JP2020527339A - 低凝集の酵素含有粒子 - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/0008—Detergent materials or soaps characterised by their shape or physical properties aqueous liquid non soap compositions
- C11D17/0013—Liquid compositions with insoluble particles in suspension
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38681—Chemically modified or immobilised enzymes
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C11D17/0039—Coated compositions or coated components in the compositions, (micro)capsules
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
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- C11D3/38609—Protease or amylase in solid compositions only
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11D3/38—Products with no well-defined composition, e.g. natural products
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
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Abstract
Description
ρc≦(ρf+31250/Dp 2)*xc/(Dc/Dp)(1/3)及び
ρc≧(ρf−31250/Dp 2)*xc/(Dc/Dp)(1/3)
[式中、ρcはg/cm3でのコアの密度であり、ρfはg/cm3での液体組成物の質量密度であり、xcは粒子中のコアの質量分率であり、DcはμMでのコアの直径であり、DpはμMでの粒子の直径である]で定義される密度を有する。
本明細書において別途定義されない限り、本明細書で使用される全ての技術及び科学用語は、本発明の属する技術分野の当業者により一般に理解される意味と同様の意味を有する。本発明の実践においては、本明細書に記載の方法及び物質に類似しているか同等である方法及び材料が利用されるが、本明細書では好ましい方法及び物質について記載する。したがって、直下に定義した用語は、本明細書全体を参照することにより、より十分に説明される。また、本明細書で使用する場合、単数形「a」、「an」、及び「the」は、文脈上特に明記されない限り、複数形を含む。他に特に指示しない限り、それぞれ、核酸は5’から3’への方向で左から右へと記載され、アミノ酸配列はアミノからカルボキシへの方向で左から右へと記載される。本発明は、当業者によって使用される状況に依存して、それらは変動する可能性があるので、本明細書に記載した特定の方法、プロトコル及び試薬には限定されないことを理解すべきである。
℃ 摂氏度
AU 活性ユニット
CaCl2 塩化カルシウム
Cm センチメートル
cm3 立方センチメートル
D(0.5) 粒子の50%が指定された直径以下である中央粒径
D(0.9) 粒子の90%が指定された直径以下である中央粒径
dH2O又はDI 脱イオン水
eq. 当量
ETOH エタノール
g又はgm グラム(注、以下)
H2O 水
hr 時間
M モル濃度
融解温度 融解温度
mg ミリグラム
min 分
mL及びml ミリリットル
mm ミリメートル
mM ミリモル濃度
MW 分子量
N 規定
Na2SO4 硫酸ナトリウム
NaOH 水酸化ナトリウム
nm ナノメートル
PE ポリエチレン
PEG ポリエチレングリコール
ppm 百万分の一
PVA ポリ(ビニルアルコール)
PVP ポリ(ビニルピロリドン)
sec 秒
TiO2 二酸化チタン
U 単位
v/v 体積/体積
w/v 重量/体積
w/w 重量/重量
(wt%) 重量パーセント
μg マイクログラム
μL及びμl マイクロリットル
μm マイクロメートル
μM マイクロモル濃度
洗浄又はその他の利益を提供するために、低水分液体洗剤中の活性剤とともに粒子を組み込むことが望ましいことが多い。残念なことに、水溶性又は親水性特性を有する物質から作製される外表面を有する従来の粒子は、製造、混合、取り扱い、輸送及び/又は貯蔵の間に、低水分洗剤への組み込み中及び/又は組み込み後に凝集することが示されている。
本組成物及び方法の重要な特長は、製造、取り扱い、輸送及び/又は貯蔵の間に、粒子が凝集するのを防ぐが、洗浄液体中で希釈された場合に容易に崩壊する、疎水性及び/又は非水溶性特性を有する最も外側のコーティングを有する粒子である。
コア(以下で説明する)は、多種多様な酵素のうちの1つ以上若しくはその他の活性物質でコーティングされるか、及び/又は任意選択でそれを含有してもよい。本記載は酵素に焦点を合わせているが、同様の粒子を使用して、無数の他の活性成分を低水分組成物で提供できることは明らかであろう。
本粒子及び方法の特定の実施形態に応じて、粒子を懸濁させることを目的とする低水分液体組成物中に存在する水から酵素及び/又はその他の活性成分層を保護するために、コア又はコーティングされたコアに適用される少なくとも1つの非水性、水溶性コーティングが含まれてもよい。コーティングは、無害且つ生分解性でなければならない。水中のコーティングの溶解度は、25℃で、1mg/mL超、2mg/mL超、3mg/mL超、4mg/mL超、5mg/mL超、6mg/mL超、7mg/mL超、8mg/mL超、9mg/mL超、又は更には10mg/mL超でなければならない。コーティングは、懸濁される低水分液体組成物が少なくとも1つの容量の水で希釈された場合に、5分以内、4分以内、3分以内、2分以内、1分以内、30秒以内、又は更には15秒以内で溶解しなければならない。
いくつかの実施形態では、外側の、疎水性又は非水溶性、水崩壊性、外側コーティングを特徴とする本粒子のコアは、本組成物及び方法にとって重要ではなく、従来の性質のものであってもよい。一般に使用される物質は、塩及び糖並びにその他の比較的安価な水溶性物質である。コアは不活性であってもよいか、又は活性成分を特徴としてもよい。他の実施形態では、コアは、上記の酵素など一部又更には全ての活性剤を含んでもよい。
粒子のコアは、1つ以上の無害且つ生分解性物質から作製してよい。好ましくは、コアは水中に溶解又は分散する。上記に記載されているように、コアは、実質的に沈降することなく担体液体に均一に懸濁したままであるように、液体を懸濁することを目的とする低水分組成物の密度と同様の密度を有し得る。ほとんどの水性液体は、溶解した溶質に応じて、1.0g/cm3〜1.3g/cm3の密度を有し、コアの密度は、液体の密度の0.5g/cm3以内、0.4g/cm3以内、0.3g/cm3以内、0.2g/cm3以内、又は更には0.1g/cm3以内でなければならない。
(|Δρpf|*Dp 2)=(0.5)*(250)2
[式中、Dpは、粒子の全径である]と同じvsを有する。このような粒子は、最大密度について差が:
|Δρpf|<(0.5)*(250)2/Dp 2
又は
|Δρpf|≦31250/Dp 2
によって与えられる場合と比べて、より速く沈降(又は上昇)しない。
沈降を避けるためには、ρp≦ρf+31250/Dp 2、
浮上を避けるためには、ρp≧ρf−31250/Dp 2
で表現される。
ρc/ρp=(mc/vc)/(mp/vp)
[式中、mc及びmpは、それぞれ、コアの質量及び全体の粒子の質量を表し、vp及びvcは、全体の粒子及びコアのぞれぞれの体積を表す]
による全体の粒子の密度に関連し得る。整理し直すと:
ρc=ρc *mc/mp *(vp/vp)
ρc=ρp *xc/(Dc/Dp)(1/3)
が得られるか、又はコア密度の観点からの粒子密度:
ρp=ρc *(Dc/Dp)(1/3)/xc
を示すことができる。
|ρp−ρf|≦18750/Dp 2
|ρc *(Dc/Dp)(1/3)/xc−ρf|≦18750/Dp 2
を得るために、上記表現を置換することによってコアと流体との最大密度差を得ることができる。
沈降を最小限に抑えるために、ρc≦(ρf+31250/Dp 2)*xc/(Dc/Dp)(1/3)
沈降を最小限に抑えるために、ρc≧(ρf−31250/Dp 2)*xc/(Dc/Dp)(1/3)
粒子の全体の密度は、密度調整剤を組み込むことによっても変更することができる。密度調整剤はコア自体に含められ得るか、又はコーティング層中で提供され得る。密度調整剤はコア自体に含められ得るか、又は酵素/活性層若しくはコーティング層中で提供され得る。酵素/活性層又はコーティング層中で密度調整剤を提供する利点は、後に適用されるコーティング中の密度調整剤の量を変えるだけで、事前に選択したコアを、所与の低水分組成物中で使用するために微調整できることである。
低密度粒子は、上記で提供される式で定義される。いくつかの実施形態では、粒子は、1.6g/cm3未満、1.5g/cm3未満、1.4 g/cm3未満、1.3 g/cm3未満、又は更には1.2g/cm3未満、例えば、1.0〜1.6g/cm3、1.0〜1.5g/cm3、1.0〜1.4g/cm3、1.0〜1.3g/cm3、及び1.0〜1.2g/cm3の全体の真密度(すなわち、開いた細孔及び閉じた細孔を除く、粒子の質量を体積で割ったもの)を有し、粒子の全体の真密度と、粒子が懸濁されることを意図した低水分液体組成物の密度との差は、±0.5g/cm3未満、±0.4g/cm3未満、±0.3g/cm3未満、±0.2g/cm3未満、更には±0.1g/cm3未満、又は更には±0.05g/cm3未満である。これにより、粒子は、従来の粒子に典型的であるように、懸濁液から落下することなく、液体組成物中に実質的に懸濁したままでいることが可能になる。真密度は、実施例3に記載されているように計算することができる。上述のように、粒子は、ヒトの目に見えるほど十分に大きく、例えば、それらが溶解されることが意図される低水分組成物の外観を補完するか、又はヒトの目に見えないほど十分に小さくすることができる。粒子が目に見えることが意図される場合、それらは染料及び顔料を含み得る。
本粒子は、流動床コーティング、粒状化、噴霧乾燥、ドラム造粒、高剪断凝集、又はこれらの技術の組み合わせが挙げられるがこれらに限定されない、粒子生成の当業者に公知の方法により作製することができる。最も好ましくは、顆粒は、流動床スプレーコーティングプロセス(以下に例示されるような)によって作製される。
本粒子は、洗浄、消毒、汚染除去、織物加工、飼料、及び食品に使用されるものなどの低水分組成物中に含まれてもよい。組成物は、5〜20重量%の水を含有してもよい。いくつかの実施形態では、酵素懸濁液を含有する組成物は、約5〜10重量%、10〜15重量%、又は15〜20重量%の水(w/w)のいずれかを含有する。粒子を懸濁させることができる例示的な液体洗濯洗剤組成物としては、限定されるものではないが、PUREX(登録商標)ULTRAPACKS(Henkel)、FINISH(登録商標)QUANTUM(Reckitt Benckiser)、CLOROX(商標)2 PACKS(Clorox)、OXICLEAN MAX FORCE POWER PAKS(Church & Dwight)、TIDE(登録商標)STAIN RELEASE、CASCADE(登録商標)ACTIONPACS、TIDE(登録商標)及びARIEL(登録商標)PODS(商標)、並びにGAIN FLINGS(Procter & Gamble)、ALL(商標)MIGHTY PACS(Sun Products)、KIRKLAND SIGNATURE(商標)ULTRACLEAN PACS(商標)が挙げられる。
本粒子は、酵素活性が使用中の少なくとも1つの容量の水で予め希釈されることを意図した低水分液体組成物から所望される任意の用途にて使用されてもよい。希釈すると、少なくとも約50、60、70、80、90、又は95%の酵素は、希釈された組成物中に溶解し、触媒的に活性である。
低水分洗剤組成物中の粒子凝集評価
10gの洗濯洗剤を、透明な15mL試験管に添加した。およそ0.2gの粒子を添加し、混合して、よく分散した懸濁液を形成した。管を転倒式ミキサーに配置し、製造及び貯蔵条件下での名目上の動きを表しながら、室温(すなわち、25℃)にて低RPMで回転させた。7日後に、凝集の程度を目視で評価した。理想的な結果は、全ての粒子が任意の他の粒子に関連付けられていない個々の粒子として残ることであった。理想的ではない結果には、少数の粒子の集団化が観察されることが含まれる。最も理想的ではない結果は、結果は、数十から数百、又はそれ以上の粒子の凝集である。
プロテアーゼ活性を測定するためのN−スクシニル−L−アラニル−L−アラニル−L−プロリル−L−フェニル−p−ニトロアニリド(AAPF−pNA)アッセイ
以下の試薬溶液を使用した:
AAPF基質ストック:ジメチルスルホキシド(DMSO)に溶解した160mM(すなわち、100mg/mL)suc−AAPF−pNA、安定性緩衝液:0.005%v/vのTween 80を含む100mM MES(pH5.5)(10mM CaCl2を任意選択的に含み得る)、活性緩衝液:0.005%v/vのTween−80を含む100mMトリス(pH8.5又は8.6)(10mM CaCl2を任意選択的に含み得る)、アッセイ溶液(活性緩衝液に1:100で希釈した基質ストック):100mMトリス(pH8.5又は8.6)中1.6mM AAPF−pNA。
粒子の作製及び試験
米国特許第6413749号明細書(これは参照により組み込まれる)で例示されているような標準流動床法を使用して様々な粒子を作製した。粒子タイプをA〜Cと標識し、表2にまとめる。コアの組成(コア)、結合剤を含む若しくは含まない、及び/又は密度調整剤を含む若しくは含まない、酵素を含有する第1コーティング層の組成(SP1)、第2コーティング層の組成(SP2)、該当する場合、第3コーティング層の組成(SP3)、を示す。全ての粒子には、表示量の変異体サブチリシンプロテアーゼ(enz)が含まれていたため、実施例2に記載の標準化されたプロテアーゼ活性アッセイを使用してタンパク質の放出及び漏出が測定されることが可能になった。
Claims (14)
- 製造及び/又は貯蔵中に凝集することなく、液体組成物中で酵素を単離し、安定化することができる粒子であって:
(a)活性成分を含むコア、及び/又はコアであって前記コア上に直ちに堆積される活性成分を含む第1コーティング層を有するコア;並びに
(b)25℃で水中約1mg/mL未満の量の水中溶解度を有する、疎水性で非水溶性の水分崩壊物質を含む最も外側のコーティング層
を含み、
前記(b)のコーティング層が、前記液体組成物を25℃にて水で1:1に希釈すると、約5分以内に完全に崩壊し、前記希釈された液体組成物への前記酵素及び/又は活性成分の溶解を可能にし、
前記粒子が、25℃で水中約1mg/mL超の溶解度を有する水溶性ポリマーを含む第3コーティング層を含み、他の点では同一の粒子と比較して、前記液体組成物中で凝集の低減を示す、粒子。 - (a)と(b)との間に、水溶性ポリマー及び活性成分を含む少なくとも1つの追加の層を含む、請求項1に記載の粒子。
- (a)と(b)との間に、活性成分を欠く水溶性ポリマーを含む少なくとも1つの追加の層を含む、請求項1に記載の粒子。
- 前記コアが活性成分を欠く、請求項1又は2に記載の粒子。
- 前記コアが活性成分を含む、請求項1又は3に記載の粒子。
- 前記粒子を含有する液体組成物を少なくとも1つの追加の容量の水と25℃で接触させた後、前記最も外側のコーティングが、5分以内、4分以内、3分以内、2分以内、1分以内、30秒以内、又は更には15秒以内で崩壊する、請求項1〜5のいずれか一項に記載の粒子。
- 前記最も外側のコーティングが、前記粒子の全体重量の8%未満、7%未満、6%未満、又は更には5%未満に相当する、請求項1〜6のいずれか一項に記載の粒子。
- 前記最も外側が、25℃で水中約1mg/mL未満の量の水中溶解度を有する、疎水性で非水溶性の水分崩壊物質から本質的になるか又はそれからなる、請求項1〜7のいずれか一項に記載の粒子。
- 前記コアが式:
ρc≦(ρf+31250/Dp 2)*xc/(Dc/Dp)(1/3)及び
ρc≧(ρf−31250/Dp 2)*xc/(Dc/Dp)(1/3)
[式中、ρcはg/cm3での前記コアの密度であり、ρfはg/cm3での前記液体組成物の質量密度であり、xcは前記粒子中の前記コアの質量分率であり、DcはμMでの前記コアの直径であり、DpはμMでの前記粒子の直径である]で定義される密度を有する、請求項1〜8のいずれか一項に記載の粒子。 - 1.6mg/mL未満、1.4mg/mL未満、又は更には1.2mg/mL未満の全体の真密度を有する、請求項1〜9のいずれか一項に記載の粒子。
- 製造及び/又は貯蔵中の粒子の凝集を低減するための方法であって、25℃で水中約1mg/mL未満の量の水中溶解度を有する、疎水性で非水溶性の水分崩壊物質を含む最も外側の層に前記粒子をコーティングすることを含む、方法。
- 前記最も外側が、25℃で水中約1mg/mL未満の量の水溶性を有する、疎水性で非水溶性の水分崩壊物質から本質的になるか又はそれからなる、請求項11に記載の方法。
- 前記粒子を含有する液体組成物を少なくとも1つの追加の容量の水と25℃で接触させた後、前記最も外側のコーティングが、5分以内、4分以内、3分以内、2分以内、1分以内、30秒以内、又は更には15秒以内で崩壊する、請求項11又は12に記載の方法。
- 前記最も外側のコーティングが、前記粒子の全体重量の8%未満、7%未満、6%未満、又は更には5%未満に相当する、請求項11〜13のいずれか一項に記載の方法。
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- 2018-06-28 EP EP18743260.4A patent/EP3645696A1/en active Pending
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US20200131456A1 (en) | 2020-04-30 |
MX2019014556A (es) | 2020-02-07 |
CN110809624A (zh) | 2020-02-18 |
BR112019027976A2 (pt) | 2020-07-07 |
WO2019006077A1 (en) | 2019-01-03 |
EP3645696A1 (en) | 2020-05-06 |
CA3067837A1 (en) | 2019-01-03 |
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