶液を、25mlの0.25Mのリン酸緩衝溶液と混合し、1675mlの水を加え、一緒に混合し、そして20mm×150mmのネジ付き試験管に9mlの部分に分配する。オートクレーブ中で394K(121℃)で20分間滅菌する。
製品のラベル上の製造業者の説明により、培養培地を調製し、そして滅菌する。中和溶液を伴う、滅菌前の標準的な方法のための寒天培養培地の場合、1リットルの標準的方法のための寒天培養培地に25mLの中和溶液を加える。
表1に示す成分を一緒に混合し、溶解が起こるまで加熱し、pHを7.2に調整し、オートクレーブに入れ、そして394K(121℃)で15分間滅菌する。
黄色ブドウ球菌(Staphylococcus aureus)(EMB寒天)
大腸菌(Escherichia coli)(EMB寒天)
緑膿菌(Psudomona aeruginosa)
サルモネラ種(Salmonella sp)(EMB寒天)
エンテロバクター種(Enterobacter sp)(E.M.B.寒天)
肺炎桿菌(Klebsiella pneumoniae)(EMB寒天)
カンジダ・アルビカンス(Candida albicans)(EMB寒天)
黒色アスペルギルス(Aspergillus niger)(EMB寒天)
試験微生物の保存
微生物株を、斜面培養培地(7mlの栄養寒天)を伴う16mm×125mmの試験管に毎週再播種することによって保存し、20時間〜24時間、308K〜310K(35℃〜37℃)の温度でインキュベートし、そして冷蔵下で保存した。
試験の前に、それぞれの試験微生物の二つの再播種物を作製し、そして20時間〜24時間、308K〜310K(35℃〜37℃)の温度でインキュベートした。
99mLの滅菌された希釈リン酸緩衝溶液を含有するエルレンマイヤーフラスコに、1mLの試験微生物懸濁液を移し、そしてそれぞれが25〜250個のコロニーを含有するプレートを得るために必要な10倍法による希釈を行った。
1.株の播種及び培養;
2.残存細胞の決定;
3.試料の調製;
必要な場合、容器のラベル上で製造業者によって推奨されている産物の濃度に達するまで水で適当な希釈を行った。
それぞれの試験微生物のために、99mLの産物又はその稀釈物を、正確に、そして二重で測定し、ネジ蓋を持つ250mLの滅菌エルレンマイヤーフラスコに移した。
減少%の決定
初期の生存数及び残存細胞のプレートからの結果の平均値を求め、次いで以下の式を使用して減少%を計算した:
減少%=100−S×100/V.C.
式中:
Sは、残存細胞のCFU/mLであり、そして
V.C.は、初期生存数である。
殺菌剤とラベルされた産物は、初期の生存数が75〜125×108CFU/mLである場合、推奨される濃度における接触の30秒以内で生存数の99.999%の減少を持たなければならない。
有効性の試験を、AOAC966.04の方法論に基づいて行い、ここで、枯草菌に対する60個のレプリカ中、59個が得られた。
Claims (16)
- a)ナノ粒子の金属酸化物支持体、
b)表面に化学的に吸着された官能基、
c)表面及びポアに物理的に吸着されたハーブ及び果実抽出物
を含む、複合ナノ材料。 - 前記ナノ粒子の金属酸化物が、約1nm〜約100nmの範囲の平均直径のサイズを有する、請求項1に記載のナノ材料。
- ヒドロキシル、ホスフェート、サルフェート、クロライド、アミノ、メチル、フォレートであり、そして前記材料に病原性微生物に対する選択性の特性を与える、請求項1に記載の官能基。
- 表面に吸着されたハーブ又は果実抽出物を伴う二酸化チタンナノ材料を調製するための方法であって、
a)ハーブ及び果実抽出物を調製し、
b)前記表面を官能基の化学的吸着によって改質し、
c)ハーブ及び果実抽出物を物理的に吸着すること
を含む、前記方法。 - 請求項6に記載の前記抽出物が、柑橘類植物及び果実の各種の部分、例えば、樹皮、幹、根、葉、皮、果肉、及び種からのものであることができる、請求項4に記載の方法。
- 前記表面の改質が、100〜400RPMの範囲の平均速度における一定の撹拌を維持しながら行われる、請求項4に記載の方法。
- 前記表面の改質が、30〜100℃の範囲内の一定の温度を維持しながら行われる、請求項4に記載の方法。
- 前記表面の改質が、請求項4に記載の官能基を与える溶液を1.4%の濃度で加え、そして請求項4に記載の順序でこれらを加えることによって官能基を化学的に吸着させることによって行われる、請求項4に記載の方法。
- 官能基の吸着による前記表面の改質が、それぞれの官能基を完全に吸着するために、請求項9に記載のそれぞれの溶液の添加後、5〜30分の平均静置時間を必要とする、請求項4に記載の方法。
- 前記表面の改質が、30〜100℃の範囲の平均温度での乾燥期間を必要とする、請求項4に記載の方法。
- 前記ハーブ及び果実抽出物の吸着が、50m2/gより大きい又はそれに等しい表面積を有する請求項1に記載のナノ材料を必要とする、請求項4に記載の方法。
- 前記ハーブ及び果実抽出物の吸着が、少なくとも70%の抽出物を含む、請求項6及び7によって得られる抽出物を必要とする、請求項4に記載の方法。
- 前記ハーブ及び果実抽出物の吸着が、100〜400RPMの範囲の平均の一定の撹拌を維持することを必要とする、請求項4に記載の方法。
- 前記ハーブ及び果実抽出物の吸着が、30〜50℃の範囲の平均温度を維持することを必要とする、請求項4に記載の方法。
- 前記ハーブ及び果実抽出物の吸着が、24時間の撹拌期間を必要とする、請求項4に記載の方法。
- a)請求項1に記載のナノ材料、
b)各種の比率の選択された原材料
を含む、殺菌剤溶液。
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AR120069A2 (es) | 2022-02-02 |
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AU2014281904A1 (en) | 2016-02-18 |
IL243239A0 (en) | 2016-02-29 |
CA2916216A1 (en) | 2014-12-24 |
EA201690030A1 (ru) | 2016-10-31 |
MX339086B (es) | 2016-05-09 |
UY35623A (es) | 2015-01-30 |
SA515370298B1 (ar) | 2017-06-07 |
WO2014204290A1 (es) | 2014-12-24 |
SG11201510528YA (en) | 2016-01-28 |
CR20160028A (es) | 2016-03-30 |
CN105517956B (zh) | 2017-09-29 |
BR112015031884A2 (pt) | 2017-07-25 |
AR096661A1 (es) | 2016-01-27 |
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JP6625051B2 (ja) | 2019-12-25 |
MY184044A (en) | 2021-03-17 |
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CU20150182A7 (es) | 2016-10-28 |
IL243239B (en) | 2020-07-30 |
CL2015003674A1 (es) | 2016-08-19 |
AP2016009043A0 (en) | 2016-02-29 |
ZA201509284B (en) | 2017-09-27 |
KR20160034299A (ko) | 2016-03-29 |
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