WO2003106607A1

WO2003106607A1 - Silicone emulsion enzyme systems

Info

Publication number: WO2003106607A1
Application number: PCT/US2003/018943
Authority: WO
Inventors: Nathaniel T. Becker; Doris Jean Brecht; Steven Patrick Christiano; Russel Allen Elms; Qian Jane Feng; Keith Quentin Ii Hayes; Meng H. Heng; Isabelle Mazeaud; Martin Kent Severance
Original assignee: Dow Corning Corporation; Genencor International, Inc.
Priority date: 2002-06-17
Filing date: 2003-06-17
Publication date: 2003-12-24
Also published as: AU2003276412A1; AU2003276412A8

Abstract

The present invention relates to the use of silicone materials to form emulsionto protect active ingredients such as enzymes in liquid formulations during storage.The present invention is directed to a multiple-emulsion enzyme system comprising:an inner aqueous phase containing an enzyme, an outer phase of a silicone fluid, acontinuous phase surrounding the outer phase, and surfactants. The present inventionis also directed to a suspension-emulsion enzyme system comprising: a silicone fluidcontaining a solid enzyme dispersion without an aqueous solution intervening betweenthe enzyme and the silicone fluid, dispersing agent that disperses the enzyme in thesilicone fluid, a continuous phase surrounding the silicone fluid, and a silicone surfactant.

Description

SILICONE EMULSION ENZYME SYSTEMS

FIELD OF THE INVENTION

The present invention relates to the protection of active ingredients during storage in environments that inactivate or degrade such active ingredients. More particularly, the invention relates to the use of silicone materials to protect enzymes as used in surfactant rich media, such as liquid laundry detergents, personal care products, and textile care products.

BACKGROUND OF THE INVENTION

Enzymes are commonly employed as stain and soil removing agents in powder detergents but their incorporation in liquid cleaning preparations such as liquid laundry detergents has hitherto presented serious problems. Those liquid formulations that is most effective for soil removal cause rapid degradation of washing enzymes, often resulting in significant loss of stain and soil removing properties after only a few days of storage, unless significant amounts of stabilizing chemicals are added to the detergents. Surfactants, especially anionic surfactants such as linear alkylbenzenesulfonates and the like, can be particularly damaging to the enzymes. The relatively high alkalinity and the chemical interaction of most of the builder systems used in liquid laundry detergents are antagonistic to detergent enzymes and limit their maintenance of activity during storage. It is also desirable in many cleaning formulations to provide combinations of enzymes, in which case degredative interaction of protease enzyme with itself (autolysis) and with other enzymes (proteolysis) contribute to the instability of the detergent enzymes. Serious deterioration of enzymes is observed even in comparatively non-alkaline compositions, which have been specially formulated to permit incorporation of enzymes. Typically enzyme-stabilizing systems are included in liquid detergents to enhance the enzyme stability. Stabilizers that are well known to the industry include calcium salts, borate species, polyols, chlorine scavengers, and the like. These stabilizers add considerable manufacturing cost, and still do not provide fully adequate stabilization. In addition, some of the most effective protease inhibitors are based on adducts of the element boron that interacts with the active site of serine proteases; such boron compounds, however, are facing increasing regulatory restrictions due to negative environmental and health effects. Active ingredients used in liquid detergents include enzymes, surfactants, alkaline sources, dyes, perfuming agents, bleaching agents, bleach activators, brighteners/fluorescers, polymers, fabric conditioners, and other detergent active components known to the art. Liquid detergents containing such active ingredients present a particular, yet unsolved challenge to protect active ingredients.

Current heavy duty liquid detergent (HDL) formulas attempt to protect active ingredients by limiting linear alkyl benzene sulphonate (LAS) surfactant content, or reducing the ratio of LAS to co-surfactants such as nonionic surfactants. Such formulas generally use less effective and more expensive substitutes, such as alkyl sulfate and ethoxylated alkyl sulfate, which are less denaturing towards enzymes, but at the same time weaker in their action towards proteinaceous or oily stains in the wash. Even such milder anionic surfactants, however, can denature enzymes formulated in detergents. In any case, there is a need for an improved and less costly enzyme stabilization system which protects enzymes from cost effective anionic surfactants, alkalinity sources, and other denaturing chemicals present in HDL formulas.

U.S. Patent No. 4,906,396 (Falholt) is directed to a protected enzyme system consisting essentially of a hydrophobic fluid which is substantially insoluble in aqueous liquid laundry detergents and a detergent enzyme dispersed in said fluid.

U.S. Patent 5,494,600 (Surutzidis) is directed to a particulate detergent additive comprising a mixture of a surfactant and an effective amount of a water-soluble or water- dispersible detergent active absorbed into the pores of a porous hydrophobic silica having an average pore diameter larger than the size of the molecules of the detergent active, said porous hydrophobic silica containing the absorbed mixture of surfactant and detergent additive being coated completely with a hydrophobic coating material or a water-insoluble water-permeable polymeric material, provided that: (a) the detergent active is selected from enzymes, bleaches, bleach activators, bleach catalysts, photoactivators, dyes, fluorescers and fabric conditioning agents, and (b) the absorbed surfactant is present in an amount sufficient to wet the hydrophobic silica and to permit the detergent additive to be readily desorbed during washing conditions. WO 96/20274 discloses a stable enzyme-containing liquid composition comprising: at least one enzyme having an insoluble form thereof, an agent for maintaining the enzyme in the insoluble form thereof and a liquid formulation, wherein the enzyme in the insoluble form thereof and the agent are combined so that the enzyme is stable and the enzymatic activity thereof is retained. The reference discloses that this composition is useful for preparation of stable enzyme-containing liquid detergent compositions.

SUMMARY OF THE INVENTION The present invention is directed to silicone emulsion systems that protect active ingredients during storage. The multiple-emulsion enzyme system of the present invention comprises: (a) an inner aqueous phase containing an enzyme, (b) an outer phase of silicone fluid, (c) a continuous phase surrounding the outer phase, wherein said continuous phase comprises water, a non-aqueous polar liquid, a non-aqueous nonionic surfactant, or a mixture thereof, (d) a first surfactant in an amount effective to maintain the enzyme in the inner phase, and (e) a second surfactant in an amount effective to maintain the silicone fluid as a fine particle dispersion in the outer phase.

The suspension-emulsion enzyme system of the present invention comprises: (a) a silicone fluid containing a solid enzyme dispersion without an aqueous solution intervening between the enzyme and the silicone fluid, (b) a dispersing agent that disperses the enzyme in the silicone fluid, wherein said dispersing agent adsorbs at the interface of the solid enzyme and the silicone fluid and is compatible with silicone, (c) a continuous phase surrounding the silicone fluid, wherein said continuous phase comprises water, or non- aqueous polar liquid, or a non-aqueous nonionic surfactant, or a mixture thereof, and (d) a silicone surfactant in an amount effective to remain adsorbed on the interface of the silicone fluid and the continuous phase.

The present invention also includes a process of forming silicone enzyme systems in a suspension- emulsion or multiple emulsion format.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a multiple-emulsion enzyme system. Figure 2 depicts a suspension-emulsion enzyme system.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to silicone emulsion systems that protect active ingredients during storage in an environment that tends to cause degradation of active ingredients. The present invention is particularly directed to an enzyme system in a surfactant-rich medium. Surfactant-rich media are commonly used in the treatment of skin, hair, fabrics or textiles, or hard surfaces to remove stains and soil. Examples of surfactant- rich liquid media are cleaners, shampoos, skin cleaning creams or gels, liquid detergent, liquid soaps, dentifrices, etc.

Enzymes in the present invention are within an outer shell of silicone fluid, which serves as a barrier to decrease the permeation rate of hostile materials such as detergents from the environment into the inner phase that contains enzymes. The present enzyme composition comprises at least one surfactant that stabilizes the emulsion of the system; the stabilizing surfactant may play a role in enhancing the barrier properties of the silicone fluid shell.

Enzymes suitable for incorporation in the silicone 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, fabrics 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 A S (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 incorporation in a silicone system because of their hydrolytic action upon other enzymes and also their autolytic or self-proteolytic action. Protection or compartmentalization of proteases into a distinct phase with low water activity and reduced permeability limits the extent of possible proteolysis during storage prior to use. One or more enzymes can be utilized in within the hydrophobic silicone system.

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. Commercially, 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 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 system utilizing a hydrophobic material, such as silicone, in a finely dispersed form such as a suspension-emulsion or a multiple-emulsion. The enzyme system can be easily diluted and dispersed into a surfactant rich medium, while maintaining the structure of the hydrophobic material around the enzymes. During use, the enzyme is efficiently released through mechanical or chemical disruption of the hydrophobic protection system. It is desirable that the hydrophobic material be in a finely dispersed and stable particulate form with enzymes located wholly within the hydrophobic particles.

Definitions In order to provide an even clearer and more consistent understanding of the specification and the claims, including the scope given herein to such terms, the following definitions are provided:

Active ingredient is any material that is to be delivered to a liquid environment. The active ingredient includes a biologically viable material, a protein, enzyme, peptide, bleach, bleach catalyst or activator, perfume, dye, fabric softener or conditioner, brightener, a cleaning or sanitizing ingredient, or other active detergent ingredient that is soluble or dispersible in water.

Dispersing agent/Dispersant is a surface-active agent added to a suspending medium to promote uniform and maximum separation of extremely fine solid particles, often of colloidal size. Dispersing agents promote wetting, efficient distribution of fine solid particles in the liquid dispersing medium and stabilization against particle aggregation. The dispersing agent is added in the dispersing medium in amount sufficient to provide complete surface coverage of the particle surface. Emulsion is a temporary or permanent dispersion of one liquid phase within a second liquid phase. The second liquid is generally referred to as the continuous phase. Surfactants are commonly used to aid in the formation and stabilization of emulsions. Not all surfactants are equally able to stabilize an emulsion. The type and amount of a surfactant needs to be selected for optimum emulsion utility especially with regards to preparation of the emulsion, physical stability in storage, and stability during dilution and mixing within a surfactant rich detergent. Physical stability refers to maintaining an emulsion in a fine dispersion form, which may be a factor in preserving chemical activity, i.e., enzyme activity. Processes such as coalescence, aggregation, adsorption to container walls, sedimentation and creaming, are forms of physical instability, and should be avoided.

Emulsions can be further classified as either simple emulsions, wherein the dispersed liquid phase is a simple homogeneous liquid, or a more complex emulsion, wherein the dispersed liquid phase is a heterogeneous combination of liquid or solid phases, such as a double emulsion or a multiple-emulsion. For example, an oil-in-water double emulsion or multiple emulsion may be formed wherein the oil phase itself further contains an emulsified aqueous phase; this type of emulsion may be specified as a water-in-oil-in water (w/o/w) emulsion. Alternatively, an oil-in-water emulsion may be formed wherein the oil phase contains a dispersed solid phase and is herein referred to as a suspension- emulsion. Other more complex emulsions can be described. For example, an oil-in-water emulsion may be formed wherein the oil phase contains an emulsified aqueous phase that is itself a suspension of solids in an aqueous solution. Because of the inherent difficulty in describing such systems, the term multiple emulsion is used to describe these complex emulsions without necessarily limiting the form of the emulsion or the type and number of phases present.

Suspension is a system in which very small particles (solid, semisolid, or liquid) are more or less uniformly dispersed in a liquid or gaseous medium.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used in the specification and claims, the singular "a", "an" and "the" include the plural references unless the context clearly dictates otherwise. For example, the term active ingredient may include a plurality of active ingredient. Multiple-emulsion

In one embodiment of the invention, the enzyme system is a multiple-emulsion. The multiple-emulsion composition comprises: (a) an inner aqueous phase containing an enzyme, (b) an outer phase of silicone fluid, (c) a continuous phase surrounding the outer phase, wherein said continuous phase comprises water, a non-aqueous polar liquid, a non- aqueous nonionic surfactant, or a mixture thereof, (d) a first surfactant in an amount effective to maintain the enzyme in the inner phase, and (e) a second surfactant in an amount effective to maintain the silicone fluid dispersed in the outer phase. The inner liquid phase of the multiple-emulsion may contain some other phase within it. For example, there may be a solid phase dispersed in the liquid. The inner liquid is immiscible or has limited miscibility with the hydrophobic silicone protectant dispersed droplets but it may be miscible with the continuous phase. The majority of the first surfactant adsorbs on the interface of the inner phase and the outer phase. The majority of the second surfactant adsorbs on the interface of the silicone fluid and the continuous phase. Figure 1 depicts a multiple-emulsion enzyme system. The specific scale or number of inner aqueous phase droplets or outer silicone phase droplets in Figure 1 is not meant to be representative or limiting, but rather to qualitatively illustrate the phase structure of the multiple emulsion.

A first surfactant is used to stabilize the inner aqueous phase droplet of the multiple- emulsion. The first surfactant may be different from the second surfactant used to stabilize the outer phase of the emulsion droplets within the continuous phase. The inner aqueous phase is an aqueous, active ingredient solution. The active ingredient solution may contain salts, buffers, and stabilizing agents as is known to the art to preserve the stability of the active ingredient. In the multiple-emulsion system, the active ingredient can be a crystalline active ingredient, a concentrated active ingredient solution, an amorphous precipitated active ingredient slurry in an aqueous salt solution, or an active ingredient adsorbed onto porous silica (silicon dioxide). A common active ingredient is an enzyme.

A preferred form of enzyme for incorporation into the silicone systems is enzyme crystal, for example, sbtilisin protease crystal, or a mixture of crystals of different enzymes, such as a mixture of protease and amylase. Enzyme crystals represent a distinct form of matter in which the enzyme is structured into a repeating ordered array, unlike other forms of solid enzyme such as amorphous precipitates, spray-dried powders or lyophilized powders. Enzyme crystals typically contain high levels of bound water and open channels. Intact enzyme crystals represent a very stable form of enzymes by themselves, but they are easily solubilized unless their solubility is reduced by suspension in a solution of precipitants (such as salts and polymers), or they are further protected by chemical or physical means. One method of protecting enzyme crystals includes protein surface crosslinking using bifunctional reagents known to a person skilled in the art, e.g, glutaraldehyde, disuccinimidyl suberate, m-maleimidobenzoyl-N-hydroxysuccinimide, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide, which react with the surface lysine or sulfhydry groups on enzyme crystals. However, crosslinked enzyme crystals suffer the disadvantage of greatly reduced solubility, which would not satisfy the need for rapid delivery in applications such as laundry detergents, h the present invention, the silicone system provides a barrier between the enzyme and the harmful ingredients in the formulation, and protects the enzyme crystals from dissolution within the bulk formulation, which is aqueous in nature and tends to favor the solubilization of the enzyme crystals. At the same time, however, this barrier is such that it can be readily removed in a wash application by means such as dilution, agitation and shear, allowing full or substantial release of enzymes from the silicone emulsion to be utilized in an application.

Crystallization of enzymes is induced by reducing the solubility of enzymes in a more controlled and limited extent than that occurs when precipitating enzyme in the amorphous form. Addition of agents such as salts and polymers to induce crystallization of enzymes in bulk is well known. See Scopes, Robert K., Protein purification:

Principles and Practice, Springer-Nerlag, New York (1984); Northrup, John H., Kunitz, Moses, and Herriot, Roger M., Crystalline Enzymes, Number XII of the Columbia Biological Series, Columbia University Press, New York (1948); Becker, et al. U.S. Patent 5,041,377; Becker, Separation and purification processes for recovery of industrial enzymes, in Enzymes in Detergency (eds.), Singh, R.K. and Rizvi, S.S.H., Marcel Dekker, New York, pp. 299-325 (1995); Gros, et al. U.S. Patent No. 6,207,437 (2001); and Johns, M. R., J. Cryst. Growth, 90: 105-111 (1988). For example, subtilisin can be crystallized by addition of a number of salts such as sodium formate, magnesium acetate, or sodium chloride. Halide salts such as sodium chloride are preferred for subtilisin crystallization due to the high yield, rapid rate, and low risk of forming amorphous precipitates.

In the crystallization of subtilisin, a preferred process is to first concentrate the enzyme to a minimum of 6% (w/w) subtilisin, more preferably greater than 8% subtilisin, maintain the pH between pH 5.0 and 6.5, and to bring the solution either to a low temperature less than 10-15 °C (U.S. Patent No. 5,041,377), or to a temperature greater than 30 °C with rapid addition of salt to induce crystallization before the protease has time to auto-digest. Salt can then be added, e.g., in the case of sodium chloride, a level of 3-5% salt is optimal for the crystallization of subtilisins. Seed enzyme crystals from previous runs can be added to improve process speed and reproducibility, as nucleation in the absence of seed crystals is not fully controllable.

After addition of salt, the solution is agitated to ensure uniformity, crystals will form and reach equilibrium over a period of approximately 3 to 48 hours, depending on the temperature and degree of seeding. After the crystals cease further growth, the aqueous crystal slurry can be stored until further processing, optionally with the addition of antimicrobial preservatives to limit growth of organisms. The slurry can be used without further processing, or it can be diluted and washed with a nonsolubilizing buffer (e.g. a salt solution) to remove impurities such as uncrystallized protein, contaminant proteins, and salts. Crystal washing can be carried out in a perforate-bowl centrifuge, a plate-and-frame filter press, or similar solid-liquid separation equipment. A crystalline enzyme suitable for the present invention has a crystal size less than about 50 μm. For optimal protection, a preferred size of the crystal is smaller than that of a silicone droplet, such as less than about 10 μm, and more preferably less than about 5 μm. The size of enzyme crystal can be controlled by controlled crystal growth. For example, protein crystal size can be minimized by rapidly decreasing protein solubility (through precipitant addition or temperature reduction) to induce nucleation, by limiting the amount of protein in excess of the solubility limit, and by limiting the time available for crystallization. The size of enzyme crystal can also be controlled by a milling process or other mechanical process to reduced the particle size. Techniques known in the art suitable to reduce crystal size are wet- grinding using ball mills, jet mills, vibratory mills, attrition mills, sand and beads mills, high speed impellers, three-roll mill and sigma-blade kneader as described in H Mollet & A. Grubenmann, Formulation technologies: emulsions, suspensions , solid form, Wiley-NCH edition, Weinhein, Germany, (2001).

The particle size can be measured by dispersing the enzyme solids into an aqueous solution (for example, 3.5 wt.% sodium chloride solution that is saturated with the enzyme) followed by any method known to a skilled person, e.g., by laser particle size analyzer. This experiment is preferably performed using strict temperature control to maintain the sample at the temperature at which saturation was attained.

Another form of enzyme in a multiple-emulsion system is an amorphous precipitate enzyme slurry in an aqueous salt solution. The enzyme is brought to saturation by addition of a precipitant, such as a salt or polymer, such that the majority of the enzyme becomes insoluble and converted to a dispersed solid phase or slurry. The suspended solids content of the enzyme slurry is optimally set as high as possible for efficient protection, yet not too high such as to impart a high viscosity to the slurry. If the viscosity is too high, it will limit or preclude emulsification into the silicone protectant phase. Preferred droplet size for the slurry is less than the size of the silicone droplet, in order to ensure effective encapsulation within the droplet.

The enzyme can also be introduced into a silicone system within the inner aqueous phase as a true solution, rather than a solid or slurry. The enzyme solution is preferred to be as concentrated as possible, i.e., with an active enzyme solids concentration of greater than 5% (w/v), more preferable greater than 10%, most preferable greater than 15%. Producing such concentrated solutions of enzyme by ultrafiltration in some cases may require prior removal of at least some of the other solution solids (e.g. salts, peptides, carbohydrates, and other contaminant proteins) by prior steps of diafiltration, precipitation or chromatography. An alternative enzyme form can be obtained by loading a solution or slurry form of the enzyme onto silica, preferably hydrophilic silica. Enzyme is easily absorbed into hydrophilic silica. A surfactant ensures the uptake and retention of silica within silicone. The ultimate release of enzyme from hydrophilic silica is more efficient than hydrophobic silica, because the former is readily wetted by aqueous solutions. Silicone fluid useful for this invention has a general formula selected of (R¹)₃SiO-

[(R¹)₂SiO]_q-Si(R¹)₃, wherein each R¹ is independently selected from the group consisting of unbranched alkyl groups having from 1 to about 30 carbon atoms, branched alkyl groups having from 1 to about 30 carbon atoms, substituted aromatic hydrocarbon groups containing from 1 to about 10 carbon atoms in one or more side chains, and cycloalkyl groups having from about 3 to about 6 carbon atoms.

Silicone fluids (polyorganosiloxane or polydiorganosiloxane) suitable for use in the composition of the present invention have a viscosity at 25 °C from about 0.001 to about 1000 Pascal seconds, preferably from about 0.02 to about 100 Pascal seconds, and more preferably from about 0.05 to about 50 Pascal seconds. Specific examples of linear silicone fluids suitable for use in the composition of the present invention include, but are not limited to the trimethylsiloxy-terminated dimethylsiloxane fluids sold by Dow Corning Corporation under the trade name "Dow Corning® 200 Fluids." These silicone fluids are manufactured to yield essentially linear oligomers and/or polymers typically having a viscosity of from 0.001 to about 50 Pa-s at 25 °C. Such fluids are primarily linear.

Another useful form of polydiorganosiloxane fluid for this invention is copolymers commonly known as alkylmethyl fluids. They are copolymers of the structure (Me)₃SiO- [MeR¹²SiO]ι[(Me)₂SiO]_m -Si(Me)₃, where Me represents methyl groups, and R¹² are alkyl groups typically containing about 8 to 30 carbon atoms.

Yet another form of silicone fluid useful for this invention is the combination of polydiorganosiloxane with polyisobutylene, especially polyisobutylene of molecular weight of less than 1,000 grams per mole and bearing non-reactive terminal groups. Silicone fluids that are silanol terminated with a structure of HO — [(R¹³)₂SiO]_n — H, where R¹³ is alkyl or aryl groups, are also of utility in this invention. Silanol terminated polydiorganosiloxane fluids suitable for use in the composition have a viscosity at 25 °C of from about 0.001 to about 1000 Pa-s, preferably from about 0.02 to about 100 Pa-s, and more preferably from about 0.05 to about 50 Pa-s. These materials may be used alone or in combination with trimethylsilyl terminated fluids.

The silicone fluid of the present invention can optionally contain cyclic silicones of the general formula [(R )₂SiO]_n , where n is from about 2 to about 10, and R is independently selected from the group consisting of unbranched alkyl groups having from 1 to about 30 carbon atoms, branched alkyl groups having from 1 to about 30 carbon atoms, substituted aromatic hydrocarbon groups containing from 1 to about 10 carbon atoms in one or more side chains, and cycloalkyl groups having from about 3 to about 6 carbon atoms.

Mixtures of the aforementioned fluids are also useful. Many of the linear, branched, and cyclic silicone fluids have melting points below about 25° C. Such materials are also commonly referred to as silicone liquids, or silicone oils. A detailed description of silicone fluids can be found in many references, including "Chemistry and Technology of Silicones" by W. Knoll, Academic Press, 1968.

Polydiorganosiloxane blended with silicone resins can also be useful for the present invention. Silicone resins of this type are generally well known and have a general formula of R₃SiOι/₂ units and SiO /₂ units, wherein R is independently selected from the group consisting of alkyl, aryl or alkenyl, and the ratio of R₃SiOι_/ units to SiO_4/2 units is 0.4: 1 to

1.2:1.

Applicants have discovered that efficient capture and retention of crystalline enzyme slurry in the form of a multiple-emulsion is dependent on the viscoelasticity rather than the viscosity of the silicone oil phase. Niscoelasicity in polymer systems is well known as is described in Aklonis, J.J., MacKnight, W.J., Introduction to Polymer Viscoelasticity, second edition, John Wiley and Sones, New York, 1983.

Some materials exhibiting viscoelastic characteristics have some of the properties of both solid and liquid characteristics. A true fluid flows when it is subjected to a shear field and motion ceases as soon as the stress is removed. Under flow, liquids have a characteristic viscosity which is their tendency to dissipate energy, oftentimes measured as a loss modulus G". In contrast, ideal solids subjected to stress recover their original state as soon as the stress is removed, in other words they respond elastically to the stress. This response is often measured as a storage modulus G'. One can characterize the relative importance of storage and loss for a material by evaluating the dynamic mechanical loss tangent (tan δ) equal to the ratio of G'VG', such that a lower tan δ corresponds to a more solid like response to shear stresses. Viscoelastic silicone fluid typically found to be appropriate for use with this invention have a storage modulus between 0.001 and 500,000 Pascal at a frequency of 0.10 hertz at 25 °C, alternatively 5 to 100,000 Pascal, or alternatively 5 to 50,000 Pascal. Further these liquids may have a dynamic mechanical loss tangent of greater than about 0.001 and less than 1, or alternatively have a dynamic mechanical loss tangent of greater than 0.01 and less than 1, or alternatively have a dynamic mechanical loss tangent of greater than 1.0. Preferred silicone fluids in the multiple-emulsion system are viscoelastic silicone fluids. Viscoelastic silicone fluids form multiple emulsions and retain aqueous enzyme solutions or aqueous slurries of solid form enzymes to a greater degree than silicone fluids possessing simple viscosity. Viscoelastic silicone fluids are generally cross linked or branched silicone polydiorganosiloxane fluids. Formation of silicone fluids of this type are well known to those practiced in the art. For example, cross linking or branching can arise through incorporation of T (monoorganotrisiloxy) and Q (tetrasiloxy) groups into the backbone of a polydiorganosiloxane. One other method to produce a branched or cross linked silicone is through the platinum catalyzed curing together of alkenyl functional polydiorganosiloxanes with organohydrogenpolysiloxanes. Another method of cross linking or branching silicones is through condensation of a silanol terminal polydiorganosiloxane with silanol groups on a hydrolyzed alkoxysilanes (see for example U.S. Patent No. 5,581,008) or hydrolyzed alkoxypolysilicates (see U S. Patent number 4,749,740), or onto reactive silanols on silicone resins (see U.S. Patent number 4,639, 489). These chemistries are given as examples and in no way are meant to limit the application of known cross linking or branching chemistries to this invention.

Crosslinking of silicones may reduce the rate of release of the internal droplets containing enzymes. For this reason, an optimal level of cross linking or branching of silicone is desired in the viscoelastic fluids used for this invention.

A continuous phase is a phase surrounding the outer phase of silicone fluid. The continuous phase comprises water, or a substantially aqueous solution, or a non-aqueous polar liquid, or a nonionic surfactant, or mixtures thereof. A substantially aqueous solution contains salts, buffers, or other water-soluble materials dissolved in water and is often present at less than about 25 wt.% of the continuous phase. Since the continuous phase is designed to be a distinct phase, its character is further restricted to liquids which are essentially immiscible with the particular silicone fluids of the composition.

The nonaqueous polar liquids can be selected from the group consisting of ethylene glycol, propylene glycol, polypropylene glycol, polyethylene glycol, copolymers of either a random or block type of polyglycol containing propylene oxide, butylene oxide, and ethylene oxide, and condensates with polyols such as glycerol. Additional nonaqueous polar liquid continuous phase of this invention includes a wide range of nonionic organic surfactants such as alcohol alkoxylates or alkylphenol alkoxylates.

Nonionic surfactants useful as continuous phase components in this invention include alkoxylated alkyphenols, alkoxylated alcohols, blocky copolymers of ethylene oxide, propylene oxide, butylene oxide, fatty acid esters such as formed with glycerol, polyglycerol, sorbitol or other sugars such as alkoxylated sugars, alkylpolyglucasides, and alkylglucamides and the like. Other nonionic surfactants well known to the industry can also be used as continuous phase. These surfactants can be used alone as a neat phase or in combination with other previously mentioned components useful in the continuous phase, with the provision that a low viscosity liquid phase be formed such that it is readily soluble and dispersible in a liquid detergent. Compositions that form highly viscous or gel phases, liquid crystalline phases, solid phases, etc., by themselves or in combination with components in a liquid detergent are not suitable for this invention. The continuous phase is selected for ease of dispersibility and solubility in a surfactant-rich liquid medium since insufficient solubility can lead to poor dispersibility and poor stability of the silicone enzyme systems in such a liquid medium. Consideration is also made for compatibility of the liquid with the nonionic silicone surfactants which may be used in preparing the dispersion-emulsion or multiple-emulsion forms of the invention described herein. The continuous phase is further selected based on the specific gravity with a close match relative to the silicone protected enzyme particles. Preferably, the continuous phase has a viscosity below about 10,000 cSt at 25°C. A closer match of the continuous phase specific gravity to the silicone droplets can be obtained by selecting and blending two or more nonaqueous liquids to make the component or by combining aqueous and nonaqueous components within the limits of being completely miscible with each other so as to form a single phase. It is preferable that about 25 to 900 parts by weight of liquid continuous phase be used per 100 parts by weight of silicone protected enzyme. It is more preferred for purposes of the present invention that 100 to 400 parts by weight of liquid continuous phase be used per 100 parts by weight of silicone protected enzyme.

The multiple-emulsion enzyme system comprises a first surfactant and a second surfactant. The first surfactant adsorbs on the interface of the inner phase and the outer phase in an amount effective to maintain the enzyme in the inner phase. The second surfactant adsorbs on the interface of the silicone fluid and the continuous phase in an amount effective to maintain the silicone fluid in the outer phase. The first and second surfactants can be the same or different. It is preferred if the second surfactant is a silicone surfactant.

The first and second surfactants in the multiple-emulsion enzyme system are organic surfactants or silicone surfactants. Preferable organic surfactants are nonionic surfactants, especially alkoxylated surfactants, sugar based surfactants, fatty acid esters and the like.

Surfactants that are polymeric are preferred. Examples of preferred organic surfactants are the blocky polyalkyleneoxides of the general formula:

H(OCH₂CH₂)x (OCH₂CH(CH₃))y(OCH₂CH₂(CH₂CH₃))zOH.

Examples of polyalkyleneoxides are Pluronic® surfactants and Reverse Pluronic® surfactants (BASF).

Preferred silicone surfactant comprises a polydiorganosiloxane compound having at least one polyoxyalkylene group, such as generally described in Silicone Surfactants, Surfactant Science Series, volume 86, Hill, R.M., editor, Marcel Dekker, 1999. Preferable silicone surfactants have the formula Me3SiO(Me2SiO)_x(MeQSiO)ySiMe3, wherein Q is represents the polyoxyalkylene group and is exemplified by polyoxyalkylene groups having the formulae -RlO(OCH₂CH₂)_gOR¹1, CH₃ CH₂CH₃

I I

-RlO(OCH₂CH2)g(OCH₂CH)_hORl 1, -RlO(OCH₂CH₂)_g(OCH₂CH)_iOR¹ !,

CH3 CH₂CH₃

-RlO(OCH2CH)h(OCH2CH)iOR^{1 1} ₎ and

CH₃ CH₂CH₃ I I

-RlO(OCH2CH2)g(OCH₂CH)_h(OCH2CH)_iOR^{1 1}, wherein R 0 is a divalent hydrocarbon group having from 1 to 20 carbon atoms, R 1 is selected from a hydrogen atom, an alkyl group, an aryl group, or an acyl group, and g, h, and i independently have an average value from 1 to 150. As used herein, various siloxane units and the oxyethylene, oxypropylene and oxybutylene units may be distributed randomly throughout their respective chains or in respective blocks of such units or in a combination of random or block distributions.

Silicone surfactant can also be a cross-linked polydiorganosiloxane polymer having at least one polyoxyalkylene group; such polydiorganosiloxane-polyoxyalkylene polymer molecules are cross-linked through a cross-linking agent joined thereto by nonhydrolyzable bonds and being free of internal hydrolyzable bonds. This class of compounds has been generally described by Bahr, et al. in U.S. Patent Nos. 4,853,474 and 5,136,068, by Tonge, et al. in EP 0 663 225 and in Lin, et al. in EP 1 167 502; the references are incorporated herein by reference. Cross-linked silicone surfactants are preferred second surfactants for this invention.

The cross-linking in the surfactants useful for this invention can be attained through a variety of mechanisms. Those skilled in the art will readily recognize the systems wherein the required components are mutually compatible to carry out the method of preparing these polydiorganosiloxanes. By way of illustration, an extensive bibliography of siloxane polymer chemistry is provided in Siloxane Polymers, S.J. Clarson and J.A. Semlyen eds., PTR Prentice Hall, Englewood Cliffs, N.J., (1993). Not to be construed as limiting this invention, it is preferred that the cross-linking bonds and the bonds to the polydiorganosiloxane-polyoxyalkylene molecules are not hydrolyzable, and that the cross- linking bridge contains no hydrolyzable bonds. Cross-linked silicone surfactant can be made by (a) crosslinking polydiorganosiloxane polymer and combining a polyoxyalkylene group therewith or by (b) first preparing a linear polyorganosiloxane having a polyoxyalkylene and crosslinking the same. Further, within the former approach, the cross-linked polydiorganosiloxane polymer is obtained by the addition reaction between the following components: (i) a polydiorganosiloxane cross-linking agent having an Si-H group at each of its terminals and a polydiorganosiloxane having an average of between one and two allyl groups in the side chains of each molecules thereof, or (ii) more preferably, an polydiorganosiloxane having at least two Si-H groups in the side chains of each molecule thereof, and a polydiorganosiloxane cross linking agent having each of its terminals blocked with an allyl group or a silanol group.

In the present invention, it is preferred that silicone surfactant is a compound having a viscosity of 100 to 100,000 mm²/s at 25 °C and having the unit formula:

- (R¹² ₂SiO)j - (R¹²SiO)k - (R¹²R¹³SiO)ι -

A

- (R¹² ₂SiO)_m - (Rl²SiO)_n - (R¹²R¹³SiO)_p -

wherein R ² is a monovalent hydrocarbon group, A is a group having the formula

(CH₂)q-(R¹⁴2SiO)_rSi(CH2)_s or the formula O(R¹⁴ ₂SiO)_r-SiO wherein Rl denotes a monovalent hydrocarbon group, q has a value of 2 to 10, r has a value of 1 to 5000, s has a value of 2 to 10, R ³ denotes a group having its formula selected from the group consisting of:

CH₃ CH₂CH₃

I I -(CH₂)_t - (OCH₂CH₂)_u - (OCH₂CH)_v - (OCH₂CH)_w - OR15,

CH₃

-(CH₂)_t - (OCH₂CH₂)_u - (OCH₂CH)_v - OR15, CH₂CH₃

-(CH₂)_t - (OCH₂CH₂)_u - (OCH₂CH)_w - OR* 5,

CH₃ CH₂CH₃

I I

-(CH₂)_t - (OCH₂CH)_v - (OCH₂CH)_w - OR¹⁵,

-(CH₂)_t - (OCH₂CH₂)_u - ORl5,

CH₃

I

-(CH₂)_t - (OCH₂CH)_v - OR¹⁵, and

CH₂CH₃

I

-(CH₂)_t - (OCH₂CH)_w - ORl5,

wherein R 5 is selected from a hydrogen atom, an alkyl group, an aryl group, or an acyl group, t has a value of 2 to 10, u has a value of from greater than zero to 150, v has a value of from greater than zero to 150, and w has a value of from greater than zero to 150, j has a value of 1 to 1000, k has a value of from greater than zero to 30, 1 has a value of 1 to 1000, m has a value of 1 to 1000, n has a value of from greater than zero to 30, p has a value of 1 to 1000. The groups R^² and R^⁴ can be the same or different as desired and are preferably alkyl groups or aryl groups and it is highly preferred that they are both methyl. In the formulae above, it is preferred that j has a value of 1 to 500 and highly preferred that j has a value of 1 to 250. It is preferred that k has a value of from greater than zero to 20 and highly preferred from 1 to 15. It is preferred that 1 has a value from 1 to 100 and highly preferred from 1 to 50. It is preferred that m has a value from 1 to 500 and highly preferred from 1 to 250. It is preferred that n has a value of from greater than zero to 20 and highly preferred from greater than 1 to 15. It is preferred that p has a value of 1 to 100 and highly preferred from 1 to 50. It is preferred that q has a value of 2 to 6. It is preferred that r has a value of 1 to 2500 and highly preferred from 20 to 1000. It is preferred that s has a value of 2 to 6. It is preferred that t has a value of 2 to 4. It is preferred that u has a value of from 1 to 100 and highly preferred from 5 to 50. It is preferred that v has a value from 1 to 100 and highly preferred from 5 to 50. It is preferred that w has a value from 1 to 100 and highly preferred that from 1 to 50. It is preferred that the cross-linked polydiorganosiloxane polymer is triorganosiloxy endblocked at each terminal of the polymer, and it is highly preferred that the polymer is trimethylsiloxy endblocked at each terminal of the cross-linked polymer.

The method used to prepare the crosslinked polydiorganosiloxane polymers is disclosed in European Patent Application No. 0663225. An example of the method for producing crosslinked polydiorganosiloxane polymers is described as the following. Preparation of a crosslinked polydiorganosiloxane polymer is done through the following steps: (I) a charging step in which a linear polysiloxane having hydrogen atoms in its side chains, a polysiloxane having vinyl groups and a catalyst for promoting the reaction, particularly platinum catalysts such as an isopropanol solution of ^PtClgβ^O with a 2% methanol solution of sodium acetate are put in a reactor, (II) an agitation/heating step in which agitation is conducted, for example, at 40°C for 30 minutes, (III) an input step in which a polyoxyalkylene and a solvent (isopropanol) are put in the reactor, (IV) a reflux step in which the isopropanol is refluxed, for example, at 80°C for 1.5 to 2 hours while monitoring the reaction rate of Si-H, (V) a stripping step in which the isopropanol is stripped, for example, at 130°C under a reduced pressure of 25 mmHg, and (VI) a final step in which the reduced pressure condition of step (V) is released and the reaction mixture is cooled to 60°C to obtain a final product.

An example of a linear polysiloxane having hydrogen atoms in its side chains suitable for step (I) is a polysiloxane having its formula selected from:

Me Me Me Me I I I I

Me - Si - O - (SiO)j-(SiO) +ι-Si - Me

Me Me H Me Me Me Me Me

Me - Si - O - (SiO)_m-(SiO)_n+p-Si - Me

Me Me H Me wherein Me hereinafter denotes methyl and j, k, 1, m, n, and p are as defined above. An example of a polysiloxane having vinyl groups suitable for step (I) is a polysiloxane having the formula:

Me Me Me Ni - Si - O - (SiO)_r - Si - Ni

Me Me Me

wherein Me denotes methyl, Ni hereinafter denotes vinyl, and r is as defined above. The reaction of these two compounds in step (II) results in a cross-linked siloxane polymer having the formula

Me Me Me Me Me

Me - Si - O - (SiO)j - (SiO)k - (SiO)ι - Si - Me

Me Me CH₂ H Me

CH₂

I

Me - Si - Me

I o I

(Me₂SiO)_r

I

Me - Si - Me

1 CH₂

I

Me Me CH₂ Me Me

Me - Si - O - (SiO)_m -(SiO)_n - (SiO)_p - Si - Me

Me Me Me H Me

Introduction of a polyoxyalkylene group into the obtained crosslinked organopolysiloxane polymer (steps III- VI) is accomplished by reacting the crosslinked polymer with a polyoxyalkylene compound having its formula selected from the group consisting of : CH₃ CH₂CH₃

I I

CH₂=CH - CH₂ - (OCH₂CH₂)_u - (OCH₂CH)_v - (OCH₂CH)_w - OH

CH₃

I

CH₂=CH - CH₂ - (OCH₂CH₂)_u - (OCH₂CH)_v - OH,

CH₂CH₃

CH₂=CH - CH₂ - (OCH₂CH₂)_u - (OCH₂CH)_w - OH,

CH₃ CH₂CH₃

CH₂=CH - CH₂ - (OCH₂CH)_v - (OCH₂CH)_w - OH,

CH₂=CH - CH₂ - (OCH₂CH₂)_u - OH,

CH₃ I

CH₂=CH - CH₂ - (OCH₂CH)_v - OH, and

CH₂CH₃

CH₂=CH - CH₂ - (OCH₂CH)_w - OH,

wherein u, v, and w are as defined above.

Preferred cross-linked silicone surfactants have the formula Me Me Me Me Me

Me - Si - O - (SiO)j - (SiO)k - (SiO)ι - Si - Me

Me Me CH₂ CH₂ Me

I I

CH₂ CH₂

Me - Si - Me CH₂ CH₃ i i I

O (OCH2CH2)_u - (OCH2CH)_v -OR¹⁵

(Me₂SiO)_r I

Me - Si - Me

CH₂

Me Me CH₂ Me Me

Me - Si - O - (SiO)_m-(SiO)_n - (SiO)_p - Si - Me

Me Me Me CH₂ Me I

CH₂

CH₂ CH₃

(OCH2CH2)_u - (OCH2CH)_v -OR¹⁵

wherein Me denotes methyl, j has a value of 1 to 250, k has a value of from 1 to 15, 1 has a value of 1 to 50, m has a value of 1 to 250, n has a value of from greater than 1 to 15, p has a value of 1 to 50, r has a value of 20 to 1000, u has a value of 5 to 50, v has a value of 5 to 50, and R¹⁵ is hydrogen, methyl, or C(O)CH .

Other silicone surfactants include nonionic silicone surfactants having a trimethylsilyl endcapped polysilicate that has been condensed with a polyalkylene glycol or diester in a solvent, or a block copolymer of polydimethylsiloxane and polyalkylene oxide. These surfactants are well known in the art and are exemplified by the "dispersing agents" disclosed by Keil in U.S. Pat. Nos. 3,784,479 and 3,984,347, the disclosures of which are hereby incorporated by reference to teach said surfactants, hi some instances the surfactants may preferably be processed from a solvent such as a polyalkylene glycol or copolymers thereof, cyclic silicones, or an organic solvent such as xylene.

The multiple-emulsion enzyme system can be prepared by first forming a silicone premix: a silicone fluid and a first surfactant are mixed together until homogeneity to form a silicone premix. Aqueous slurry containing a solid enzyme or an aqueous solution of the enzyme (inner phase) is then mechanically agitated with the silicone premix (outer phase) to form an aqueous enzyme phase-in-silicone emulsion. A continuous phase is mixed with a second surfactant to form a substantially homogeneous mixture. The aqueous enzyme phase-in-silicone emulsion and the continuous phase mixture are mechanically agitated to provide for emulsification of the silicone into the continuous phase to produce silicone droplets of less than 100 μm, and preferably less than 20 μm. The enzyme is contained substantially within the silicone phase and mostly contained within the internal aqueous droplets. Care is taken during the preparation of emulsion to minimize shearing on the emulsion such that 50%), preferably 70%, and more preferably 80% or greater of the enzyme mass is retained within the multiple emulsion droplets.

The amount of enzyme crystals trapped inside the silicone can be qualitatively determined by a microscopic examination of the sample. The amount of enzyme crystals outside the silicone drops is determined based on their tendency to sink to the bottom of the continuous phase or dissolve within the continuous phase. A small drop (for example, about 0.02 g) of enzyme in silicone multiple-emulsion is diluted with about 5 drops of the continuous phase on a microscope slide. After about 5 minutes, the bottom of the sample is examined to assess the amount of enzyme crystals visible (not dissolved).

A quantitative measure of enzyme captured can be determined based on a protein assay or an enzyme activity assay. This approach measures the amount of enzyme that is outside of the multiple-emulsion drops. The initial enzyme activity is determined by knowing the total enzyme activity added or by breaking the emulsion to measure the total enzyme activity present. Then the percent enzyme retained in the multiple-emulsion is calculated relative to the initial enzyme activity.

Suspension-Emulsion

In another embodiment of the invention, the enzyme system is a suspension- emulsion comprising: (a) a silicone fluid containing a solid enzyme dispersion without an aqueous solution intervening between the enzyme and the silicone fluid, (b) a dispersing agent that disperses the enzyme in the silicone fluid, wherein said dispersing agent adsorbs at the interface of the solid enzyme and the silicone fluid and is compatible with silicone, (c) a continuous phase surrounding the silicone fluid, wherein said continuous phase comprises water, or nonaqueous polar liquid, or a non-aqueous nonionic surfactant, or a mixture thereof, and (d) a silicone surfactant in an amount effective to remain adsorbed on the interface of the silicone fluid and the continuous phase. Figure 2 depicts a suspension- emulsion enzyme protection system. The specific scale or number of dispersed enzyme particles in Figure 2 is not meant to be representative or limiting, but rather to qualitatively illustrate the phase structure of the suspension-emulsion.

In the suspension-emulsion system, the silicone fluid, the continuous phase and the silicone surfactant are the same as those described in the multiple-emulsion system.

In the suspension emulsion system, dry enzyme solids without the presence of intervening solution can be directly dispersed into the silicone fluid. Solid form of crystalline or amorphous enzymes is treated to remove excess or unbound aqueous solution. This can be accomplished by any of the methods known to the art to separate finely divided solids from a supernatant solution such as freeze-drying, filtration, centrifugation, evaporative drying, wicking removal of solution, or combinations of these techniques.

A dispersing agent that interacts with the surface of the enzyme solids and aids in compatibilization of the solids with the silicone protectant is used to disperse the enzyme in the silicone fluid. Many dispersants are polymeric in nature. The useful dispersing agents, tend to have chemical groups that can interact strongly with the enzyme solids while having a portion of the molecule that can interact favorably with the silicone.

A variety of agents are useful to aid in dispersing the enzyme solids into the silicone fluid. These dispersing agents are either silicone types or non-silicone organic types. Silicone types are polydiorganosiloxanes that contain functional groups that interact with the surface of the enzyme solids. One mode of interaction is through chemisorption. Chemisorption based on interaction of charged groups on the silicone with oppositely charged groups on the enzyme surface. Anionic charges on the enzyme crystal surface can arise from carboxylic acid moieties at a pH above their pKa. Carboxylic acid moieties are present from aspartic acid and glutamic acid amino acid residues, and from the carboxy terminus of the protein. Cationic charges will be present from lysine, arginine, and histadine amino acid residues, and from the N-terminus of the protein.

Silicone based dispersants useful for the purpose of this invention are polyorganosiloxanes that are cationically charged through the presence of functional groups containing primary, secondary, or tertiary amine groups, ethylenediamine groups, including substituted ethylenediamine groups. Anionically charged polyorganosiloxanes useful for the present invention contain functional groups such as carboxylic acid, pyrrolidone carboxylic acids, silianoate groups, and phosphate and phosphonate groups. Silicone dispersants containing nonionic functional groups are also useful for the present invention. Polyorganosiloxanes containing polyether functional groups are useful. The polyether groups contain ethylene oxide, propylene oxide, or combinations of ethylene oxide and propylene oxide polyglycols. Polyorganosiloxanes containing polyhydroxy functional groups, including sugar molecules, are also useful dispersants.

Organic dispersants useful for the present invention are those well known to the art; they are organic polymers selected to contain anionically charged, cationically charged groups and non-ionic organic molecules. Interactions with the crystal surface can be achieved in a similar manner as described for silicone based surfactants. Further, in order to be useful for the present invention, the dispersants must be sufficiently compatible with silicone in order to disperse the enzyme solids into the silicone. The organic dispersants useful for the invention are polymeric dispersants, block copolymers composed of hydrophobes and hydrophiles. The hydrophile strongly adsorbs on the enzyme crystal surface. The hydrophobe has good affinity for the dispersing medium (Silicone). The hydrophobes include poly(12-hydroxy stearic acid): C₆H₁₃CHOHCιoH₂oCOOH, and long chain alkylene: CH _n. The hydrophiles include nonionic such as PEG, cationic such as groups containing a quaternary amine, and anionic such as groups containing sulfonate, carboxy and phosphate. Examples of organic dispersants are Solsperse 16,000, Solsperse 21,000, Atlox LP1, Hypermer Kd-10, Zethrym SDE 1121. Using these dispersants results in high entrapment efficiency of the enzyme crystals because the dispersants minimize the tendency of enzyme crystals to pop-out from he silicone droplets into the continuous phase during emulsification and on storage stability. Good affinity for crystal surface might also be obtained when the dispersant is any of the following co-polymers: Styrene/octadecyl methyacrylate/methacrylic acid copolymer, octadecyl methyacrylate/methacrylic acid, octadecyl methacrylate/ methyl methacrylate/acrylic acid, acrylonitrile/lauryl acrylate/acrylic acid, lauryl methacrylate/styrene/acrylic acid, styrene/docosaryl acrylate/methacrylic acid, and octadecyl methacrylate/vinyl acetate/methyl methacylate/methacrylic acid.

One way to make the dispersant sufficiently compatible with silicone, thus forming dispersion of solid enzyme in silicone fluid, is to include silicone groups in the molecule as pendant groups, at the polymer termini, or as part of a blocky type of a polymer where combinations of organic groups and organosiloxane constitute contiguous portions of the polymer chain. Examples of these silicone modified polymers are given in Japanese patent 63291971 (Nitto Electric hid. KK) or Japanese patent 01146983 (Nitto Denko Corp) or World Patent 9207014 (Du Pont de Nemours & Co. E I). The dispersion method and chemistry depend on the nature of the solids to be dispersed. High shear processing can be used alone or in concert with chemical dispersants to help promote the distribution of enzyme solids into silicone.

The dried solid enzyme is added to a silicone fluid with mixing agitation to provide for rapid and uniform dispersion in the silicone fluid. This process can be aided by the correct selection of the silicone fluid, or fluid with dispersants that interact with the surface of the solid enzymes to promote their compatibility and wetting by silicone fluid. Examples are silicones that are amine functional, diethylamine functional, carboxy functional, pyrrolidone carboxy functional, silanol functional, or containing combinations of the above, such that these functionalized silicones remain miscible with the bulk silicone fluid selected. Particle size adjustment occurs in the silicone through milling or other mechanical means to break crystalline or amorphous solids into particles less than 50 μm, and preferably less than 10 μm, on average. Preferably, milling can be carried out directly upon suspended or hydrated enzyme solids, without the need to first remove excess or unbound water. The suspension or paste of milled particles can then be further dried to remove excess water. It may be desirable in some cases not to remove all the free water, in order to maintain the structure of the particles. For example, removal of too much water from enzyme crystals may lead to the loss of their ordered structure. Alternatively, particle size reduction can occur by first providing a dry form of the enzyme, as a cake, precipitate, or powder, and then dry milling or grinding of dried enzyme solids using a dry grinding mill or other means of pulverizing the enzyme solids, or by a technique, such as spray-drying, that directly forms dry particles within a defined size range. The reduced size solid enzyme particles are then dispersed into the silicone fluid.

The hydrophobic silicone protectant in the multiple-emulsion and suspension- emulsion system is thought to protect in two ways. First, the silicone protectant acts as a barrier to block or limit the penetration of substances that are damaging to enzymes. In the case of solid form of enzymes, especially crystalline enzymes, the enzymes are more stable against surfactant-rich detergent systems. If maintained in a solid form, especially a crystalline form, the enzyme is greatly stabilized and protected. Shetty (WO 96/20274) has demonstrated that enzyme in a crystalline form can dramatically enhance its stability in a surfactant rich detergent formula. The second way in which the silicone protectant acts is to maintain the local chemical environment around the enzyme in such a state that the solid enzyme forms, particularly crystals, will not dissolve or degrade. Thus, the protection is based on maintaining the local salt, water, and buffer conditions around the crystal so as to preserve its crystallinity.

Density of the silicone particle relative to the continuous phase of the emulsion and to the environment (such as a liquid detergent) into which it is dispersed impacts the rate of sedimentation or creaming of the particle. A number of methods are known to the art to minimize the rate of sedimentation and creaming. One of the useful methods that limit the rate of sedimentation or creaming of the particle is to match its density to the density of the liquid surrounding it. This is accomplished through the inclusion of optional additives to any of the liquid phases in the particle to manipulate the overall particle density. In many cases, the density of the particle needs to be increased to minimize creaming. One well known method is to modify the density of the silicone phase through the addition of non- reinforcing filler particles as described in EP 0 638 346, incorporated herein by reference. Density matching can also occur through balancing the relative make-up of the particle such as varying the ratio of silicone phase to the internal disperse enzyme solids or the enzyme containing aqueous phase. The difference in density between the droplet and the liquid detergent is preferably less than about 0.25 g/mL and more preferably less than about 0.1 g/mL.

In one aspect of the invention, a multiple-emulsion system is used to protect the enzyme. In this aspect, a first emulsion, or inner aqueous phase, contains the active ingredient and forms a droplet when added to a second emulsion. The liquid interface between the first and second emulsions forms a protective barrier for the active ingredient. When the multiple emulsion is added to a surfactant-rich media, the second emulsion serves as a protective liquid/liquid interface with other harmful ingredients in the surfactant-rich media. The multiple-emulsion aspect provides two, or more, protective barriers between the active ingredient and the intended environment for use. In another aspect of the invention, a suspension-emulsion system is used to protect the enzyme. In this aspect, the emulsion generally includes one or more dispersants to aid distribution of a solid form of the active ingredient throughout the emulsion, which forms a protective liquid interface with the solid surface of the active ingredient. When the suspension-emulsion is added to a surfactant-rich media, the single emulsion serves as a protective liquid/liquid interface with other harmful ingredients in the surfactant-rich media.

A further advantage of both the above-defined silicone based emulsion systems is that the enzyme is in a form that has a reduced tendency to form sensitizing dusts, because the enzyme is not susceptible to drying out into a friable solid residue. When liquid enzyme solutions are stored, shipped, blended or otherwise handled in the production of detergents and cleaning products, there is a significant risk of spills or splashes which could dry into solid residue deposits and become airborne as sensitizing dusts. Silicones remain liquids over the full range of normal handling temperatures and the present emulsions are efficient in entrapping enzymes, reducing the likelihood and extent of enzyme dust formation.

In formulations, the present invention provides advantages in that some of the other active ingredients contained in the continuous phase are protected from the entrapped enzyme during storage. For example, other active enzymes in the formulation are protected from digestion by the entrapped protease activity during storage. During use, the entrapped enzyme is easily released through mechanical or chemical disruption of the hydrophobic protection barrier.

Stabilization of the entrapped enzymes can be impacted by the rate at which chemical species migrate through the silicone fluid phase. Migrating species might include water and components of the detergent such as surfactants, nonaqueous solvents, and bleaching agents. Control of permeability may be important in retaining enzyme stability against inactivation by certain of these species. The relative importance of permeability to retention of enzyme activity depends on the form of the enzyme. Crystalline forms are less susceptible to inactivation by detergent ingredients than other forms such as true solutions, precipitates, amorphous solids, etc. The present invention is also directed to a liquid detergent formulation comprising the multiple-emulsion enzyme system or the suspension-emulsion enzyme system as described above. The detergent formulation maintains the multiple-emulsion form or the suspension-emulsion form for at least one day, two days, three days, or five days, and preferably one, two, three, or four weeks at temperature between 22-28°C. In one embidoment, the detergent formulation has a physical stability and is stable against sedimentation, coalescence, creaming, flocculation, and/or aggregation. Sedimentation, coalescence, creaming, flocculation, and/or aggregation in an enzyme-containing detergent formulation produce a visible oil layer on the top of a detergent, typically causing the release of internalized enzymes or causing the non-uniform delivery of enzymes during pouring and dosing of the detergent. Therefore, it is important to maintain the emulsion form and the uniformity of the dispersion in a detergent formulation.

Assessment of the physical state of dispersion of the multiple-emulsion or suspension-emulsion in a detergent formulation can be made using particle size measurements, by eye or by microscopic examination. For example, assessment by eye can be performed under uniform lighting on unmixed samples equilibrated to room temperature. Microscopic examination can be performed using a microscope fitted with a computer interfaced black and white video camera for image capture. The volume average particle size can be measured for each sample using a Coulter LSI 30 laser light scattering instrument.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES The following materials are listed for ease of reference for materials used in the Examples.

POLYORGANOSILOXANE A is a trimethylsilyl end-blocked polydimethylsiloxane having a viscosity of 1,000 mm²/s (cS) at 25 °C. Fluids of this type are sold under the trade name Dow Corning® 200 Fluid (Dow Corning Corporation, Midland, MI).

POLYORGANOSILOXANE B is a hydroxyl-terminated polydimethylsiloxane having a viscosity of 13,500 mm²/s (cS) at 25°C.

SILICONE SURFACTANT 1 is a block copolymer of polydimethylsiloxane and polyalkylene oxide having the average structure, shown below, were used alone or as present in a solvent: Me₃SiO(MeQSiO)_j(Me₂SiO)_kSiMe₃,

where Q represents: -(CH₂)₃(OCH₂CH₂)_m(OCH₂CH)_nOZ

Me

wherein Me denotes methyl radical and the values of k:j ratio is about 100:1, and m:n is about 1 with m+n less than 50, and Z equal to H. The total molecular weigh of this surfactant is about 40,000.

SILICONE SURFACTANT 2 is a nonionic silicone surfactant of trimethylsilyl end-capped polysilicate prepared according to methods described in US-A 3,784,479. A mixture of 7 parts of RESIN I (supra), 15 parts of a propylene oxide condensate with glycerol having a number average molecular weight of 4,000 and 38 parts of xylene was reacted at reflux for 8 hours with 0.2 part of a stannous octoate, 0.1 parts of phosphoric acid was added and the product was blended with 40 parts of a polyethylene glycol-polypropylene glycol copolymer. The product was stripped at 5.3 kPa (40 mm Hg) at 140°C. to remove xylene and filtered.

RESIN I =A70% xylene solution of a hydroxy- unctional siloxane resin copolymer comprising (CH3)3SiOl/2 and Si02 units having a (CH3)3SiOl/2 /Si02 ratio of 0.75:1.

SILICONE SURFACTANT 3 is a cross-linked organopolysiloxane polymer having at least one polyoxyalkelyene group prepared according to the method described in Tong et al. in EP 0 663 225 as follows. 12.8 parts of Al and 2.6 parts of Bl were placed into a reactor, mixed, and treated to 80 °C. Then 0.001 part of E was added, and the mixture was allowed to react for 60 minutes. 60.2 parts of CI and 24.4 parts of D were then added. The mixture was then heated to 90 °C. An additional 0.001 part of E was added. The mixture was allowed to further treat at 90 °C for 2 hours, followed by vacuum stripping to remove isopropanol. The final mixture was cooled and filtered. Component Al was a linear polysiloxane having the formula: Me Me Me Me

I I I I

Me - Si - O - (SiO)e - (SiO)_f+g - Si - Me

Me Me H Me

wherein Me is a methyl group, e has a value of 108 and f+g has a value of 10.

Component Bl was a polysiloxane having the formula:

Me Me Me Vi - Si - O - (SiO)_r- Si - Vi

I I I

Me Me Me

wherein Me is a methyl group, Ni is a vinyl group and r is such that Bl has a molecular weight of 11,000.

Component CI was a polyoxyalkylenen having the formula:

Ni-CH₂-O-(EO)_u - (PO)_v-H wherein Ni is a vinyl group, EO is an ethylene oxide residue, PO is a propylene oxide residue, and u and v are such that CI has a molecular weight of 3,100 with the ratio of u:v being 1:1.

Component D was isopropanol (as a solvent).

Component E was a 2 weight percent isopropanol solution of H₂PtCl₆ ^• 6H₂0.

ORGANIC SURFACTANT 1 is an alcohol ethoxylate organic nonoinic surfactant with an average linear carbon chain length of 12 to 13 carbon atoms and an average of 6.5 ethyleneoxides polymerized onto available as Tomadol ™ 23-6.5 from Tomah Products (Milton, Wisconsin).

ORGANIC SURFACTANT 2 is an aqueous solution of alkylpolyglucaside organic nonionic surfactant available as Glucopon ™ 625 (Henkel Corporation). ORGANIC SURFACTANT 3 is an aqueous solution containing roughly 45 wt.% actives of linear alkylbenenzensulfonate (LAS) available as Witconate 45 from Witco Chemical Corporation (New Jersey).

ORGANIC SURFACTANT 4 is a 50% active aqueous solution of alkylpolyglucaside organic nonionic surfactant available as Glucopon ™ 600 (Henkel Corporation).

AMINO FUNCTIONAL SILICONE DISPERSANT is an essentially 100% active functionalized silicone with a linear polysiloxane structure :

Me Me Me Me

Me - Si - O - (SiO)h - (SiO - Si - Me Me Me R Me

wherein Me is a methyl group, R has the structure -(CH₂)₃NHCH₂CH₂NH₂, h has a value of approximately 43 and i has a value of 1.

Example 1. Measurement of Particle Size of Crystalline Enzyme.

Particle sizes of a standard polystyrene latex, the unmodified subtilisin protease enzyme crystals, and the ball-milled enzyme crystals were measured. A saturated enzyme solution equilibrated at room temperature was prepared and filtered (Whatman filter paper #1450 090) to remove undissolved crystals. To 2 mL of the filtered clear tan solution, 1 small drop (about 0.02 g) of the enzyme crystal slurry was added. The samples were placed in a 3 mL acrylic cuvette, stirred about one minute, then inserted into the cuvette holder on the Brinkman model 2010 laser particle size analyzer. The particle size was typically measured in less than 2 minutes. The particle size standard material used was Polybead® Polystyrene 10.0 micron microspheres (Polysciences, Inc., Warrington, PA, Catalog #17136) with a stated size of 10.568 μm and a standard deviation of 1.034 μm. The size measured for the standard is consistent with its stated particle size.

The particle size measured for the untreated enzyme crystals is consistently larger than for the milled enzyme crystals. The untreated enzyme crystals have an area based median diameter of 8.13 μm. The milled enzyme has an area based median particle size of 3.53 μm.

Example 2. Multiple-emulsion Enzyme Systems (crystalline enzyme)

In this example, the enzyme is contained within an aqueous environment surrounded by silicone fluid, which is emulsified in a continuous phase.

Formation of water in silicone emulsion

Silicone Premix

Polyorganosiloxane A 24.75 g

Silicone Surfactant 1 2.50 g

Polyorganosiloxane B 2.75 g

Enzyme slurry in Silicone Emulsion

Reduced Size Crystalline Enzyme (1907-19-2) 20.0 g

Silicone Premix 30.0 g

A silicone premix was prepared by stirring together until homogeneity a polydimethylsiloxane fluid of between 5 and 100,000 cSt, and a silanol end capped polydimethylsiloxane fluid and a cross linked silicone polyether surfactant. To this silicone premix was added a slurry containing a crystalline containing 27.5 wt.% crystalline enzyme of crystal size of less than about 10 μm dispersed in 3.5 wt.% sodium chloride solution. Mixing provided an emulsion of the aqueous enzyme slurry in silicone, where the aqueous phase particles were less than 10 μm in size. Care was taken to minimize the shearing on the emulsion to retain greater than 70% of the enzyme mass within the aqueous phase droplets.

Multiple-emulsion

Continuous phase

Organic Surfactant 1 16.5 g

Organic Surfactant 2 67.0 g

Deionized Water 16.5 g Multiple-emulsion

Continuous phase 27.5 g Silicone Surfactant 3 2.5 g

Enzyme slurry in Silicone Emulsion 20.0 g

The continuous phase was combined with stirring with the silicone polyether to make a substantially homogeneous mixture and then the enzyme slurry-in-silicone emulsion was combined with that mixture with stirring agitation to provide for emulsification of the silicone into the continuous phase to produce silicone droplets of less than 20 μm, wherein the crystalline enzymes were contained substantially within the silicone phase and greater that 80% were contained within the internal aqueous droplets.

Detergent Containing Protected Enzymes

The multiple-emulsion protected enzyme was diluted into a liquid laundry detergent by simple mixing. The detergent formula contains a high level of linear alkyl benzene sulfonate.

Wt.(°/o

Deionized water 21.0

Sodium Citrate 8.3

Propylene glycol 7.0

Coconut fatty acid 1.0

Organic Surfactant 3 43.5

Organic Surfactant 4 15.7

Organic Surfactant 1 2.6

Multiple-emulsion protected enzyme 1.0

Example 3

Example 2 is repeated with the enzyme in the form of an amorphous precipitate slurry in aqueous salt solution.

Example 4

Example 2 is repeated with the enzyme in the form of a solution containing between 0.1 and 40 wt % active enzyme in solution within an aqueous salt solution. Example 5

Example 2 is repeated by replacing the linear Polyorganosiloxane A with a viscoelastic polydimethylsiloxane with a maximum viscosity 1,000,000 mPa-s at a shear rate of 0.01 s^"1. Niscoelasticity of this type arises due to branching or crosslinks of the molecules.

Example 6 Example 2 is repeated by replacing the Polyorganosiloxane A with a polyorganosiloxane fluid, wherein that polyorganosiloxane fluid is a polydialkylsiloxane, a polydiarylsiloxane, a polyalkylmethylsiloxane, or a lyarylmethylsiloxane, or copolymers of these with dimethylsiloxane. The polyorganosiloxane fluid may contain functional organic groups such as polyglycols, carboxylic acid groups, amine groups, diamine groups, amide groups, unsaturated organic groups such as vinyl groups, polyhydroxy alkylgroups. The polyorganosiloxane in this Example contains diorganofunctionalsiloxane monomers, or organofunctionahnethylsiloxane monomers polymerized into homopolymers or into copolymers with dimethylsiloxane.

Example 7

Example 2 is repeated by replacing the continuous phase of the multiple-emulsion with a nonaqueous moderately polar liquid such as polyglycol (polyethyleneglycol, polypropyleneglycol), or copolymers of polyethyleneglycol with polypropyleneglycol, including cross linked versions thereof.

Example 8

Example 2 is repeated by replacing continuous phases of the multiple-emulsion with nonionic surfactants such as alcohol ethoxylates, or fatty acid esters that are liquid during the emulsification step of the processing.

Example 9

Example 2 is repeated by replacing the Silicone Surfactant 1 in the Silicone Premix with Silicone Surfactant 2. Example 10. Physical Stability Protocol of encapsulated protease in liquid detergents.

Multiple emulsion enzyme systems were stored in detergent to assess stabilization against sedimentation and coalescence in aqueous or semi-aqueous detergent containing formulas. Sedimentation and coalescence produce a visible oil layer on the top of a detergent, typically causing release of internalized enzymes or causing non-uniform delivery of enzymes during pouring and dosing of the detergent.

The following materials were used in this Example to prepare Formulae I-IV:

ORGANIC SURFACANT 5: a polyglycol block copolymer surfactant of average molecular weight of 3650g/mole with a blocky EO/PO/EO structure containing 20 weight percent ethylene oxide. Surfactants of this type are sold under the trade name Pluronic® L92 (BASF Corporation Mount Olive, New Jersey).

ORGANIC SURFACANT 6: a polyglycol block copolymer surfactant of average molecular weight of 2900g/mole with a blocky EO/PO/EO structure containing 40 weight percent ethylene oxide. Surfactants of this type are sold under the trade name Pluronic® L64 (BASF Corporation Mount Olive, New Jersey).

POLYORGANOSILOXANE C: a hydroxyl-terminated polydimethylsiloxane having a viscosity of 42 mm²/s (cS) at 25°C.

POLYORGANOSILOXANE D: viscoelastic polydimethylsiloxane fluids formed through cross-linking or branching condensations of a silanol terminal polydiorganosiloxane with silanol groups on hydrolyzed alkoxypolysilicates (see U S. Patent number 4,749,740). This fluid has a viscosity of about 20,000 mPa s at a shear rate of 10 s^"1 at 25°C.

POLYORGANOSILOXANE E: a polydiorganosiloxane blended with silicone resin. The polyorganosiloxane is an ethyhnethyl, methyl 2-phenylsiloxane copolymer and the silicone resin with the general formula of R₃SiOι_/2 units to SiO_4/2 units is about 0.22:1, with Rbeing methyl. The mixture contains approximately 77wt.% of the polydiorganosiloxane and has a viscosity of 2000 mm²/s. POLYORGANOSILOXANE F: a polydiorganosiloxane blended with silicone resin. The polyorganosiloxane is an ethyhnethyl, methyl 2-phenylsiloxane copolymer and the silicone resin with the general formula of R₃SiOι_/2 units to SiO /₂ units is about 0.22: 1, with R being methyl. The mixture contains approximately 74wt.% of the polydiorganosiloxane and has a viscosity of 5000 mm Is.

The following formulae I-IV were made in a manner consistent with the approach defined in Example 2. The formulations of formulae I-IV are summarized in Table 1.

Table 1. Formulation Summary

Silicone Premix Grams of I nεredient

Ingredient Formula I Formula II Formula III Formula IV

1 POLYORGANOSILOXANE A 19.7

2 POLYORGANOSILOXANE B 2.2

3 POLYORGANOSILOXANE C 2.2 2.2 2.2

4 POLYORGANOSILOXANE D 19.8

5 POLYORGANOSILOXANE E 19.8

6 POLYORGANOSILOXANE F 19.8

7 SILICONE SURFACTANT 1 2.2

8 SILICONE SURFACTANT 2 2.0

9 ORGANIC SURFACTANT 5 2.0 2.0

Enzyme slurry in silicone emulsion

Reduced size crystalline enzyme (1907-19-2) 15.9 16.0 16.0 16.0

Multiple Emulsion

Continuous phase

SILICONE SURFACTANT 3 55.0 55.0 55.0 55.0 SILICONE SURFACTANT 6 5.0

100.0 100.0 100.0 100.0

A silicone premix was prepared by stirring together until homogeneity the silicone and organic surfactant phases. To this silicone premix was added slurry containing 27.5 wt.% crystalline enzyme of crystal size of less than about 10 μm dispersed in 3.5 wt.% sodium chloride solution. Mixing provided an emulsion of the aqueous enzyme slurry in silicone, where the aqueous phase particles were less than 10 μm in size. Care was taken to minimize the shearing on the emulsion to retain greater than 70% of the enzyme mass within the aqueous phase droplets. The continuous phase was combined with stirring with the silicone polyether to make a substantially homogeneous mixture. The multiple emulsion was subsequently prepared by adding the enzyme slurry-in-silicone emulsion. Stirring agitation was applied during and after the addition for emulsification of the silicone phase into the continuous phase to produce multiple droplets of average of less than 20 μm, wherein the crystalline enzymes were contained substantially within the silicone phase.

Physical Stability Testing of Multiple-Emulsion Enzyme System in Detergent The multiple-emulsion enzyme system was diluted into a liquid laundry detergent as defined in Example 2 by mechanical stirring agitation. The uniformly mixed samples were then split into 800-gram samples for storage stability testing at room temperature (22°C) and in a 37°C oven. The results are summarized in Table 2.

Table 2 Summary of Physical Stability Testing

* collar describes the visible collection of material in the meniscus around the top perimeter of the sample Assessment of the physical state of dispersion of the multiple-emulsion was made using particle size measurements, assessment by eye using a single trained observer, and through microscopic examination. Assessments were made after 1 day and at 1, 2, and 4 weeks. Assessment by eye was performed under uniform lighting on unmixed samples equilibrated to room temperature. Samples were stored in transparent polycarbonate jars with wide mouths to afford easy assessment of distribution of the multiple emulsion droplets in the bulk and on the surface of the samples. Microscopic examination of portions of the unmixed samples removed from the top, middle, and bottom was performed using a Zeiss Axioskope microscope fitted with a computer interfaced black and white video camera for image capture.

Microscopic examination demonstrated retention of multiple emulsion droplet form in all samples after one day, in all samples except Formula I after 1 week, and up to four weeks for Examples II, III and IN. Differences in physical stability of these systems in liquid detergent were observed. The volume average particle size was measured for each storage sample after remixing on a portion of the sample pulled from the middle and diluted into water using a Coulter LSI 30 laser light scattering instrument.

The size variations were not believed to be statistically significant.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A multiple-emulsion enzyme system comprising:

(a) an inner aqueous phase containing an enzyme;

(b) an outer phase of silicone fluid, (c) a continuous phase surrounding the outer phase, wherein said continuous phase comprises water, or a non-aqueous polar liquid, or a non-aqueous nonionic surfactant, or a mixture thereof, (d) a first surfactant adsorbed on the interface of the inner phase and the outer phase in an amount effective to maintain the enzyme in the inner phase, and (e) a second surfactant adsorbed on the interface of the silicone fluid and the continuous phase in an amount effective to maintain the silicone fluid in the outer phase.

2. The multiple-emulsion enzyme system according to Claim 1, wherein said first and second surfactant is independently a silicone surfactant or an organic surfactant.

3. The multiple-emulsion enzyme system according to Claim 2, wherein said second surfactant is a cross-linked silicone surfactant.

4. The multiple-emulsion enzyme system according to Claim 2, wherein said silicone surfactant comprises at least one polydiorganosiloxane compound having at least one polyoxyalkylene group.

5. The multiple-emulsion enzyme system according to Claim 4, wherein said silicone surfactant has the formula Me₃ SiO(Me₂SiO)_x(MeQSiO)ySiMe₃ , wherein Q is selected from the group consisting of

CH₃

I -(CH2)_z(OCH₂CH₂)gOH, -(CH₂)_z(OCH2CH2)g(OCH₂CH)_hOH,

CH₃

I

-(CH₂)_z(OCH₂CH₂)_gOCH₃, -(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOCH₃, CH₃

I

-(CH₂)_z(OCH₂CH₂)gOC(O)CH₃, and -(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOC(O)CH₃ , wherein Me denotes methyl, x has an average value from 100 to 500, y has an average value from 1 to 50, z has an average value of 2 to 10, g has an average value of 1 to 36, and h has an average value of 1 to 36.

6. The multiple-emulsion enzyme system according to Claim 5, wherein Q is

CH₃ I

-(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOH, and Y is between 1 and 10.

7. The multiple-emulsion enzyme system according to Claim 4, wherein said polyoxyalkylene group has the fonnulae -R 0(OCH₂CH₂)gORl * ,

CH₃ CH₂CH₃

I I

-Rl °(OCH₂CH2)g(OCH₂CH)_hORl 1 , -Rl 0(OCH2CH₂)_g(OCH₂CH)iOR¹1 ,

CH₃ CH₂CH₃

I I

-R¹⁰(OCH₂CH)_h(OCH2CH)_iOR¹¹, and

CH₃ CH₂CH₃

I I

-R¹0(OCH₂CH2)_g(OCH2CH)_h(OCH₂CH)iOR¹1 , wherein R¹⁰ is a divalent hydrocarbon group having from 1 to 20 carbon atoms, R^u is a hydrogen atom, an alkyl group, an aryl group, or an acyl group, and g, h, and i independently have an average value from 1 to 150.

8. The multiple-emulsion enzyme system according to Claim 2, wherein said organic surfactant is a polyalkyleneoxide.

9. The multiple-emulsion enzyme system according to Claim 8, wherein said polyalkyleneoxide is H(OCH₂CH₂)x (OCH₂CH(CH₃))y (OCH₂CH₂(CH₂CH₃))zOH.

10. The multiple-emulsion enzyme system according to Claim 1, wherein said silicone fluid is polyorganosiloxane or polydiorganosilixane.

11. The multiple-emulsion enzyme system according to Claim 10, wherein said silicone fluid is linear, branched, or crosslinked.

12. The multiple-emulsion enzyme system according to Claim 10, wherein said silicone fluid is viscoelastic.

13. The multiple-emulsion enzyme system according to Claim 10, wherein said polydiorganosiloxane is polydialkylsiloxane, polydiaryllsiloxane, polyalkylmethylsiloxane, polyarylmethylsiloxane, or copolymers thereof with dimethylsiloxane.

14. The multiple-emulsion enzyme system according to Claim 10, wherein said polydiorganosiloxane contains functional groups.

15. The multiple-emulsion enzyme system according to Claim 1, wherein said enzyme is in the form of a crystalline enzyme.

16. The multiple-emulsion enzyme system according to Claim 15, wherein said crystalline enzyme has a crystal size of less than about 10 μm.

17. The multiple-emulsion enzyme system according to Claim 16, wherein said crystalline enzyme has a crystal size of less than about 5 μm.

18. The multiple-emulsion enzyme system according to Claim 1, wherein said enzyme is in the form of an amorphous precipitated slurry in an aqueous salt solution.

19. The multiple-emulsion enzyme system according to Claim 18, wherein said aqueous salt solution contains between 0.1-40 % (w/w) enzyme.

20. The multiple-emulsion enzyme system according to Claim 1, wherein said enzyme is adsorbed onto silica.

21. The multiple-emulsion enzyme system according to Claim 20, wherein said silica is hydrophilic.

22. The multiple-emulsion enzyme system according to Claim 1, wherein said enzyme is a detergent enzyme used in laundry detergents, fabrics care products, or dishwasher detergents.

23. The multiple-emulsion enzyme system according to Claim 22, wherein said detergent enzyme is selected from the group consisting of a protease, an amylase, a lipase, a cellulase, a mannanase, a ligninase, a pectinase, a peroxidase, and a catalase.

24. The multiple-emulsion enzyme system according to Claim 23, wherein said protease is a subtilisin.

25. A suspension-emulsion enzyme system comprising:

(a) a silicone fluid containing a solid enzyme dispersion without an aqueous solution intervening between the enzyme and the silicone fluid,

(b) a dispersing agent that disperses the enzyme in the silicone fluid, wherein said dispersing agent adsorbs at the interface of the solid enzyme and the silicone fluid and is compatible with silicone,

(c) a continuous phase surrounding the silicone fluid, wherein said continuous phase comprises water, or nonaqueous polar liquid, or a non-aqueous nonionic surfactant, or a mixture thereof, and (d) a silicone surfactant in an amount effective to remain adsorbed on the interface of the silicone fluid and the continuous phase.

26. The suspension-emulsion enzyme system according to Claim 25, wherein said dispersing agent is a silicone based dispersant or an organic dispersant.

27. The suspension-emulsion enzyme system according to Claim 26, wherein said silicone based dispersant is a polyorganosiloxane that contain functional groups capable of interacting with the surface of the solid enzyme.

28. The suspension-emulsion enzyme system according to Claim 27, wherein said functional group is cationically charged, anionically charged, or non-ionic.

29. The suspension-emulsion enzyme system according to Claim 26, wherein said organic dispersant is a polymer or a block copolymer of hydrophobes and hydrophiles.

30. The suspension-emulsion enzyme system according to Claim 29, wherein said hydrophobes are poly(12-hydroxy stearic acid) and long chain alkylene.

31. The suspension-emulsion enzyme system according to Claim 29, wherein said hydrophiles are polyethyleneglycols, quaternary amines, sulfonates, carboxylates or phosphates.

32. The suspension-emulsion enzyme system according to Claim 26, wherein said organic dispersant is a copolymer selected from the group consisting of styrene/octadecyl methyacrylate/methacrylic acid, octadecyl methyacrylate/methacrylic acid, octadecyl methacrylate/methyl methacrylate/acrylic acid, acrylonitrile/lauryl acrylate/acrylic acid, lauryl methacrylate/styrene/acrylic acid, styrene/docosaryl acrylate/methacrylic acid, and octadecyl methacrylate/vinyl acetate/methyl methacylate/methacrylic acid.

33. The suspension-emulsion enzyme system according to Claim 25, wherein said silicone fluid is polyorganosiloxane or polydiorganosilixane.

34. The suspension-emulsion enzyme system according to Claim 33, wherein said silicone fluid is linear, branched, or crosslinked.

35. The suspension-emulsion enzyme system according to Claim 25, wherein said silicone fluid is viscoelastic.

36. The suspension-emulsion enzyme system according to Claim 33, wherein said polydiorganosiloxane is a polydialkylsiloxane, a polydiaryllsiloxane, a polyalkylmethylsiloxane, a polyarylmethylsiloxane, or copolymers thereof with dimethylsiloxane.

37. The suspension-emulsion enzyme composition according to Claim 33, wherein said polyorganosiloxane contains functional groups.

38. The suspension-emulsion enzyme system according to Claim 25, wherein said silicone surfactant comprises at least one polydiorganosiloxane compound having at least one polyoxyalkylene group.

39. The suspension-emulsion enzyme system according to Claim 38, wherein said silicone surfactant has the formula Me₃SiO(Me2SiO)_x(MeQSiO)ySiMe₃, wherein Q is selected from the group consisting of

CH₃

-(CH2)_z(OCH₂CH₂)gOH, -(CH2)_z(OCH2CH2)g(OCH₂CH)_hOH,

CH₃

I

-(CH₂)_z(OCH₂CH2)_gOCH₃, -(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOCH₃,

CH₃

-(CH₂)_z(OCH₂CH₂)_gOC(O)CH₃, and -(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOC(O)CH₃ , wherein Me denotes methyl, x has an average value from 100 to 500, y has an average value from 1 to 50, z has an average value of 2 to 10, g has an average value of 1 to 36, and h has an average value of 1 to 36.

40. The suspension-emulsion enzyme system according to Claim 39, wherein Q is

CH₃ I

-(CH₂)_z(OCH₂CH₂)_g(OCH₂CH)_hOH, and Y is between 1 and 10.

41. The suspension-emulsion enzyme system according to Claim 38, wherein said polyoxyalkylene group has the formula -Rl 0(OCH₂CH₂)_gORl 1 , CH₃ CH₂CH₃

-RlO(OCH₂CH₂)_g(OCH₂CH)_hOR¹ !, -RlO(OCH₂CH₂)_g(OCH₂CH)_iOR¹1,

CH₃ CH₂CH₃

-RlO(OCH CH)_h(OCH₂CH)iOR^π, or

CH₃ CH CH₃

-Rl 0(OCH₂CH₂)g(OCH₂CH)_h(OCH₂CH)_iOR¹1 , wherein R¹^ is a divalent hydrocarbon group having from 1 to 20 carbon atoms,

R¹1 is a hydrogen atom, an alkyl group, an aryl group, or an acyl group, and g, h, and i independently have an average value from 1 to 150.

42. The suspension-emulsion enzyme system according to Claim 25, wherein said enzyme is a crystalline or amorphous precipitated enzyme.

43. The suspension-emulsion enzyme system according to Claim 42, wherein said crystalline or amorphous precipitated enzyme has been treated to remove excess aqueous solution.

44. The suspension-emulsion enzyme system according to Claim 42, wherein said crystalline or amorphous enzyme has a particle size of less than 50 μm.

45. The suspension-emulsion enzyme system according to Claim 44, wherein said crystalline or amorphous enzyme has a crystal size of less than about 5 μm.

46. The suspension-emulsion enzyme system according to Claim 42, wherein said enzyme is a detergent enzyme used in laundry detergents, fabrics care products, or dishwasher detergents.

47. The suspension-emulsion enzyme system according to Claim 46, wherein said detergent enzyme is selected from the group consisting of a protease, an amylase, a lipase, a cellulase, a mannanase, a ligninase, a pectinase, a peroxidase, and a catalase.

48. The suspension-emulsion enzyme system according to Claim 47, wherein said protease is a subtilisin.

49. A liquid detergent formulation comprising the multiple-emulsion enzyme system according to Claim 1, wherein said formulation maintains the multiple-emulsion form for at least one day at temperature between 22-28°C.

50. A liquid detergent formulation comprising the multiple-emulsion enzyme system according to Claim 1, wherein said formulation is stable against sedimentation, coalescence, creaming, flocculation, and/or aggregation for at least one day at temperature between 22- 28°C.