WO2005028603A1 - Sols-gels derives du silicate sensibles aux changements de teneur en eau - Google Patents

Sols-gels derives du silicate sensibles aux changements de teneur en eau Download PDF

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Publication number
WO2005028603A1
WO2005028603A1 PCT/US2004/030989 US2004030989W WO2005028603A1 WO 2005028603 A1 WO2005028603 A1 WO 2005028603A1 US 2004030989 W US2004030989 W US 2004030989W WO 2005028603 A1 WO2005028603 A1 WO 2005028603A1
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Prior art keywords
gels
sol
enzymes
enzyme
gel
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PCT/US2004/030989
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English (en)
Inventor
Nathaniel T. Becker
Dave C. Bakul
Kiranmayi Deshpande
Mark C. Gebert
Joseph C. Mcauliffe
Wyatt Charles Smith
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Genencor International, Inc.
Southern Illinois University
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Publication of WO2005028603A1 publication Critical patent/WO2005028603A1/fr

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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
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38672—Granulated or coated enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0052—Preparation of gels
    • B01J13/0056—Preparation of gels containing inorganic material and water

Definitions

  • the present invention relates to silicate sol-gels having proteins such as enzymes entrapped, wherein the proteins are released when the sol-gels are placed in a solution having a high water percentage by weight.
  • the present invention is useful in detergent and fabric care applications.
  • the present invention is applicable to cleaning products such as liquid detergents, soaps, and shampoos.
  • BACKGROUND OF THE INVENTION The detergent industry has been actively advocating the use of the enzyme technology in detergent formulations. Proteases are used for protein-based stains such as blood, egg, milk and grass. They bring about hydrolysis of peptide linkages within protein molecules.
  • ⁇ -Amylases which hydrolyse ⁇ -1, 4-glycosidic bonds, have been used to break down stubborn starch based stains.
  • Lipases which hydrolyze ester bond linkage, are used to fight triglyceride based stains.
  • Cellulases the most recent introduction to the detergent industry, are used mainly for their antipilling and color revival properties.
  • Use of enzymes in the detergent industry is now an accepted technology. The recent endeavors have been focused on developing methods to stabilize the protein structure of enzyme molecules in a detergent environment. Linear alkylbenzene sulfonates and other anionic surfactants that are commonly used in detergents have a tendency to unfold proteins, thereby deactivating them.
  • a protein such as an enzyme is usually deactivated by denaturation, unfolding, catalytic site inactivation, or proteolysis.
  • Denaturation is usually caused by thermal unfolding, or oxidation, or covalent modification of reactive groups. Maintenance of a certain temperature and pH range and absence of chemical denaturants in the detergent environment is found helpful in preventing the protein unfolding.
  • Catalytic site deactivation can be prevented by maintaining a sufficient level of cofactors such as a metal ion.
  • the rate of proteolysis has been lowered by using water sequestering agents such as sugars, sugar alcohols such as sorbitol, and polyols such as propylene glycol or polyethylene glycol.
  • Proteolytic inhibitors which include series of small molecular weight carboxylic acid salts such as formate, acetate, propionate and butyrate and boric acid, have also been used.
  • carboxylic acid salts such as formate, acetate, propionate and butyrate and boric acid
  • calcium salts are known to stabilize subtilisin type proteases and Bacillus ⁇ - amylases (Becker, et al., "Enzymes in detergency", Eds., VanEe, Misset, and Baas, 69: 299).
  • Granulation techniques have been used to separate enzymes from each other and from the surfactants of the detergent. Polyethylene glycol and polyvinyl alcohol are useful in this regard.
  • Other attempts to separate enzymes from detergent involved the formation of liquid emulsions.
  • the present detergent technology is faced by many challenges.
  • Zeolites, phosphonic acids, polyacrylic acids, and polycarboxylate polymer builders which are used in detergent compositions, sequester calcium ions from the active sites of the enzymes thus deactivating them.
  • Linear alkyl benzene sulfonates and alkyl sulfonates are amongst the most harmful chemicals for enzyme stability, followed by alkyl ethoxysulfonates.
  • Reactive oxygen species are another problem. Few enzyme species are known yet to overcome the presence of chlorine bleach.
  • Another challenge faced by the industry is development of dust- free enzyme granules for improved hygiene of workers involved in product manufacture. Stability of multi-enzyme formulations, preventing autolysis of proteases and proteolysis of other enzymes represent additional hurdles.
  • 6,495,352 discloses a method to encapsulate molecules, comprising: forming a silica sol from a solution of a silicon oxide and alkali metal oxide in water; adjusting the pH to a pH value less than approximately 7 to stabilize the silica sol, forming a silica sol matrix solution; adding a solution containing an organic compound to be encapsulated to form a silica sol matrix encapsulating said organic compound; aging said silica sol matrix encapsulating said organic compound; and forming a material selected from the group selected of a thin film and a gel.
  • EP 0676 414 Bl discloses a process for the preparation of immobilized lipases, characterized in that lipases are entrapped in a hydrophobic silica matrix containing organic substituents attached through Si-C bonds.
  • Santos, et. al (Biomaterials, 20: 1695-1700 (1999)) report that silica xerogels containing trypsin inhibitor made from tetramethylorthosilicate (TMOS) slowly release 20- 40% of the entrapped trypsin inhibitor in a diffusion controlled manner over nine weeks.
  • TMOS tetramethylorthosilicate
  • 6,756,217 discloses a porous glass composite material comprising (1) at least one alkoxodisilane precursor having the general formula (OR ⁇ Si- spacer-Si(OR ) , where R and R may be the same or different and may comprise hydrogen, alkyl, alkenyl, alkynyl, or aryl groups as defined herein below, and the two silicon atoms are bridged by a spacer unit comprising an organic unit, an inorganic unit, biological unit and combinations thereof; and (2) water.
  • None of the references have disclosed a sol-gel formulation wherein the entrapped proteins are stable and their release from the gel is triggered when the formulation is placed in a solution having a high water percentage.
  • a stable protein formulation wherein the protein release can be triggered when the formulation is diluted in water.
  • Such formulations are useful in cleaning products such as liquid detergents, soaps, and shampoos.
  • the present invention is directed to a method for preparing enzyme-entrapped silica sol-gels, wherein one or more enzymes are stably entrapped within the gels and are released from the gels when the gels are placed in a solution having water percentage by weight of 86% or more.
  • the method comprises the steps of: (a) mixing a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to reduce the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors selected from the group consisting of dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, or aminoalkylsilane to form a second sol; (c) mixing the first sol, the second sol, and an aqueous buffered solution containing one or more enzymes; and (d) incubating (c) to form gels.
  • the present invention provides an enzyme-entrapped sol-gel composition that is sensitive to water percentage change.
  • the present invention further provides a liquid detergent, a liquid soap or a shampoo formulation comprising an enzyme-containing sol-gel system.
  • the enzymes are stable during storage in the formulation. The enzymes stay within the gels without significant leakage during the storage and are released from the gels upon diluting the formulation in water within less than an hour.
  • the enzymes in the formulation are released when the formulation is diluted in water to a water percentage by weight of 86% or higher, preferably 90% or higher, more preferably 95% or higher, more preferably 98% or higher, and most preferably 99% or higher.
  • the present invention is based on use of silica sol-gels for the controlled release of proteins in detergent and fabric care applications.
  • the present invention is applicable to cleaning products such as liquid detergents, soaps, and shampoos.
  • the silica gel matrices not only improve the thermal stability of enzymes but also prevent the enzyme denaturation due to proteolysis/autolysis or interference from other components of the cleaning products.
  • Figure 1 shows the percentage of weight change at different time intervals after immersed in water or detergent.
  • Figure 2 shows the amount of enzyme released from gels (a) when the gel is soaked in pH 7.85 buffer, and (b) after the gel is soaked in TIDE ® for one day, then subjected to a dilution trigger.
  • Figure 3 shows the amounts of enzyme (mg/ml) released from gels having different additives (starch, pectin, alginate, and carageenan) after a dilution trigger at different time intervals.
  • Figure 4 shows the percentage of enzyme released from gels that contain different additives (starch, pectin, alginate, and carageenan), at 10 minutes and 60 minutes after a dilution trigger.
  • Figure 5 shows the amount of enzyme released from different gel systems.
  • Figure 6 shows the percentage of enzyme released from silicate/siliconate (JA) and sodium silicate/starch (SS) gels upon a 10-fold dilution with water within an hour, after the gels had been stored in TIDE ® for one, two, three or four weeks.
  • JA silicate/siliconate
  • SS sodium silicate/starch
  • the present invention is based on use of silica sol-gels for the controlled release of proteins in detergent and fabric care applications.
  • the present invention is applicable to cleaning products such as liquid detergents, powder detergent, soaps, shampoos, and fabric cleaning agents.
  • the general strategy of the present invention is based on encapsulation of proteins in a porous silica sol-gel matrix, which does not release the proteins when the matrix is placed in a solution having low water content (percentage by weight) but releases the proteins when the solution is diluted with water.
  • the protein is an enzyme.
  • the solution is a liquid detergent such as a laundry detergent.
  • sol-gels are used both for stabilizing the protein activity and for controlling the release of proteins.
  • Proteins such as enzymes are encapsulated and stabilized in sol-gels.
  • the external environment of sol-gels changes to a higher water percentage by weight (when the sol-gel containing matrix is diluted with water)
  • the enzymes are released from the gels.
  • Silica sol-gels refer to silicon dioxide based materials made through a sol-gel process.
  • Sols are formed first, which consist of a colloidal solution of very small (nanometer sized) polysiloxane particles formed through hydrolysis of organofunctional silane precursors or neutralization of alkali metal silicates or alkyl siliconate salts. Further polymerization/chemical reaction/hydrolysis converts the sols into gels by chemically linking together the individual colloidal sol particles into monolithic gels.
  • a sol is first formed by hydrolysis of an alkoxysilane precursor followed by condensation to yield a polymeric oxo-bridged SiO 2 network. In the process, molecules of the corresponding alcohol are liberated. The initial hydrolysis and polycondensation reactions in a localized region lead to formation of colloidal particles.
  • a suspension containing these colloidal particles is called a sol.
  • the viscosity of the sol starts to increase and leads to the formation of a solid gel.
  • the nature of individual events is somewhat random and the geometry and pore-size distribution of the product gel are difficult to control, the nature of the final polymeric gel can be regulated to a certain extent by controlling the rates of the individual steps.
  • the protein to be encapsulated is added to the sol after partial hydrolysis or neutralization of the precursor. As the degree of cross-linking from polycondensation increases, the gel becomes viscous and solidifies.
  • Protons or hydroxide ions are required for catalysis in silica sol-gel formation; therefore, the pH of the reaction medium is an important factor that affects the stoichiometry of the final gel.
  • Both acid and base catalysis lead to the hydrolysis of alkoxysilane functions and the production of silanol (Si-OH) groups.
  • An acidic medium e.g. pH 1-4
  • a more basic medium e.g. pH 5-10
  • the polycondensation process continues during aging, and a porous matrix is formed around the protein molecule, trapping it inside.
  • Such physical entrapment of the protein is functionally non-invasive and preserves the integrity and directional homogeneity of the protein surface microstructure.
  • the mode of entrapment of biomolecules in sol-gels is primarily physical in that the pore size of the sol-gels is equal to or smaller than the protein, there are other interactions that also contribute to the interaction of the protein with the sol-gel matrix. These include ionic interaction between charged groups of proteins with ionizable groups in the matrix, hydrogen bonding, non-polar interactions (Van Der Waals forces) and even covalent bonds.
  • the entrapped protein is stabilized not only by a physical barrier, but also by immobilizing the protein in a silica gel matrix. Protein stabilization in the silica gel matrix is a consequence of prevention or minimization of the interaction between protein segments due to immobilization, the collision between protein segment and the hydrated silica surface on which the protein is immobilized, and the electrostatic interaction between proteins and other counter-charged macromolecules.
  • the present invention is directed to a method for preparing enzyme-containing silica sol-gels, wherein one or more enzymes are stably entrapped within the gels and are released from the gels within less than an hour when the gels are placed in a solution having water percentage by weight of 86% or more.
  • the method comprises the steps of: (a) mixing a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to reduce the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors selected from the group consisting dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, or aminoalkylsilane so as to promote hydrolysis and form a second sol; (c) mixing the first sol, the second sol, and a third aqueous solution containing one or more enzymes; and (d) incubating (c) to form gels.
  • the method further comprises the steps of hydrolyzing one or more negatively charged organosilane precursors to form a third sol, and mixing the third sol with the first sol, the second sol, and the aqueous solution containing one or more enzymes.
  • the first aqueous solution in step (a) which reduces the pH of the alkali metal silicates or alkyl siliconate salts to 12 or lower, can be an acid, e.g., phosphoric acid, citric acid, acetic acid, and hydrochloric acid, an acidic solution, or a low pH buffer.
  • the organofunctional silane precursors or the negatively charged organofunctional silane precursors can be hydrolyzed to form a second sol or a third sol at either an acidic pH (pH 1-6) or a basic pH (pH 8-13).
  • a preferred acidic pH is, for example, pH 1-5, or pH 1.5-4.
  • a preferred basic pH is, for example, pH 9-12.
  • the method comprises the steps of: (a) mixing (i) a first aqueous solution, (ii) one or more alkali metal silicates or alkylsiliconate salts, and (iii) one or more organofunctional silane precursors such as dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, and aminoalkylsilane to form sols; (b) mixing the sols with a second aqueous solution containing one or more enzymes; and (c) incubating (b) to form gels.
  • step (a) further comprises mixing (iv) one or more negatively charged organosilane precursors together with (i), (ii), and (iii).
  • Step (a) in general is carried in an acidic pH (e.g., pH 1-6) or a basic pH (e.g., pH 8-13) to facilitate the formation of sols.
  • a preferred acidic pH is for example is pH 1-5, or pH 1.5-4.
  • a preferred basic pH is for example is pH 9-12.
  • An aqueous solution that contains one or more enzymes is usually buffered at a pH that stabilizes the enzymes or does not denature the enzymes.
  • an aqueous enzyme solution buffered at pH 4-10, preferably pH 5-9, is suitable for use in this invention.
  • a silicate sol is defined as a stable colloidal solution of silicate oligomers where the particle size is in the nanometer range.
  • Silicate sols can undergo gelation or precipitation when exposed to a change in pH or a catalyst (Her, R.K. 'The Chemistry of Silica' (Wiley, 1979); Brinker, C.J. and Scherer, G.W. 'Sol Gel Science: The Physics and Chemistry of Sol- Gel Processing' (Academic press, 1990)).
  • the order for mixing sols or enzymes to form gels is not important. However, it is important that the enzymes are not exposed to an extreme pH such as outside of pH 4-10 or 5-9 during the process.
  • sols can be accelerated by sonicating the precursors in the acidic solution, for example, for 30 minutes to an hour.
  • the time to form gels varies from a few minutes to many hours. The gelation time usually takes 20 minutes to 1 hour.
  • prolonged curing and drying of sol-gels often result in a reduction in both enzyme leakage during storage and enzyme release upon dilution into water.
  • Sol-gels that are optimally cured and dried have low enzyme leakage during storage and high enzyme release upon dilution into water.
  • sol-gels of the present invention are often cured for about 1 day to 1 week before formulated into a detergent formulation.
  • Sol-gels can be in various forms.
  • sol-gels are in the form of a fine powder, slurry, a microemulsion, or an emulsified suspension.
  • sol-gels can be suspended in liquids.
  • sol-gels can be incorporated into granules for addition to powdered product.
  • Sol-gels can be stored as crushed powders or pastes or any other as form mentioned above. Controlled monodispersed sol-gel microparticles can be prepared based on emulsion techniques known to a skilled person, for example, see Osseo-Asare, K. et al.
  • Sol-gel microparticles can also be prepared by mechanically crushing a sol-gel monolith, which usually results in a highly polydisperse distribution in particle size.
  • Silicate precursors useful for the present invention include alkali metal silicates and alkyl siliconate salts.
  • Alkali metal silicates include sodium silicates (e.g. sodium metasilicate, sodium orthosilicate and sodium silicate solutions), potassium silicates, and cesium silicates.
  • Preferred alkali metal silicates are sodium silicates and potassium silicates.
  • alkali metal silicates are sodium silicates.
  • Sodium silicates are commercially available.
  • sodium metasilicate and sodium orthosilicate can be obtained from Gelest Inc. (Morrisville, PA).
  • Sodium silicate solution (a solution of SiO and LiO).
  • Alkyl siliconate salts include sodium alkyl siliconate, potassium alkyl siliconate, and cesium alkyl siliconate.
  • Preferred alkyl siliconate salts are sodium alkyl siliconate and potassium alkyl siliconate.
  • the most preferred alkyl siliconate salt is sodium methyl siliconate.
  • the Si-OH groups capable of condensation with gel formation are generated by the protonation of Si-O-metal groups, such as an alkyl siliconate, e.g. sodium methylsiliconate, MeSi(ONa) .
  • Organofunctional silane precursors useful for the present invention include dialkyldialkoxysilanes, alkyltrialkoxysilanes, alkylsilanes, and aminoalkylsilanes.
  • An example of an dialkyldialkoxysilane is dimethyldimethoxysilane (DMDS).
  • DMDS dimethyldimethoxysilane
  • MTMOS methyltrimethoxysilane
  • An alkylsilane is aminopropylsilanetriol.
  • aminoalkylsilanes are aminoalkyltrialkoxysilane, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, bis(trialkoxyalkylsilane)amine, and aminoalkylsilanetriol or its salts.
  • An example of an aminoalkyltrialkoxysilane is 3-aminopropyltrirnethoxysilane (3-APTS).
  • An example of a bis(trialkoxyalkylsilane)alkylenediamine is bis[(trimethoxysilyl)propyl]ethylenediamine
  • An example of a bis(dialkyldialkoxysilane)amine is bis(methyldiethoxysilylpropyl)amine.
  • An example of a bis(trialkoxyalkylsilane)amine is bis[3-trimethoxysilyl)-propyl]amine (ATMOS).
  • the negatively charged organosilane precursors useful for this invention include trialkoxysilylalkyl succinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkyl sulfonic acid or carboxyalkylsilanetriol.
  • Preferred negatively charged organosilane precursors are 3-(triethoxysilyl)propyl succinic anhydride, 3- trihydroxysilylpropylmethylphosphonate, 3-trialkoxysilylpropylmethylphosphonate, and 3-
  • one or more additives such as disaccharides, polysaccharides, water soluble or dispersable polymers, i.e., polyvinyl alcohols, polyethylene glycol, or polypropylene glycol, can be added during the preparation of sol-gels.
  • the additives can be added in any step prior to the gelation.
  • Polysaccharides for example, include starch, pectin, sodium, alginate, or carrageenan.
  • a useful additive is sucrose. Sucrose helps formation of the gel network by hydrogen bonding.
  • Sucrose also adds hydrophilicity to the gel system.
  • the moisture retention helps in releasing the encapsulated enzyme, upon the dilution of the gel system in water.
  • the starting materials for enzyme-containing silica gels that are responsive to water percentage change include a combination of one or more silicate precursors and one or more organofunctional silane precursors. Pure alkoxysilane sol-gels derived from silicate precursors such as tetramethylorthosilicate (TMOS), and tetraethylorthosilicate (TEOS), do not release the entrapped protein in response to a triggering event of increasing the water content. Adding an organofunctional silane precursor makes the gel responsive or sensitive to water percentage changes.
  • TMOS tetramethylorthosilicate
  • TEOS tetraethylorthosilicate
  • Swelling and shrinking capacity of the silica gel matrices is an important criterion on which this invention is based.
  • One of the several mechanisms for enzyme release from sol- gels responsive to environmental water percentage is that upon dilution in water, swelling of the gel results in expansion of the micro pores in which the enzyme molecules are entrapped. This expansion of the micro pores allows for the diffusion and subsequent release of the enzyme from the sol-gel into the surrounding medium.
  • Swelling is the volume expansion of a monolilthic gel through absorption of water
  • Swelling and shrinking can be determined by weighing the gel before and after its exposure to water. Swelling can be reported as an increase in mass and shrinking can be reported as a decrease in mass. Alternatively, swelling and shrinking can also be determined by measuring the volume of the gel before and after its exposure to water. Measurement by weight is easier than measurement by volume. Swelling/shrinking of sol-gels is one of several mechanisms that allow for the release of enzyme from sol-gels upon a change in the surrounding fluid's water activity. Other mechanisms for controlling protein's release from sol-gels include hydrophobic interactions, hydrophilic interactions (i.e. hydrogen bonding) and ionic interactions, between the protein and sol-gels.
  • Sol-gels obtained from the starting materials of the present invention exhibit bulk volume changes and generate active mechanical responses when the surrounding environment changes.
  • the enlarged pores obtained due to aminoalkoxysilane precursors such as bis[3-(trimethoxysilyl)propyl]ethylenediamine (enTMOS), or (CH 3 O) 3 Si(CH 2 ) 3 NH(CH 2 ) 3 Si(OCH 3 ) 3 (ATMOS), with long chain spacer units, help in retaining aqueous phase in the porous network, which in turn makes the swelling and shrinking mechanism more pronounced.
  • enTMOS bis[3-(trimethoxysilyl)propyl]ethylenediamine
  • ATMOS alkyloxysilane precursors
  • the hydrophilic side chain of the precursor endows pH sensitivity to the gel system.
  • Enzymes suitable for entrapment in the sol-gel system can be any enzymes. Enzymes include but are not limited to commercially available types, improved types, recombinant types, wild types, variants not found in nature, and mixtures thereof. Preferred enzymes are detergent enzymes used in laundry detergents, fabric care products, or dishwasher detergents.
  • Suitable enzymes include hydrolases, cutinases, oxidases, transferases, reductases, hemicellulases, esterases, isomerases, pectinases, lactases, peroxidases, laccases, pectinases, catalases, and mixtures thereof.
  • Hydrolases hydrolyze substrates, e.g., stains, and are used in laundry detergents, dish detergents, and fabric care products. Hydrolases include, but are not limited to, proteases (bacterial, fungal, acid, neutral or alkaline), amylases (alpha or beta), lipases, mannanases, cellulases, and mixtures thereof.
  • Suitable enzymes for this invention also include those sold by Genencor International under the trade names Purafect, Purastar, Properase, Puradax, Clarase, Multifect, Maxacal, Maxapem, and Maxamyl (U.S. Patent No. 4,760,025 and WO 91/06637); and those sold by Novo Industries AS (Denmark) under the trade names Alcalase, Savinase, Primase, Durazyme, Duramyl, Lipolase, and Termamyl.
  • Suitable proteases are subtilisins, produced by Bacillus species.
  • Another suitable enzyme is cellulase and particularly cellulase or cellulase components isolated from Trichoderma reesei, such as found in the product Clazinase.
  • Amylases such as alpha amylases obtained from Bacillus licheniformis are also suitable enzymes.
  • Proteases are especially suitable for entrapment in a sol-gel system because of their hydrolytic action upon other enzymes and also their autolytic or self-proteolytic action.
  • Proteases and other enzymes are typically produced by aerobic fermentation of bacteria or fungi. These enzymes are generally secreted as extracellular proteins, but in some cases, enzymes can be isolated from the cell membrane or from within the cell by chemical, enzymatic or physical disruption.
  • the cells and cell debris are removed by processes such as centrifugation or filtration through porous media, often with the aid of flocculation agents.
  • enzymes Prior to the sol-gel process, enzymes are preferably concentrated by removing water and low molecular weight species such as salts or peptides, e.g., by ultrafiltration, evaporation, precipitation or extraction. Generally, ultrafiltration or tangential flow filtration through polymeric or ceramic membranes is a preferred practical or economical route.
  • the present invention provides an enzyme-entrapped sol-gel composition that is responsive to water percentage change. Water percentage as used herein is the percentage of water by weight in a solution.
  • a solution having a water percentage of 100% is pure water; a solution having a water percentage of 60% is a solution having 60% water by weight.
  • One or more enzymes can be entrapped in the sol-gel system.
  • the entrapped enzymes in general have an improved stability over the free, non-entrapped enzymes.
  • the enzymes in the sol-gel composition are stable during storage in that they maintain at least 60%, 70%, or 80%, preferably at least 85%, and more preferably at least 90%, of activities for 7 days at room temperature.
  • the enzymes are thermally stable during storage in that they maintain at least 60%, 70%, or 80%, preferably at least 85%, and more preferably at least 90%), of activities for 2 days at elevated temperature such as 37 °C.
  • the enzymes also stay within the sol-gels with less than 20%, preferably less than 15%, preferably less than 10%, and more preferably less than 5% leakage out of sol-gels for 7 days.
  • the enzymes Upon dilution of the sol- gel into a solution having a water percentage greater than 86%, the enzymes are quickly released into the solution. For example, at least 30% of enzymes are released from the sol- gel into the solution within one hour.
  • the present invention provides a liquid detergent, a liquid soap or a shampoo formulation comprising the enzyme-entrapped sol-gel system.
  • the formulation is prepared by mixing the enzyme-containing sol-gels with the liquid detergent, the liquid soap or the shampoo.
  • sol-gels are crushed and a detergent is added to the crushed powders to form a viscous detergent/sol-gel paste.
  • the enzymes are stable during storage in the formulation and are quickly released from the gels upon diluting the formulation in water.
  • the enzymes are stable during storage in the formulation in that they maintain at least 60%o, 70%), or 80%o, preferably at least 85%, and more preferably at least 90%>, of activities for 7 days at room temperature.
  • the enzymes are thermally stable during storage in the formulation in that they maintain at least 60%>, 70%>, or 80%, preferably at least 85%, and more preferably at least 90%, of activities for 2 days at elevated temperature such as 37 °C.
  • the enzymes also stay within the sol-gels with less than 20%, preferably less than 15% 0 , preferably less than 10%, and more preferably less than 5% leakage out of sol-gels into the liquid detergent, liquid soap or shampoo formulation for 7 days.
  • the liquid detergent, liquid soap or shampoo formulation in general has a water percentage (by weight) of 70% or lower, or 40% or lower, or 30%> or lower.
  • a "concentrated” or “compact” liquid detergent typically has 30-45 %> water and a "dilute” liquid detergent typically has greater than 50-60% water, sometimes 70% water by weight.
  • a common liquid detergent, TIDE ® Procter & Gamble, Cincinnati, OH
  • TIDE ® Procter & Gamble, Cincinnati, OH
  • the enzymes in the formulation are released when the formulation is diluted in water to a water percentage by weight of 86%> or higher, preferably 90%) or higher, more preferably 95%> or higher, more preferably 98% or higher, and most preferably 99% or higher.
  • the formulation is diluted at least 5 fold, often 10, 100, or even 1000 fold in water.
  • Table 1 shows the water percentage of a formulation before and after dilution in water.
  • the present formulation comprises a stable enzyme-entrapped gel system.
  • the enzyme Upon dilution of the formulation into water, at least 30% (preferably 40%>, 50%>, 60%, 70%>, or 80%) or the enzyme is released from the gel system into water within 1 hour, preferably 30 minutes, more preferably 10 minutes, more preferably 5 minutes, more preferably 2 minutes, and most preferably 1 minute.
  • the present invention further provides a powder detergent formulation comprising the enzyme-containing sol-gels.
  • enzymes are more stable in a powder detergent form than in a liquid detergent form.
  • a bleach-containing detergent powder is harmful to the enzymes.
  • some detergent powders are hygroscopic and have a tendency to absorb water during storage in humid climates; which creates stability problems for the enzymes.
  • a powder detergent formulation comprising the enzyme-containing sol-gels improves the enzyme stability.
  • Sol-gels can be granulated by a variety of enzyme granulation processes, e.g., using fluid bed technology to spray-coat a slurry of the sol-gel as a layer on a core; mixing the sol-gel entrapped enzyme into a paste prior to drum granulation, wet granulation or extrusion, etc.
  • the sol-gel granules are then mixed with the detergent powder to form a powder detergent formulation comprising the enzyme-containing sol-gels.
  • the enzymes are stable during storage in such a formulation and are released from the gels upon diluting the formulation in water.
  • silica gel matrices are used as vehicles for controlling the release of proteins such as enzymes used in cleaning products such as liquid detergents, soaps, and shampoos; the protein release is triggered by a dilution event that increases the water content.
  • the gel matrices not only improve the thermal stability of enzymes but also prevent the enzyme denaturation due to proteolysis/autolysis or interference from other components of the cleaning products.
  • Multi-enzyme detergent formulations are prepared by including different enzyme- entrapped silica gel matrixes in the detergent formulations, in which different enzymes are separated from each other by the gel matrixes, thus the proteolysis of the enzymes are prevented.
  • a control over the release of the enzyme into the detergent formulation during the wash cycle can be achieved.
  • cellulases that are used as fabric softeners can be released during the last few minutes of the wash cycle.
  • Example 1 Preparation of sodium silicate gels (Slaa) Preparation ofMTMOSsol. To 6 mL of H 2 O, 0.1 mL of HC1 (0.04 M) and 9 mL of methyltrimethoxysilane were added and the mixture was sonicated for 45 minutes to obtain a MTMOS sol. Silicate sol. To a solution of 1.2 ml H 3 PO 4 (3M) and 0.5 ml H 2 O, aqueous sodium silicate solution containing 0.75 ml sodium silicate solution (27%> w/w silica and 14%> w/w NaOH, Aldrich, WI) and 1.05 ml H 2 O was added to form a silicate sol.
  • Example 2 Swelling/Shrinking experiment
  • the blank Slaa gels were subjected to the swelling and shrinking experiments.
  • the gels were weighed at regular time intervals after keeping them immersed in water/detergent.
  • the % weight change is calculated as the percentage change in the weight of the gel in the medium for that time interval as compared to the starting weight.
  • a negative % weight change indicates shrinking of the gel matrix in that particular medium while a positive weight change indicates swelling.
  • Figure 1 indicates similar extent of % weight change when the gels were immersed in water and detergent. The results indicate that the gels shrank in both the water and detergent media.
  • Example 3 Release of enzyme from the Slaa gel upon a dilution trigger in water
  • the enzyme-containing S 1 aa gel was soaked for a day in TIDE ® liquid detergent, which had been heated at 90°C for one hour to deactivate the enzymes contained therein.
  • the gel-containing detergent was diluted in water, under mechanical agitation over a period of two hours.
  • the amount of the enzyme released from gel at each time interval was determined by assaying the enzyme activity in the medium in which the enzyme release was carried out.
  • the enzyme activity was determined by measuring the hydrolysis of substrate: N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-P-nitroanilide (Suc-AAPF- pNA).
  • Example 4 Effect of additives to the Slaa gels
  • Polysaccharides such as starch, pectin, sodium alginate and carrageenan ( ⁇ or type IV) were added to the silicate sol (Slaa gel).
  • the enzyme-containing silicate sol was prepared according to Example 1.
  • To the obtained sol 0.5 ml of 2% aqueous solution of starch/pectin/sodium alginate/carrageenan ( ⁇ ) was added, followed by the addition of 0.45 ml MTMOS sol.
  • Translucent gels were obtained within 45 minutes.
  • the gels obtained are referred as Slaa-starch, Slaa-pectin, Slaa-alginate and Slaa-Car(IV).
  • Example 5 Release of enzyme from Slaa - additive gels • (K) The enzyme containing gels were soaked for a day in TIDE detergent, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. Then the release experiments were carried out by diluting the detergent that contains the gels into water. The enzyme released from different gel systems at different time intervals is given in Figure 3. The addition of polysaccharides appears to facilitate the enzyme release from the Slaa gels. The percentage of the enzyme released from the Slaa-additives gel systems after the detergent is diluted by water, are 10 minutes and 60 minutes are given in Figure 4. Addition of pectin appears to enhance the release of enzyme in the first 10 minutes.
  • pectin, alginate and carrageenan all seem to enhance the enzyme release in an hour after the dilution in water.
  • the cuvette was filled with buffer (pH 5.5) and heated at 60°C in an oven for 4 h.
  • a control experiment was also carried out where the gel with the same composition was left at room temperature under same conditions. After 4 h of heating, the buffer solution was changed from pH 5.5 to pH 8.6
  • the assay was carried out by adding 20 ⁇ l suc-AAPF-pNA substrate to the activity buffer contained in the cuvette.
  • thermal stability of the unencapsulated stock enzyme was also carried out.
  • a solution of stock protease in buffer (pH 5.6) was heated at 60°C for 4 h. Control experiment under room conditions was also carried out.
  • Example 7 Preparation of Sodium Meta Silicate gels Preparation of MTMOS sol To 6 mL of H 2 O, 0.1 mL of HCl (0.04 M) and 9 mL of methyltrimethoxy silane were added and the mixture was sonicated for 45 minutes to obtain MTMOS sol.
  • Example 8 Release of enzyme from the metasilicate gels
  • TIDE ® detergent was heated at 90°C for one hour to deactivate the enzymes contained therein.
  • the enzyme release experiment was carried out from (a) MS(metasilicate)la gel in pH 7.85 buffer without prior soaking in TIDE ® , (b) MS(metasilicate)la after soaking in TIDE ® , (c) MS(metasilicate)lad after soaking in TIDE ® , and (d) MS(metasilicate)ladl after soaking in TIDE .
  • the results are shown in Figure 5.
  • the results show that 86%> of enzyme was released from MS(metasilicate)la in pH 7.85 buffer (without soaking in TIDE ), which indicates the absence of loss of activity of the enzyme during the gelation process.
  • MSla gel showed 38% leakage of the enzyme from the matrix when the gel was soaking in the detergent for one day.
  • MS lad gel which was derived from DMDS sol along with the MTMOS sol, had a decrease in leakage to 27%.
  • MSladl gel which was derived from DMDS sol (higher amount than that of MS lad gel) along with the MTMOS sol, had a further decrease in leakage to 11%.
  • Example 9 Preparation of Silicate/siliconate (JA) gels
  • 1.0 mL of an aqueous sodium methyl siliconate solution (30%> w/w, Gelest, NJ) and 2.0 mL H 2 O was added with stirring to 2.0 mL H 3 PO 4 solution (3M).
  • the resulting mixture had a pH of about 6.
  • Example 10 Preparation of Sodium Silicate Gels encapsulating a Dye Silicate sol-gel containing a dye was prepared. This is a visual model that can easily demonstrate the release of the active component from the gel.
  • MTMOS sol To 6 mL of H 2 O, 0.1 mL of HCl (0.04 M) and 9 mL of
  • Methyltrimethoxysilane were added and the mixture was sonicated for 45 minutes to obtain MTMOS sol.
  • Silicate sol To a solution of 1.2 ml H 3 PO 4 (3M) and 0.5 ml H 2 O, aqueous sodium silicate solution containing 0.75 ml sodium silicate solution (27% > w/w silica and 14% w/w NaOH, Aldrich, WI) and 1.05 ml H 2 O was added to form a silicate sol. To the silicate sol, 0.75 ml of 0.02 mg/mL Chicago Sky Blue dye (in water) was added, followed by 0.5 mL of 2%> aqueous solution of starch. This was " followed by addition of 0.45 ml MTMOS sol.
  • Example 11 Enzyme leakage during storage in detergent and enzyme release upon dilution in water.
  • Silicate/siliconate sol-gels were prepared according to Example 9.
  • Silicate- starch gels (Slaa-starch or SS) were prepared according to Example 4 where the starch used was PURE-COTE ® B790 (Grain Processing Corp., Muscatine, LA).
  • PURE-COTE ® B790 Gin Processing Corp., Muscatine, LA
  • TIDE ® detergent was heated at 90°C for one hour to deactivate the enzymes contained therein.
  • Each silicate/siliconate (JA) and silicate/starch gel (SS) monolith was immersed in a TIDE ® and stored for 1-4 weeks. There was no rotation during storage.

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Abstract

L'invention concerne une composition sol-gel comprenant à enzymes piégés dans laquelle un ou plusieurs enzymes sont piégés de façon stable dans les gels et libérés hors des gels en réponse à une augmentation du pourcentage d'humidité ambiant (en poids). Le procédé permettant de préparer ladite composition consiste : (a) à mélanger une première solution aqueuse avec un ou plusieurs silicates de métal alcalin ou siliconate d'alkyle afin de réduire le pH à 12 ou moins afin de former un premier sol ; (b) à mélanger une seconde solution aqueuse avec un ou plusieurs précurseurs de silanes organofonctionnels afin de former un second sol ; (c) à mélanger le premier sol, le second sol et une troisième solution aqueuse contenant un ou plusieurs enzymes afin de former des gels. L'invention concerne également un détergent liquide, un savon liquide ou une formulation de shampooing comprenant un système sol-gel à enzymes piégés, les enzymes étant libérés lorsque la formulation se dilue dans l'eau. Non seulement les matrices de gel de silice améliorent la stabilité thermique des enzymes, mais encore elles préviennent la dénaturation enzymatique due à la protéolyse/autolyse ou à l'interférence d'autres composants de la formulation.
PCT/US2004/030989 2003-09-19 2004-09-17 Sols-gels derives du silicate sensibles aux changements de teneur en eau WO2005028603A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1721881A1 (fr) * 2005-05-10 2006-11-15 Northrop Grumman Corporation Propergol gélifié par polymère et son procédé de fabrication
DE102007056166A1 (de) 2007-11-21 2009-05-28 Henkel Ag & Co. Kgaa Granulat eines sensitiven Wasch- oder Reinigungsmittelinhaltsstoffs
WO2011005913A1 (fr) 2009-07-09 2011-01-13 The Procter & Gamble Company Composition catalytique de détergent pour lessive comprenant des taux relativement bas d'électrolyte soluble dans l'eau
WO2011005730A1 (fr) 2009-07-09 2011-01-13 The Procter & Gamble Company Composition catalytique de détergent pour le linge comprenant des taux relativement faibles d'électrolyte soluble dans l'eau
EP2365053A1 (fr) 2010-03-12 2011-09-14 The Procter & Gamble Company Compositions liquides de détergent comprenant des gélifiants amido à pH réglable et procédés de fabrication
EP2365051A1 (fr) 2010-03-12 2011-09-14 The Procter & Gamble Company Compositions liquides de détergent comprenant un gélifiant di-amido et procédés de fabrication
EP3067410A2 (fr) 2008-02-15 2016-09-14 The Procter and Gamble Company Compositions de nettoyage
EP3275990A1 (fr) * 2016-07-28 2018-01-31 The Procter and Gamble Company Procédé pour re-mélanger une première composition de détergent liquide dans une seconde composition de détergent liquide
US10047329B2 (en) 2013-09-27 2018-08-14 Rohm And Haas Chemicals Llc Water dispersible films for packaging high water containing formulations
US10400114B2 (en) 2013-09-27 2019-09-03 Rohm And Haas Company Ionic strength triggered disintegration of films and particulates

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Publication number Priority date Publication date Assignee Title
US5378399A (en) * 1990-01-31 1995-01-03 Industrial Progress, Inc. Functional complex microgels with rapid formation kinetics
US6337089B1 (en) * 1998-02-06 2002-01-08 Seiwa Kasei Company, Limited Microcapsule containing core material and method for producing the same
US20040127393A1 (en) * 2002-10-23 2004-07-01 Valpey Richard S. Process and composition for producing self-cleaning surfaces from aqueous systems

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5378399A (en) * 1990-01-31 1995-01-03 Industrial Progress, Inc. Functional complex microgels with rapid formation kinetics
US6337089B1 (en) * 1998-02-06 2002-01-08 Seiwa Kasei Company, Limited Microcapsule containing core material and method for producing the same
US20040127393A1 (en) * 2002-10-23 2004-07-01 Valpey Richard S. Process and composition for producing self-cleaning surfaces from aqueous systems

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1721881A1 (fr) * 2005-05-10 2006-11-15 Northrop Grumman Corporation Propergol gélifié par polymère et son procédé de fabrication
DE102007056166A1 (de) 2007-11-21 2009-05-28 Henkel Ag & Co. Kgaa Granulat eines sensitiven Wasch- oder Reinigungsmittelinhaltsstoffs
EP3067410A2 (fr) 2008-02-15 2016-09-14 The Procter and Gamble Company Compositions de nettoyage
WO2011005913A1 (fr) 2009-07-09 2011-01-13 The Procter & Gamble Company Composition catalytique de détergent pour lessive comprenant des taux relativement bas d'électrolyte soluble dans l'eau
WO2011005730A1 (fr) 2009-07-09 2011-01-13 The Procter & Gamble Company Composition catalytique de détergent pour le linge comprenant des taux relativement faibles d'électrolyte soluble dans l'eau
WO2011112886A1 (fr) 2010-03-12 2011-09-15 The Procter & Gamble Company Compositions détergentes fluides comprenant un gélifiant di-amido et leurs procédés de fabrication
EP2365051A1 (fr) 2010-03-12 2011-09-14 The Procter & Gamble Company Compositions liquides de détergent comprenant un gélifiant di-amido et procédés de fabrication
WO2011112910A1 (fr) 2010-03-12 2011-09-15 The Procter & Gamble Company Compositions détersives liquides comprenant des gélifiants d'amidon à ph réglable, et leurs méthodes de fabrication
EP2365053A1 (fr) 2010-03-12 2011-09-14 The Procter & Gamble Company Compositions liquides de détergent comprenant des gélifiants amido à pH réglable et procédés de fabrication
US10047329B2 (en) 2013-09-27 2018-08-14 Rohm And Haas Chemicals Llc Water dispersible films for packaging high water containing formulations
US10400114B2 (en) 2013-09-27 2019-09-03 Rohm And Haas Company Ionic strength triggered disintegration of films and particulates
EP3275990A1 (fr) * 2016-07-28 2018-01-31 The Procter and Gamble Company Procédé pour re-mélanger une première composition de détergent liquide dans une seconde composition de détergent liquide
WO2018022271A1 (fr) * 2016-07-28 2018-02-01 The Procter & Gamble Company Procédé pour remélanger une première composition détergente liquide dans une seconde composition détergente liquide
US10323219B2 (en) 2016-07-28 2019-06-18 The Procter & Gamble Company Process for reblending a first liquid detergent composition into a second liquid detergent composition

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