AU630880B2 - Stabilized enzyme dispersion - Google Patents

Description

Le ;r, 311~-11 Il i i. 1 I OPI DATE 05/02/90 APPLN. ID 39780 89 P r AOJP DATE 22 /3/90 PCT NU R PCT/DK89/00172 INTERNATIONAL APPLICATION

L

I D T Af T COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/00593 CUlD 3/386, C12N 9/96 Al (43) International Publication Date: 25 January 1990 (25.01.90) (21) International Application Number: PCT/DK89/00172 (74) Common Representative: NOVO-NORDISK A/S; Patent Department, Novo All6, DK-2880 Bagsvard (DK).

(22) International Filing Date: 11 July 1989 (11.07.89) (81) Dsignated States: AT, AT (European patent), AU, BB, BE Priority dat*': (European patent), BF (OAPI patent), BG, BJ (OAPI 8816443.9 11 July 1988 (11.07.88) GB patent), BR, CF (OAPI patent), CG (OAPI patent), CH, PCT/DK/00164 7 October 1988 (07.10.88) WO CH (European patent), CM (OAPI patent), DE, DE (34) Countries for which the regional (European patent), DK, FI, FR (European patent), GA or international application (OAPI patent), GB, GB (European patent), HU, IT (Euwas filed: DK et al. ropean patent), JP, KP, KR, LK, LU, LU (European patent), MC, MG, ML (OAPI patent), MR (OAPI patent), MW, NL, NL (European patent), NO, RO, SD, SE, SE (71) Applicants (for all designated States except US): NOVO- (European patent), SN (OAPI patent), SU, TD (OAPI NORDIK A/S [DK/DK]; Novo All6, DK-2880 Bags- patent), TG (OAPI patent), US.

vaird ALBRIGHT WILSON LIMITED [GB/ GB]; 210-222 Hagley Road West, Oldbury, Warley, West Midlands Published With international search report.

(72) Inventors; and Before the expiration of the time limit for amending the Inventors/Applicants (for US only) HAWKINS, John [GB/ claims and to be republished in the event of the receipt of GB]; 15 Oakbank Avenue, Whitehaven, Cumbria CA25 amendments.

CHADWICK, Philip [GB/GB]; Glenfield House, Glenfield Place, Barepot, Workington, Cumbria CA14 1JR MESSENGER, Edv.ard, Tunstall [GB/ GB]; Ramsey House, Camerton, Workington, Cumbria CA14 INB LYKKE, Mads [DK/DK]; Korsorgade 33/3 tv, DK-2100 Koebenhavn 0 (DK).

(54)Title: STABILIZED ENZYME DISPERSION (57) Abstract A convenient method for preparing a stabilized aqueous enzyme dispersion comprises: 1) precipitating a water-soluble polymer from aqueous solution to form an aqueous dispersion, and 2) before, during or after contacting the dissolved or dispersed polymer with an aqueous solution or fine aqueous dispersion of enzyme. Using this method, substantial improvement of the enzyme stability during storage can be obtained with surprisingly little polymer (relative to enzyme). And enzyme stabilization can, surprisingly, even be obtained by contacting precipitated polymer with dissolved enzyme. The stabilizing effect therefore appears not to be due (or at least not primarily due) to encapsulation.

V

WO 90/00593 PCT/DK89/00172 1 STABILIZED ENZYME DISPERSION TECHNICAL FIELD The present invention relates to stabilized enzyme dispersions.

BACKGROUND ART Ensuzing sufficient enzyme stability during storage represents a problem in the formulation of liquid enzymatic systems such as liquid enzymatic detergents, particularly those containing a detergent builder. The problem has received considerable attention in the prior art. One approach has been incorporation of various chemicals as enzyme stabilizers.

Another approach has been to coat or encapsulate the enzyme with a suitable coating agent and disperse the coated enzyme in the liquid detergent.

Thus, the method described in EP.-A-0238216 entails dispersing enzymes as particles in liquid detergent which has a structure which prevents sedimentation of the particles, after coating the particles with a hydrophobic, waterinsoluble substance such as a silicone which isolates the particles from the aggressive medium. US 4,090,973 describes encapsulating the enzyme in a water-soluble, solid surface active agent, such as polyvinyl alcohol or polyethylene glycol before addition to the liquid detergent. JP-A 63- 105,098 describes coating of enzymes with polyvinyl alcohol to form microcapsules and dispersing the capsules uniformly in a liquid detergent to improve storage stability.

The methods according to said publications involve physically surrounding a particle or droplet containing the enzyme with a barrier which isolates the enzyme more or less effectively from the detergent medium. To ensure effective coating or encapsulation of the enzyme with a protective material, a relatively high amount of the latter is required.

One method, described il EP-A 0,238,216, is to protect the enzyme by dispersing i in a hydrophobic liquid _11 11 I) -2 which is insoluble in the detergent, such as silicone oil, and dispersing the liquid in the detergent. Another proposed method is to encapsulate the enzyme in non-ionic surfactant (US 4,090,973) or polyvinyl alcohol (GB 1,204,123, JP-A 63-105,098, FR 2,132,216) by physically coating solid particles of enzyme with the encapsulant. JP-A 61-254,244 describes dispersing an enzyme in an aqueous polymer solution, dispersing the latter in a hydrocarbon and precipitating the polymer to form the micro capsules.

SUMMARY OF THE INVENTION We have found that when water soluble polymers are precipitated from aqueous solution to form a dispersion in the water and either the precipitation is effected in the presence of dissolved or finely dispersed enzyme, or the precipitate is subsequently contacted with dissolved or finely dispersed enzyme, so as to form a codispersion in water of the enzyme and polymer, substantial improvement of the enzyme stability during storage can be obtained with surprisingly little polymer (relative to enzyme). Our observation that enzyme stabilization can, surprisingly, even be obtained by contacting precipitated polymer with dissolved enzyme, leads us to believe that the stabilizing effect is not due (or at least not primarily due) to encapsulation.

According to a first embodiment of this invention, there is provided a method for the preparation of a stabilized aqueous enzyme dispersion comprising: precipitating a water-soluble polymer from aqueous solution to 25 forn an aqueous dispersion, and before, during or after contacting the dissolved or S" dispersed polymer with sufficient of an aqueous solution or fine aqueous dispersion of enzyme such that the ratio of said polymer to said enzyme is in the range of 0.03 to According to a second embodiment of this invention, there is provided a clear solution when used in the method of claim 11, said solution comprising an enzyme and a water soluble polymer wherein the polymer is polyvinylpyrrolidone and the enzyme is a protease, an amylase, a cellulase or a lipase.

According to a third embodiment of this invention, there is provided a stabilized enzyme dispersion, characterized in that the dispersed enzyme particles contain polyvinyl pyrrolidone or salt of I 2A polymeric carboxylic acid wherein the weight ratio of polyvinyl pyrrolidone or salt of polymeric carboxylic acid to enzyme is from 0.03 to According to a fourth embodiment of this invention, there is provided an aqueous based liquid detergent composition comprising water, surfactant and electrolyte according to the third embodiment.

A particularly preferred method of the invention comprises coprecipitation of enzyme and polymer from a solution comprising both of these or precipitation of the polymer in the presence of the dissolved enzyme. The stabilized enzyme dispersion according to the invention may in particular be an i: i r

B

i: i; i ri i STA/0964Z .WO 90/00593 PCT/DK89/00172 3 enzymatic liquid detergent- or an enzymatic detergent additive.

DETAILED DESCRIPTION OF THE INVENTION Enzyme Typically the enzyme used in the invention is a protease, lipase, cellulase, amylase or other stain and/or soil removing enzyme. Mixtures of enzymes may be employed.

For use in a liquid detergent the enzyme is preferably selected for stability at alkaline pH.

Polymer The polymer to be used in the invention is preferably a water-soluble polymer that can be precipitated by electrolyte or organic solvent. This choice of polymer allows the enzyme to be released by diluting the enzyme dispersion with water.

We particularly prefer a water soluble polyvinyl pyrrolidone. We can also use a polyvinyl alcohol or a cellulose derivative such as carboxymethyl cellulose, methyl cellulose or hydroxypropyl cellulose, a gum such as guar gum, gum benzoin, gum tragacanth, gum arabic or gum acacia, a protein such as casein, gelatin or albumin, or polycarboxylates such as polyacrylates, polymaleates or copolymers of acrylate and methacrylate. For obvious reasons we prefer not to use protein to stabilize proteases or cellulose derivatives to stabilize cellulases.

Where polyvinyl pyrrolidone is used we prefer to use a polymer with a molecular weight of 1,000 to 1,500,000.

For good stabilization we prefer molecular weights below 1,000,000, e.g. below 800,000, especially below 200,000 and most preferably below 100,000. We generally prefer to use molecular weights above 5,000, especially above 10,000, more particularly above 20,000, e.g. above 25,000.

In the case of polyvinyl alcohol we particularly prefer polymers with a molecular weight of 18,000 to 140,000, preferably 50,000 to 120,000, e.g. 80,000 to 100,000. Prefer- WO 90/00593 pCT/DK89/0017 4 ably any polyvinyl alcohol used according to our invention is a partially hydrolysed polyvinyl ester of a lower C 1

C

4 carboxylic acid, especially polyvinyl acetate, which has a degree of hydrolysis of greater than 25%, and desirably less than 95%, especially 50 to 90%, more preferably 60 to e.g. 70 to To obtain sufficient stabilization we generally prefer an amount of polymer corresponding to a weight ratio of polymer enzyme (pure enzyme protein) above 0.03, e.g.

above 0.1, especially above 0.4 and particularly above 1. If the polymer is used only for enzyme stabilization we prefer a polymer enzyme ratio below 5, especially below 2, but a larger amount of polymer may be used if it also serves another function PVA or CMC for antiredeposition in detergent).

Precipitation The method of the invention for preparing an enzyme dispersion involves precipitation of a water soluble polymer to form an aqueous dispersion, which is preferably nonsedimenting. Coprecipitation of enzyme and polymer or precipitation of the enzyme in the presence of dissolved polymer are preferred embodiments.

In one preferred embodiment, the precipitation is effected by contacting a solution containing the polymer (and optionally the enzyme) with an effective amount of a precipitant. Conventional measures may be used to obtain a suitably small particle size to form a dispersion, e.g. slow addition of precipitant with agitation.

The precipitant may be an electrolyte, i.e.

precipitation by salting out. Examples of suitable electrolytes are sodium sulphate, sodium citrate, sodium carbonate, sodium nitrilotriacetic acid, sodium tripolyphosphate, sodium nitrate, sodium borate and ammonium sulphate. Solid electrolyte or an electrolyte solution may be added to the polymer solution.

Alternatively, the prec.pitant may be an organic solvent. The solvent should be partly or fully miscible with SWO 90/00593 PCT/DK89/00172 water and should be able to precipitate the polymer. Examples of suitable solvents are, in the case of PVP: acetone, and in the case of PVA: acetone or ethanol.

In an alternative embodiment, the precipitation of polymer (and optionally the enzyme) may be effected by evaporation of a solution, e.g. an aqueous solution. Spray drying is preferred, e.g. the polymer may be dissolved in a concentrated aqueous solution of enzyme and the mixture spray dried.

In order to obtain a non-sedimenting dispersion of the water soluble polymer it is preferred that the precipitation of the polymer is effected in the presence of a dispersant. The dispersant may be a surfactant capable of maintaining the precipitated polymer in stable dispersion. In particular a structured surfactant formed by the interaction with electrolyte is preferably present. Alternatively solvents such as polyglycols, present in the enzyme solution, may act as the dispersant.

Contacting polymer with enzyme A preferred embodiment of the invention comprises coprecipitation of enzyme and polymer, especially from a clear solution. Such a clear solution containing polyvinyl pyrrolidone as the polymer and a protease, an amylase, a cellulase or a lipase as the enzyme is novel and is provided by the invention.

Particularly advantageously, the coprecipitation may take place in situ by contacting the enzyme/polymer solution with a precipitant to directly form the stabilized enzyme dispersion. This reduces the cost of preparing the dispersion and gives a reliable stabilization.

As an alternative to in-situ preparation, the coprecipitated polymer and enzyme, formed e.g. by precipitation by contacting with a precipitant or by evaporation, may be collected as a finely divided solid, e.g.

by filtration or spray drying, optionally followed by comminution, e.g. by ginding. The solid coprecipitate can WO 90/00593 PCT/DK89/00172, 6 then be dispersed in liquid to form the stabilized enzyme dispersion.

Enzyme solutions for use in coprecipitation according to the preferred embodiment of our invention may conveniently contain 0.1-10% of enzyme (pure enzyme protein, by weight), especially The solution may contain up to by weight of the solution, of an enzyme stabilizing water-miscible organic solvent, especially a water-miscible alcohol or glycol such as propylene glycol or glycerol. The alcohol is preferably present in proportion of from 10 to by weight of the solution, e.g. 25 to 75% by weight. Other enzyme stabilizers that may be present include lower mono- or dicarboxylic acids and their salts, such as formates, acetates and oxalates, borates and calcium salts. The solution typically' contains from 0.5% to 10%, e.g. 1 to 5% by weight organic enzyme coating material. We prefer, however, that the enzyme solution be substantially free of polyglycols which may tend to disperse the polymer used in the invention.

The solution of the polymer before coprecipitation may conveniently have a concentration of from 0.5% by weight of polymer (based on the weight of the solution) up to saturation. Preferably the concentration is sufficiently low for the enzyme and the polymer to be mixed to form a stable, clear, mobile mixed solution. Concentrations from 1 to 20% of polymer, depending on the solubility are usually preferred, especially 2 to 10%, e.g. 3 to by weight of the solution.

A solution of enzyme and polymer suitable for use in preparing dispersions of the invention may be prepared by dissolving solid polymer in aqueous enzyme.

In the case of preparing a liquid detergent by coprecipitation preferably a concentrated aqueous surfactant at substantially neutral pH and containing sufficient electrolyte to form a structured system is mixed with a solution of enzyme and polymer. Part of the electrolyte may optionally be premixed with the enzyme and polymer immediately less than 2 minutes) prior to addition thereof to the sirfactant. The resulting dispersion of enzyme and polymer may be stored and subsequently added to an WO 90/00593 PCT/DK89/00172 7 alkaline aqueous liquid detergent, preferably together with alkaline and/or solid builders such as sodium tripolyphosphate and/or zeolite.

As an alternative to coprecipitation, precipitated, dispersed polymer may be contacted with dissolved enzyme. Or alternatively dissolved polymer may be contacted with finely divided solid dispersed) enzyme. These alternatives provide effective stabilization and may be convenient if the po.,.ymer or enzyme is available in solid form.

Enzyme dispersion The stabilized enzyme dispersion according to the invention should have a high enough content of precipitant electrolyte) to prevent complete dissolution of the dispersed particles of enzyme and polymer. The content of precipitant is not necessarily high enough to precipitate the enzyme in the absence of polymer.

The stabilized enzyme dispersion may additionally comprise stabilizers or activators for the enzyme. For example enzymes may be stabilized by the presence of calcium salts.

Depending on the intended use of the enzyme dispersion it may be desirable, or even essential, that the dispersion does not sediment during storage, but a sedimenting system may be acceptable if the sediment can be re-dispersed e.g. by stirring or shaking. A non-sedimenting system can be formulated according to principles known in the art.

aAs mentioned above, the invention is particularly amenable to the preparation of liquid enzymatic detergent and to preparation of liquid enzymatic detergent additive for use in liquid detergent.

A stabilized enzyme dispersion wherein the dispersed enzyme particles contain polyvinyl pyrrolidone or polycarboxylic acid is novel and is provided by the invention.

WO 90/00593 PCT/DK89/00172 8 Enzymatic licuid detergent In the case of a liquid detergent, the enzyme dispersion should preferably be non-sedimenting. The liquid detergent compositions may be of the type in which an electrolyte interacts with aqueous surfactant to form a structured dispersion of lamellar or spherulitic surfactant, as described in GB 2,123,846 or GB 2,153,380. The suspending properties of a structured liquid detergent assist in preventing the particles of enzyme and polymer from undergoing agglomeration and sedimentation. The electrolyte also prevents the dissolution of the water soluble particles.

The latter protect the enzyme until the detergent is introduced into wash liquor, where the electrolyte is diluted sufficiently for the particle to dissolve and release the enzyme, so that it is available to act on stains. Physical shearing associated with washing may also contribute to the release of the enzyme.

Thus, preferably the liquid detergent composition comprises a surfactant desolubilising electrolyte, said electrolyte being present in 2 concentration at which said surfactant forms a structure capable of stably suspending the enzyme/polymer particles and sufficient to prevent or inhibit dissolution of the water soluble polymer. Typically, the polymer is a hydrophilic polymer which is soluble in dilute wash liquor but insoluble in concentrated liquid laundry detergent.

Preferably the dispersed enzyme is added to, or formed by precipitation in, a liquid detergent which comprises an aqueous phase, surfactant and sufficient 30 electrolyte dissolved in the aqueous phase to form, with the surfactant, a structure capable of supporting suspended particles.

Preferably the composition contains an effective amount of a detergent builder. Suitable builders include condensed phosphates, especially sodium tripolyphosphate or, less preferably, sodium pyrophosphate or sodium tetraphosphate, sodium metaphosphate, sodium carbonate, sodium silicate, sodium orthophosphate, sodium citrate, i VVO 90/00593 PC/DK9/00172 9 sodium nitrilotriacetate, a phosphonate such as sodium ethylenediamine tetrakis (methylene phosphonate), sodium diethylenetriamine pentakis (methylene phosphonate), sodium aceto diphosphonate or sodium aminotris (methylene phosphonate), sodium ethylenediamine tetraacetate or a zeolite. Other less preferred builders include potassium or lithium analogues of the above sodium salts.

The proportion of builder is typically from about to about 40% by weight of the liquid detergent composition. Usually 10% to 35%, preferably 15-30%, more preferably 18 to 28%, most preferably 20 to 27%. Mixtures of two or more builders are often employed, e.g. sodium tripolyphosphate with sodium silicate and/or sodium carbonate and/or with zeolite; or sodium nitrilotriacetate with sodium citrate.

Preferably the builder is at least partly present as solid particles suspended in the composition.

The invention is also applicable to the preparation of unbuilt cleaning compositions or compositions in which all the builder is present in solution.

The surfactant may be an anionic, nonionic, cationic, amphoteric, zwitterionic and/or semi polar surfactant which may typically be present in concentrations of from 2 to 35% by weight of the composition, preferably to 30%, more usually 7 to 25%, e.g. 10 to Usually the composition contains an alkyl benzene sulphonate together with one or more other surfactants such as an alkyl sulphate and/or alkyl polyoxyalkylene sulphate and/or a non-ionic surfactant. The latter may typically be an alkanolamide or a polyoxyalkylated alcohol.

Other anionic surfactants include alkyl sulphates, alkane sulphonates, olefin sulphonates, fatty acid ester sulphonates, soaps, alkyl sulphosuccinates, alkyl sulphosuccinamates, taurides, sarcosinates, isethionates and sulphated polyoxyalkylene equivalents of the aforesaid categories of anionic surfactant.

The cation of the anionic surfactant is preferably sodium but may alternatively be, or comprise, potassium, WO 90/00593 PCT/DK89/00172 ammonium, mono-di- or tri C1-4 alkyl ammonium or mono-di- or tri- C1-4 alkanolammonium, especially ethanolammonium.

The surfactant may be wholly or predominantly non ionic, e.g. a polyoxyalkylated alcohol alone or in admixture with a polyoxyalkylene glycol. Other non-ionic surfactants which may be used include polyoxyalkylated derivatives of alkylamines, carboxylic acids, mono or dialkylglycerides, sorbitan esters, or alkylphenols, and alkyloamiOes. Semipolar surfactants include amine oxides.

All references herein to polyoxyalkylene groups are preferably to polyoxyethylene groups, or less preferably to polyoxypropylene or mixed oxyethylene oxypropylene copolymeric or block copolymeric groups or to such groups with one or more glyceryl groups. Preferably the polyoxyalkylene groups from 1 to 30, more usually 2 to e.g. 3 to 15, especially 3 to 5 alkyleneoxy units.

Cationic surfactants for use according to our invention include quaternised or unquaternised alkylamines, alkylphosphines, or amido amines or imidazolines. Examples include mono- or di- (C 8 -22 alkyl) tri- or di- (Cl_ 4 alkyl) ammonium salts, mono (C822 alkyl) di (C1-4 alkyl) mono phenyl or benzyl ammonium salts, alkyl pyridinium, quinolinium or isoquolinium salts, or mono- or bis- (C 8 22 alkylamidoethyl) amine salts or quaternised derivatives, and the corresponding imidazolines formed by cyclising such amido amines. The anion of the cationic salts may be chloride, sulphate, methosulphate, fluoride, bromide, nitrate, phosphate, formate, acetate, lactate, tartrate, citrate, tetrachloroacetate or any other anion capable of conferring water solubility. Amphoteric surfactants include betaines and sulphobetaines e.g. those formed by quaternising any of the aforesaid cationic surfactants with chloroacetic acid.

In every case the surfactant for use herein has an alkyl group with an average of from 8 to 22 preferably 10 to 20, e.g. 12 to 18 carbon atoms. Alkyl grour: are preferably primary and straight chain, however wc do not exclude branched chain or secondary alkyl groups. In the case of r_ i dispersed polymer with sufficient of an aqueous solution or fine aqueous dispersion of enzyme such that the ratio of said polymer to said enzyme i is in the range of 0.03 to S./2 WO 90/00593 PCT/DK89/00172 11 alcohol based non-ionics the branched chain are sometimes preferred.

In general any surfactant referred to in GB 1,123,846, or in "Surface Active Agents and Detergents" by Schwartz, Perry and Berch, may be used.

Preferably the pH of the liquid detergent composition is alkaline, e.g. above 7.5, especially 7.5 to 12 typically 8 to 11, e.g. 9 to 10.5.

The liquid detergent composition contains dissolved, surfactant-desolubilising electrolyte. This may comprise a dissolved portion of the builder and/or any other salt, inorganic or organic, which is not itself a surfactant and which salts out the encapsulant, and also preferably the surfactants present, from solution (including micellar solution). Examples include sodium chloride, sodium nitrate, sodium bromide, sodium iodide, sodium fluoride, sodium borate, sodium formate, or sodium acetate, or corresponding potassium salts. Preferably, however, the electrolyte is a salt. which is required to perform a useful function in the wash liquor. The selection of electrolyte will to some extent depend on the encapsulant and the surfactant, since certain of the above electrolytes may desolubilise some compounds but not others.

The electrolyte may comprise sodium sulphate in minor concentrations, but electrolyte mixtures containing concentrations of sodium sulphate of about 3% or over based on the total weight of the detergent composition, are preferably not used because they may give rise to undesirable crystallization on standing.

The amount of dissolved electrolyte needed to provide a suspending structure depends upon the nature and amount of surfactant present as well aa the capacity of the electrolyte to salt out the surfactant. The greater the concentration of surfactant, and the more readily it is salted out by the electrolyte in questio the less the amount of electrolyte which is required. Generally, concentrations of electrolyte in solution of greater than 3%, more usually greater than 5% by weight, are required, i -e WO 90/00593 PCT/DK89/00172, 12 typically 6 to 20%, especially 7 to 19%, preferably 8 to 18%, more preferably 9 to 17%, most preferably 10 to 16%, e.g. 11 to 15% by weight of electrolyte in solution, based on the weight of the composition, or enough to contribute at least 0.5, preferably at least 1.0 more preferably at least most preferably from 2 to 4.5 gm ions of alkali metal per litre to the aqueous phase left after any suspended solid has been separated e.g. by centrifuging.

In order to determine the optimum amount of electrolyte required for a particular formulation any one or more of a number of indications may be employed. The concentration of dissolved electrolyte may be raised progressively in an aqueous surfactant, until the electrical conductivity falls to a minimum with addition of more electrolyte and a stable, turbid, spherulitic system is observed. The amount of electrolyte may then be optimised within this region by preparing samples with different concentrations of electrolyte in the region of the conductivity minimum and centrifuging for 90 minutes at 20,000 G until a concentration is identified at which no clear lye phase separates.

The electrolyte content is preferably 'adjusted to provide at least three months storage stability at ambient, at 0°C and at 40°C. Behaviour on shearing is another characteristic which is controllable by adjusting the electrolyte concentration. Where the concentration is too low the formulations, all of which are usually thixotropic, tend not only to become less viscous with increasing shear, but to retain the greater fluidity after the applied shear has been withdrawn instead of reverting to their original higher viscosity. Such formulations are often unstable after shearing thus they may undergo separation after high shear mixing, centrifugal deaeration, or high speed bottling.

Increasing the concentration of dissolved electrolyte will generally avoid such shear instability by providing a more robust structure.

Electrolyte concentrations just above the minimum required to prevent shear instability sometimes cause the SWO 90/00593 PCT/DK89/00172 13 opposite problem. After shearing, the viscosity of the composition recovers to a higher value than that before shearing. This can result in the composition becoming too viscous after being agitated or shaken. This problem too can usually be cured by increasing the electrolyte content.

If difficulty is encountered obtaining a stable spherulitic composition the concentration of surfactant may be increased, or the proportion of less "soluble" surfactant raised, e.g. increasing the amount of sodium alkyl benzene sulphonate or of low HLB non-ionic surfactant, i.e. having an HLB less than 12, preferably less than 10 e.g. less than 8 more usually 2 to Alternatively, if larger concentrations of electrolyte are used a lamellar, G-phase or hydrated solid structure may be obtained. This may be obtained for any desired detergent surfactant or surfactant mixture by adding enough electrolyte to salt out the surfactant so that the majority is centrifuged off at 800 g leaving a clear lye phase. If the composition is then not sufficiently stable to storage, it may be rendered non-sedimenting by decreasing the proportion of water. Alternatively if the composition obtained in this way is not mobile it may be progressively diluted with water until it is capable of being poured, or until an optimum balance of mobility and stability has been struck.

Additionally, but less preferably, our -nvention covers liquid detergent compositions having suspending power which is provided or contributed to by components other than the salted out surfactants, e.g. high concentrations of car- 1 30 boxymethyl cellulose or the presence of poly electrolyte dispersants, soluble gums or emulsifiers or bentonite.

The detergent composition may contain any of the usual minor ingredients such as soil suspending agents (e.g.

carboxymethyl celluose), preservatives such as formaldehyde 33 or tetrakis (hydr xymethyl) phosphonium salts, bentonite clays, or any of the enzymes described herein, protected according to the invention. Where a bleach is to be employed it may be convenient to encapsulate the bleach e.g. with a One method, described i7 EP-A 0,238,216, is to protect the enzyme by dispersing it in a hydrophobic liquid WO 90/00593 PCT/DK89/00172.

14 hydrophilic encapsulant, or in a hydrophobic medium, such as, for instance a silicone or hydrocarbon as described in EP-A- 0238216 or GB-A-2200377.

Particularly preferred liquid detergents are those containing: long chain C 1 0-14) linear alkyl benzene sulphonates in an amount of 5-12%, long chain alkyl, or alkyl ether, sulphates, e.g. with 0-5 ehtyleneoxy units, in an amount of fatty acid alkanolamides, and/or alcohol ethoxylates having HLB of less than 12 in an amount of mixtures of mono-and di-long chain alkyl phosphates in an amount of e.g. sodium tripolyphosphate (preferably pre-hydrated with from 0.5 to 5% by weight of water) in an amount of 14-30%, e.g. 14-18% or 20-30%; optionally sodium carbonate in an amount of up to 10%, e.g.

5-10% with the total of sodium tripolyphosphate and carbonate being preferably 20-30% antiredeposition agents such as sodium carboxymethyl cellulose in an amount of 0.05-0.5%; optical brightening agents in an amount of chelating agents, e.g. amino phosphonates such as methylene phosphonates of di- and polyamines, especially sodium ethylenediamine tetra[methylene phosphonate] or diethylene triamine hexa[methylene phosphonate] optionally present in an amount of 0.1-15%; together with conventional minor additives such as perfume colouring preservatives, the remainder being water, the percentages being by weight of the total liquid detergent. The liquid detergent may have a pH after dilution to 1% of 6 to 13, preferably 7 to 12, more usually 8 to ,i e.g. 9 to 10.5.

The invention is by no means exclusively applicable to the preparation of laundry detergents. Any liquid aqueous surfactant system in which particulate additives can be suspended and which require the presence of enzymes which are chemically incompatible with the aqueous surfactant medium may be prep red according to the invention. For example enzymes, esecially proteases, lipases and amylases are useful in dish washing detergents, both for manual and automatic use.

L 1 _i llIIY~~ provided a stabilized enzyme dispersion, characterized in that the dispersed enzyme particles contain polyvinyl pyrrolidone or salt of A A/0964Z WO 90/00593 PCT/DK89/00172

EXAMPLES

The invention will be illustrated by the following examples in which all storage tests were performed at unless otherwise noted.

Example 1 2 parts by weight of a 2% protease solution in an 80:20 wt/wt mixture of propylene glycol and water, having an activity of 8,000 Novo Protease Units gm-l sold by Novo- Nordisk A/S under the registered trademark ESPERASE, and ona part by weight of a 4% by weight aqueous solution of polyvinyl alcohol having a mean molecular weight of 80,000- 100,000 and being 88% hydrolysed were mixed to give a clear mobile liquid which was stable to storage.

The enzyme/P.V.A-containing liquid was added to a liquid detergent formulation to give a final composition.: wt% Sodium linear C 1 2-14 alkylbenzene sulphonate 9.3% Sodium linear C 1 2-18 alkyl 3 mole ethoxy sulphate 1.85% Coconut diethanolamide 1.85% Sodium tripolyphosphate 16.7% Sodium carbonate 6.7% Sodium carboxymethylcellulose 0.9% Optical brightening agent 0.1% Enzyme/PVA solution Water balance pH 10.5% After two weeks storage the stain removing power of the above formulation was superior to that of a control formulation containing a silicone protected enzyme at equivalent initial protease activity.

WO 90/00593 PCT/DK89/00172.

16 Example '2 ESPERASE 8.OL protease solution was mixed with various aqueous polymers.

The mixtures were added to a liquid detergent formulation comprising: sodium C 1 0-14 linear alkyl benzene sulphonate triethanolamine C 1 2-14 alkyl sulphate

C

1 2-13 alkyl 3 mole ethoxylate sodium tripolyphosphate 25.0% sodium ethylenediamine tetrakis (methylene phosphonate) Optical brightener 0.2% Silicone antifoam 0.2% sodium carboxymethyl cellulose 0.1% perfume 0.2% formaldehyde 0.05% Enzyme activity was determined by comparing soil and stain removal with that of an enzyme free, control formulation.

The retention of activity after storage was the percentage improvement after storage compared with the control, expressed as a percentage based on the percentage improvement of the freshly prepared sample.

The results are indicated in the following table: i molecular weights above 5,000, especially above 10,000, more particularly above 20,000, e.g. above 25,000.

In the case of polyvinyl alcohol we particularly prefer polymers with a molecular weight of 18,000 to 140,000, preferably 50,000 to 120,000, e.g. 80,000 to 100,000. PreferjI *WO 90/00593 PC/DK89/00172 weight ratio enzyme solution :polymer solution by weight additive system added to detergent Polymer added 4% aqueous P.V.A.

MW 80,000-100,000 88% hydrolysed 4% polyvinyl pyrrolidone MW 700,000 4% aueous gelain 1% "Emulgum," 200 guar gum 1% "Emulgum," 200 S guar gum residual performance residual performance J 4 2:1 2:1 2:1 1:2 -1:2 0.5% 0.5% 0.5% 1% 1% 0.33% 73% 100% 60% after 47% after 21 days 23 days after 85% after 21 days 151 days after 53% after 21 days 26 days 64% after 17 days 77% after 21 days None 69% after days 31% after 50 days The final result in the above table was obtained using "ESPERASE" 8.0L without added polymer. The percentage retention appeared remarkable for an unprotected enzyme, and contradicted earlier results obtained with other unprotected enzyme systems in which activity was lost totally after 2 to 3 days.

It was noted, however, that the particular sample of liquii enzyme used in the above experiment contained about 2% of adventitious carbohydrate which may have functioned as a stabilizing polymer in accordance with our invention and to which the high retention of activity of the "unprotected" sample has now been ascribed.

The performance of polyvinyl pyrrolidone was especially marked.

L.upouypnospnate, sodium nitrate, sodium borate and ammonium sulphate. Solid electrolyte or an electrolyte solution may be added to the polymer solution.

Alternatively, the precipitant may be an organic solvent. The solvent should be partly or fully miscible with WO 90/00593 PCT/DK89/00172 18 Example 3 Example 2 was repeated using 8 different PVA compositions. The detergent samples were tested at intervals and the stain removal compared with that of a detergent containing a commercial silicone protected enzyme according to our EP-A-0238216, and a non-enzymatic control.

The retention of the activity of the enzymatic formulations, compared with the non-enzymatic formulation is recorded in Table 2.

Table 2 Encap- I I% retention of activity after: sulant MW hydrolysis 2 weeks 4 weeks 8 weeks PVA 3,000 75 82 64 64 PVA 2,000 75 84 58 PVA 10,000 88 88 70 64 PVA 90,000 88 83 72 61 PVA 125,000 88 82 70 64 PVA 95,000 96 81 56 PVA 16,000 98 88 58 53 PVA 88,000 98 70 58 41 PVA 126,000 98 92 64 PVA 14,000 100 72 39 PVA 155,000 100 78 39 Silicone 58 3 5 23 The results indicate that the more sparingly soluble PVA polymers having a degree of hydrolysis less than are more effective then the polymers which are more soluble than 90% hydrolysed PVA.

Example 4 Acetone precipitated PVP-protease was prepared as follows: 15 g of polyvinyl pyrrolidone having a mean molecular weight of about 38,000 was dissolved in 150 ml of a 2% protease solution with about 10% total dry substance prepared according to US 3,723,250 and sold by Novo-Nordisk A/S under the registered trade mark "SAVINASE" to give a jI I L .L II t:U e.g. DY I precipitation by contacting with a precipitant or by evaporation, may be collected as a finely divided solid, e.g.

by filtration or spray drying, optionally followed by comminution, e.g. by gi nding. The solid coprecipitate can WO 90/00593 PCT/DK89/00172 19 clear solution. 300 ml of acetone was added slowly with vigorous stirring, causing precipitation and heating from room temperature to about 30-35*C. The dispersion was left with stirring for 10-15 minutes and then filtered on a Buchner funnel, washed with acetone, sucked dry and left to air dry. The PVP:protease ratio was calculated as Salt precipitated PVP-protease was prepared as follows: 2 g of PVP (MW 38,000) was dissolved in 22 g of SAVINASE solution. The solution was heated to 35*C, and 6 g of sodium sulphate was added slowly with vigorous stirring., causing precipitation. The suspension was filtered and air dried. The PVP:protease ratio was 2% of each PVP-protease sample was added to the detergent of Example 1 instead of the Enzyme/PVA at a level Ratio Prcpt. 0 days 3 d 7 d 14 d 21 d acetone 100 88.3 79.2 70,3 58.8 2.5 salt 100 85.7 73.2 56.9 37.9 0 reference 100 83.3 61.5 34.0 16.5 It is seen that samples prepared according to the invention provide substantial stabilization.

Example Samples of salt precipitated PVP-protease were prepared as in Example 4, but with varying PVP:protease ratio and PVP molecular weight, as indicated below.

A spray dried PVP-protease sample was prepared as follows: 226 g of PVP was dissolved in 26 kg of a 7% protease solution (Savinase), pH was adjusted to 6.5 (dilute sulfuric acid), and the solution was spray dried on a Standard Unit 1 from A/S Niro Atomizer with the atomizing wheel at 2000 rpm and with an air throughput of approx. 1000 cubic meters per L .1 solution of enzyme and polymer. Part of the electrolyte may optionally be premixed with the enzyme and polymer immediately less than 2 minutes) prior to addition thereof to the sdrfactant. The resulting dispersion of enzyme and polymer may be stored and subsequently added to an WO 90/00593 PCT/DK89/00172'hour. The air temperature was inlet 170 C and outlet 65 C.

The spray dried product contained 17 of protease.

All samples were tested by storage tests as in Example 4. A protease solution was included as reference.

Method MW PVP:enz 0 days 3 d 7 d 14 d 28 d Salt 38,000 0.75 100 63.7 49.7 35.5 21.5 0.5 100 64.2 51.7 41.9 28.3 0.25 100 59.8 45.1 34.7 22.2 0.033 100 33.3 14.5 7.8 4.8 630,000 0.033 100 30.8 12.8 8.3 5.4 Spray 38,000 0.125 100 75.8 55.8 41.4 22.9 Reference 0 100 15.3 4.9 0.0 0.0 It is seen that the invention provides stabilization even at dosages as low as polymer:enzyme 0.033:1 with both molecular weights tested. Increasing amounts of PVP provide increasing stabilization. Enzyme Preparations made by spray drying and by salt precipitation appear to provide a similar degree of stabilization.

Example 6 Detergent containing PVP (MW 700,000) and protease was prepared and tested as in Example 1. The type of protease and the enzyme dosage in the detergent are indicated below; a protease solution was used in the case of Alcalase.

Washing tests were made before and after storage with standard soiled cloths EMPA 116 and 117, and results express residual washing performance after 56 days storage. Liquid proteases without PVP were used as references.

dispersed enzyme particles contain polyvinyl pyrrolidone or polycarboxylic acid is novel and is provided by the invention.

~i i R lh I I Ii I i~-I WO 90/00593 PCT/PDK89/00172 Protease Esperase 11 Alcalase

I

PVP Dosage .375% .25% .375% .15% .375% .1875% .125% retention 77% 17% 73% 23% 17% 71% 58% 0% Savinase It Example 7 The experiment in Example 6 was repeated with Alcalase and varying ratios PVP:protease. The enzyme dosage in the detergent was 0.28% in each case. Liquid Alcalase was used as reference.

PVP:protease 0 (reference) .016 .08 .4 retention 0% 38% 62% 56% Stabilization according to the invention is observed even with extremely low amounts of PVP.

Example 8 This experiment was similar to Example 7, but the order of mixing was varied. In each case 0.28% of a Alcalase solution and 0.14% of a 4% PVP solution were added (PVP:protease In one case the two solutions were premixed before adding to the detergent (as in Example in another case PVP was added first, then protease; and in yet another first protease, then PVP. In the reference, PVP was omitted.

Enzyme stabilization was observed both in the case of coprecipitation, in the case of contacting dispersed PVP I L amount of a detergent builder. Suitable builders include condensed phosphates, especially sodium tripolyphosphate or, less preferably, sodium pyrophosphate or sodium tetraphosphate, sodium metaphosphate, sodium carbonate, sodium silicate, sodium orthophosphate, sodium citrate, i4: ii d r- WO 90/00593 PCT/DK89/00172 with dissolved protease and in the case of contacting dissolved PVP with dissolved protease.

Claims (19)

1. A method for the preparation of a stabilized aqueous enzyme dispersion comprising: precipitating a water-soluble polymer from aqueous solution to form an aqueous dispersion, and before, during or after contacting the dissolved or dispersed polymer with sufficient of an aqueous solution or fine aqueous dispersion of enzyme such that the ratio of said polymer to said enzyme is in the range of 0.03 to

2. A method according to claim 1 wherein said enzyme is a protease, amylase, cellulase or lipase.

3. A method according to claim 1 or claim 2, whereby said polymer is polyvinyl alcohol, polyvinyl pyrrolidone, polycarboxylic acid salt, carboxymethyl cellulose salt, gelatin or guar gum.

4. A method according to claim 3 whereby said polyvinyl alcohol is a partially hydrolyzed polyvinyl ester of a CI-4 carboxylic acid having a degree of hydrolysis of from 25 to A method according to claim 3 whereby said polyvinyl pyrrolidone has an average molecular weight in the range of about 1,000 to 1,500,000.

6. A method according to any one of claims 1 to 5 comprising precipitation of polymer by contacting with an effective amount of a precipitant.

7. A method according to claim 6 wherein the precipitant is an 25 electrolyte or an organic solvent.

8. A method according to claim 7 whereby said electrolyte is Ssodium sulphate, sodium citrate, sodium tripolyphosphate, sodium carbonate or ammonium sulphate. S" 9. A method according to claim 7 whereby said solvent is acetone 30 or ethanol.

10. A method according to any one of claims 1 to 5 comprising precipitation of polymer by evaporation, preferably by spray drying.

11. A method according to any one of claims 1 to 10 comprising coprecipitation of said enzyme and said polymer.

12. A clear solution when used in the method of claim 11, said solution comprising an enzyme and a water soluble polymer wherein the polymer is polyvinylpyrrolidone and the enzyme is a protease, an amylase, a cellulase or a lipase. 3S, A/0964Z salted out by the electrolyte in question, the less the amount of electrolyte which is required. Generally, concentrations of electrolyte in solution of greater than 3%, more usually greater than 5% by weight, are required, St. 24

13. A method according to claim 11 comprising contacting a solution containing said polymer and said enzyme with a precipitant to directly form an enzyme dispersion.

14. A method according to claim 11 wherein finely divided coprecipitate is dispersed in water. A method according to any one of claims 1 to 10 wherein precipitated, dispersed polymer is contacted with dissolved enzyme.

16. A method according to any one of claims 1 to 10 wherein dissolved polymer is contacted with finely divided solid enzyme.

17. A method according to any one of claims 1 to 11 or 13 to 16 for the preparation of an aqueous based liquid detergent composition comprising water and surfactant.

18. A method according to claim 17 comprising a spherulitic or lamellar surfactant structure and having suspended particles of solid builder.

19. A method according to claim 18 wherein said solid builder comprises sodium tripolyphosphate and/or zeolite. A method according to any one of claims 1 to 11 or 13 to 16 for the preparation of an enzymatic detergent additive.

21. A stabilized enzyme dispersion, characterized in that the dispersed enzyme particles contain polyvinyl pyrrolidone or salt of polymeric carboxylic acid wherein the weight ratio of polyvinyl pyrrolidone or salt of polymeric carboxylic acid to enzyme is from 0.03 to 25 22. An aqueous based liquid detergent composition comprising water, surfactant and electrolyte according to claim 21.

23. A method for the preparation of a stabilized aqueous enzyme dispersion substantially as hereinbefore described with reference to any ne of the examples excluding the comparative examples.

24. A stabilized enzyme dispersion substantially as hereinbefore described with reference to any one of the examples excluding the comparative examples. DATED this TWENTY-FIFTH day of AUGUST 1992 Albright Wilson Limited Novo Nordisk A/S T Patent Attorneys for the Applicant SSPRUSON FERGUSON STA/0964Z 3. or tetrakis (hydrC~ymethyl) phosphonium salts, bentonite clays, or any of the enzymes described herein, protected according to the invention. Where a bleach is to be employed it may be convenient to encapsulate the bleach e.g. with a INTERNATIONAL SEARCH REPORT International Application No PCT/DK 89/00172 I. CLASSIFICATION OF SUBJECT MATTER (it several classification symools aDDly, indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC IPC5 C D 3/386, C 12 N 9/96 IPC C 11 D 3/386, C 12 N 9/96 II. FIELDS SEARCHED Minimum Documentation Searched Classification System I Classification Symbols IPC C 11 D, C 12 N Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Flelds Searched Ill. DOCUMENTS CONSIDERED TO BE RELEVANT* Category Citation of Document, i with indication, where appropriate, of the relevant passages a Relevant to Claim No. A EP, A, 0238216 (ALBRIGHT et al.) 23 Septem- 1 ber 1987, see the claims cited in the application A US, A, 3660563 GOULD et al.) 2 May 1 1972, see column 1, lines 16-52; claims A EP, A, 0206718 (CLOROX) 30 December 1986, 1 see the claims i A EP, A, 0177183 (CLOROX) 9 April 1986, see 1 the claims A US, A, 4090973 MAGUIRE et al.) 1 23 May 1978, see the claims A Chemical Abstracts, vol. 110, no. 2, 1 January 1989 (Columbus, Ohio, US), Special categories of cited documents: ei later document published after the International filing date document defining the general staii. c the art which is not 'o priority date and not In conflict with the application but considered to b of particular relevance cited to understand the principle or theory underlying the invention earlier document but published on or after the International document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an Inventive step which is cited to establish the publication date of another document of particular relevance;* the claimed invention citation or other special reason (as specified) cannot be considered to involve an Inventive step when the document referring to an oral disclosure, use, exhibltlon or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document publlahed prior to the international filing cate but In the art. later than the priority date claimed document member of same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 1st November 1989 28 MOV 1989 International Searching Authority Signature of Authorized Offcer EUROPEAN PATENT OFFICE WILLIS Form PCTIISA/210 (aecond sheet) (January 196) L enzymes, esiecially proteases, lipases and amylases are useful in dish washing detergents, both for manual and automatic use. I PCT/DK 89/00172 lrittrnatlomal Acitlicstion No, 2- Ill. DOCUMENTS CONSIDERED O at RELEVANT (CONTINUED FROM THlE SECOND SHEET) Category Citationt of Oecumerit, wIth 0'nio, Wherie aooroorita. of tne ruieverif passsg~it Releanit to Clam No see page 101, abstract nio. 10112s, CS, A, 251377 PROCHAZKA) 15 March 1988 Form PCT ISA,210 (extra shot) (aWiliuiil 19411 I' j c~ ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. DK 8900172 SA 30108 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The memhers are as contained in the European Patent Office EI)PI file on 20/11/89 The European Patent Office is in no nay liable for these particulars ohich are merely given fur the purpose of information. Patent document Publication Patent family Publication cited in search report date rembner(s) date EP-A- 0238216 23-09-87 AU-A- 6911187 27-08-87 GB-A- 2186884 26-08-87 JP-A- 62248486 29-10-87 US-A- 3660563 02-05-72 AT-A,B 312930 15-12-73 AT-A,B 304724 15-12-72 AT-A,B 306229 15-02-73 AT-A,B 306191 15-02-73 BE-A- 701813 02-01-68 BE-A- 735240 29-12-69 CA-A- 1007395 22-03-77 CH-A- 555865 15-11-74 CH-A- 532118 31-12-72 CH-A- 537204 13-07-73 CH-A- 537961 31-07-73 DE-A- 1745187 23-03-72 FR-A- 2022100 31-07-70 FR-A- 1604129 12-07-71 GB-A- 1205764 16-09-70 GB-A- 1205765 16-09-70 GB-A- 1205766 16-09-70 GB-A- 1205767 16-09-70 GB-A- 1205768 16-09-70 GB-A- 1205769 16-09-70 GB-A- 1205770 16-09-70 NL-A- 6710346 29-01-68 NL-A- 6910624 13-01-70 SE-B- 366214 22-04-74 SE-B- 366213 22-04-74 US-A- 3849185 19-11-74 US-A- 3520949 21-07-70 US-A- 3574822 13-04-71 US-A- 3914405 21-10-75 US-A- 3635756 18-01-72 IS-A- 3660071 02-05-72 US-A- 3674901 04-07-72 US-A- 3697643 10-10-72 US-A- 3761286 25-09-73 US-A- 3941858 02-03-76 For more details about this annex see Oficial Journal of the European Patent Office, No. 12/82 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. Page 2 OK 8900172 SA 30108 This annex lists; thc patent family members relating to the patent documents cited in the abo.e-mcntioned international search report. The members are as contained in the Furopean Patent Office FD)1 Wje on 20/1118X9 The European Patent Office is in no isay liable for these particulars ishich are merely gi%en for the purpose of information. EP-A- 0206718 30-12-86 None EP-A- 0177183 09-04-86 AU-B- 577148 15-09-88 AU-A- 4710385 20-03-86 JP-A- 61072100 14-04-86 US-A- 4801544 31-01-89 US-A- 4090973 23-05-78 CA-A- 1081148 08-07-80 For mvre details about tils annex see Official Journal of the European Patent Office, No. i z/8z