OA10056A - Detergent composition - Google Patents

Abstract

A shaped detergent corn position comprises a detergent active, water and a mutant subtilisin enzyme in which the amino acids in positions 195 and 222 in the native enzyme have been replaced with different amino acids (preferably with glutamic acid and alanine, respectively).

Description

10056 1

DETERGENT COMPOSITION

The présent invention relates to detergent compositions inthe fora of bars, tablets, sticks and the like for directapplication to fabrics, hard surfaces or any othersurface. In particular, it relates to soap or soap/synthetic compositions in bar form which bars alsoinclude protease enzymes.

It is known to incorporate protease enzymes into soapbars. However, enzymes tend to be unstable, that is theylose activity, in such bars. According to British Patent265 024 this disadvantage may be overcome by dehydratingthe enzyme and the soap bar to a water content of 10% or1 e s s .

As an alternative, and in order to produce bars whichcontain greater than 10% of water, the use of stabilisingagents for protease enzymes hâve been proposed. Manystabilising agents cannot readily be used in bars or othersimilar shaped solid products because of the adverseeffect they hâve on the structure and firmness of theproduct.

According to British Patent 2 186 883, it is proposed touse a mixture of a boron compound, a polyol, an organicacid or its alkali métal sait and an alkali métal sait ofan inorganic acid which is not a boron compound as astabilising system for protease enzymes, such as Savinase,in soap bars.

It is also known to use mutant subtilisin proteases which.hâve been modified by substitution at an amino acid site.US 4 760 025 (Genencor), for example, daims subtilisinmutants with amino acid substitutions at amino acid sites32, 155, 104, 222, 166, 64, 33, 169, 189, 217 or 157. 1 00 56 2 A mutant, protease whereby méthionine at position 22 2 hasbeen replaced by alanine, is shown to hâve an improvedoxidation stability in the presence of a bleaching agent.

We hâve now found it is possible to préparé detergentcompositions in bar or other shaped solid form witt. arelatively high level of water, typically 10 to 30? byweight, with mutant subtilisin enzymes which hâve beenmodified at positions 195 and 222 in the protease. Suchcompositions, particularly in the form of soap bars, hâveimproved enzyme storage stability compared to soap barscomprising other commonly used protease enzymes such asSavinase, everi in the absence of a bleaching agent.Furt.hermore, as a resuit of this increased enzymestability, there are improvements in the performance ofthe bars.

Thus, accordingly the présent invention provides a detergent composition in shaped solid form comprising adetergent active, water, and a mutant subtilisin enzyme inwhich the amino acid sequence has been altered at positions 195 and 222 by substitution with another aminoacid.

Preferably, the detergent composition will also contain astabilising System comprising a boron compound and apolyol.

The stabilising System may also additionally containat least oae further component selected from i) an organic acid, an alkali métal sait of an organicacid, an inorganic acid and mixtures thereof; and ii) an alkali métal sait of an inorganic acid not being aboron compound. C3470 10 15 20 25 1 0056 3

An advantage of the compositions according to the invention is that they can be stored for much longerperiods of time, even at conditions of high températureand relative humidity, without any significant loss inenzyme activity, than similar compositions previouslyproposed. This is important since such detergentcompositions in bar or other shaped solid form aretypically used in countries where such conditions prevail.

Boron compounds suitable for the stabilising Systeminclude boric acid, boric oxide and alkali métal boratesand, in particular, borax. The polyol may be an aliphaticpolyol with 2 to 6 carbon atoms and 2 to 6 hydroxylgroups, especially propylene glycol, glycerol andsorbitol. The organic acid may be an aliphatic mono-,di-, or tricarboxylic acid, and in particular formic acid,succinic acid, adipic acid, glutaric acid, citric acid orcommercially available mixtures thereof, for exampleSokalan DCS (Trade Mark), and soap acids such as tallow,coconut, palm kernal and babassu fatty acids. Suitablealkali métal salts of organic acids include sodiumformate, sodium citrate and sodium propionate. A suitableinorganic acid is phosphoric acid. Alkali métal salts ofinorganic acids, not being a boron compound, includesodium sulphate, sodium chloride, sodium carbonate, sodiumbicarbonate and sodium phosphate.

The boron compound is preferably présent in an amount of0.1 to 10%, most preferably 0.5 to 5%; the polyolpreferably 0.1 to 15%, most preferably 0.5 to 10%; theorganic acid or sait thereof or inorganic acid in amountof 0.1 to 10%, preferably 0.5 to 5% ; and the alkali métalsait of the inorganic acid which is not a boron compound,in an amount 0.1 to 5%, preferably 0.25 to 2.5%; where ailpercentages are by weight of the composition. 347 0 10056 4

Preferably bars ;.n accordance with this invention alsocomprise : i) 25 to 80%, most preferably 25 to 70%, by weightof detergent active which is soap or a mixtureof soap and synthetic detergent active, reckonedas anbydrous; ii) 0 to 35 % and, most preferably, 10 to 30% by10 weight of water; iii) 0 to 35% and, most preferably, 0,1 to 30% byweight filler. 15 When a synthetic detergent active is included in the bar it will preferably be présent at a level between 0.1 and10%, most preferably 0.5 to 5% by weight based on thetotal .level of detergent active. 20 Fatty acid soaps suitable for use herein can be obtained from natural sources such as, for instance, plant oranimal esters (e.g. palm oil, coconut oil, babassu oil,soybean oil, castor oil, tallow, whale or fish oils,grease, lard and mixtures thereof). The fatty acid soaps 25 can also be synthetically prepared (e.g. by the oxidation of Petroleum, or by the hydrogénation of carbon morioxideby the Fischer-Tropsch process). Resin acids, such asthose présent in tall oil, may be used. Naphthenic acidsare also suitable. 3 0

Tallow fatty acids can be derived from various animalsources and generally comprise about 1% to 8% myristicacid, about 21% to 32% palmitic acid, about 14% to 31%stearic acid, about 0% to 4% palmitoleic acid, about 36%

35 to 50% oleic acid and about 0% to 5% linoleic acid. A typical distribution is 2.5% myristic acid, 29% palmitic C3470 1 0056 5 acid, 23% stearic acid, 2% palmitoleic acid, 41.5% oleicacid, and 3% linoleic acid.

Coconut oil refers to fatty acid mixtures having an5 approximate carbon chain length distribution of : 8% C8, r,% C10, 48% C12, 17% C14, 8% C16, 2% C18, 7% oleic and 2%linoleic acids (the first six fatty acids listed beingsaturated) . Other sources having similai' carbon chainlength distributions, such as palm kernel oil and babassu 10 kernel oil, are included within the term coconut oil.

Coconut oil fatty acids ordinarily hâve a sufficiently lowcontent of unsaturated fatty acids to hâve satisfactorykeeping qualifies without further treatment. Generally,however, fatty acids are hydrogenated to decrease the 15 amount of unsaturation (especially polyunsaturation) of the fatty acid mixture.

Babassu oil refers to fatty acid mixtures havincj anapproximate carbon chain length distribution of: 7.8% C3, 20 6.5% C10, 44% C12, 15.2% C14, 7.9% C16, 3.2% C18, 12% C18;1, 2.3% C16;2. (The last two being mono and double unsaturatedrespectively)

It is also possible to include small amounts of non-soap 25 decergent active in compositions of the invention such as branched alkyl benzene sulphonates, linear alkyi benzenesulphonates and nonionic ethoxylated alcohols.

The filler is preferably selected from kaolin, bentonite, 30 silicate, talc, sodium sulphate and sodium carbonate.

The mutant subtilisin enzymes used in the composition ofthe invention are disclosed in WO-A-89/06279(Novo/Nordisk). They differ from the native subtilisin 35 enzyme in that they contain a different amino acid at positions 195 and 222. The native enzyme contains a 1 0056 6 glycine residue at position 195 and a méthionine atposition 222. A particularly preferred mutant enzyme isone which contains a glutamic acid residue; at position 195and an alanine residue at position 222. A commerciallyavailable material with the aforementioned mutations isDurazym (ex Novo/Nordisk). The enzyme may also be mutâtedat sites other than those specified hereinbefore.

In general, the amount of mutant subtilisin enzymeincluded in the composition of the invention is such tha.tit corresponds with a proteolytic activity of 0.1 to 10CGU/'ing based on the composition, preferably 0.5 to 20GU/mg,most preferably 1.0 to lOGU/mg, where GU/mg is glycineunit per milligram. A glycine unit is defined as the enzyme activity whichunder standard conditions, during a 15 minute incubationat 40°C with N-Acetyl casein as substrate, produces anamount of terminal NH:;-groups équivalent to 1 microgramme/ml of glycine.

The compositions of the invention may contain otherproteolytic enzymes in addition to the mutant subtilisinenzyme hereinbefore defined. Further subtilisin proteasescan be of vegetable, animal or microorganism origin.Preferably it is of the latter origin, which incluclesyeast, fungi, moulds and bacteria. Particularly preferredare bacterial subtilisin type proteases, obtained frome.g. particular strains of B. subtilis and B. licheniformis. Examples of suitable commercially available proteases are Alcalase, Savinase, Esperase, ailof Novo/Nordisk A/S; Maxatase and Maxacal of Gist-Brocades; Kazusase of Showa Denko; Subtilisin BPN'proteases and so on.

The compositions of the invention may also contain enzymes 1 0056 7 such as lipases, for example Lipolase (Trade Nane exNovo/Nordisk), a lipase extracted from HumicoluLanuginosa; amylases, a commercially available material isscld under the Trade Mark Termamyl ex Novo/Nordisk; andcellulases, a commercially available material is sold asCelluzyme (Trade Mark) ex Novo/Nordisk.

Enzymes may be incorporated in the detergent compositionof the invention in the form of a concentrated aqueousliquid or a slurry.

In addition to the components described above the detergent bars of the présent invention may contain a widevariety of optional materials. For example builders suchas water-soluble phosphate salts, water soluble carbonatesand organic builders may be added. Other ingrédients suchas starch, sodium carboxymethylcellulose, colouringmaterials, fluorescers, opacifiers, germicides, perfumes,preservatives for example ethylene diamine tetra-aceticacrd (EDTA), ethane 1-hydroxy-l,1'-diphosphonic acid(EHDP) , forrnaldehyde and sodium hyphochlorite, sucrose andsorbitol may also be included.

Bars according to the invention may be prepared onconventional eguipment and in a conventional mariner. Themutant subtilisin enzyme, any other additional enzymeand the components of the stabilising System may be addedat any stage during the manufacturing process. Preferablythe stabilising components are added before the enzyme.

The resuit inc; mixture of components may then be plodded inconventional manner and, if necessary, stamped, left toâge wrapped.

The invention will now be described further and exemplified in the following examples in which percentagesare by weight. 23470 ίο 20 1 0056

Example I

Hard soap bars were produced from conventional soap (82?tallow, 18% coconut oil) produced by pan saponification.The soap was neutralised with 0.15% orthophosphoric acicand then mixed for approximately 20 minutes at atempérature of 80°C. Thereafter, the stabilising Systemwas added and mixing continued for a further hour whilethe température was maintained at 80°C. The water contentof the resulting mixture was reduced by vacuum drying tethe desired moisture content and the enzyme added. Theresulting mixture was then plodded on a soap productionline in a conventional manner.

In each case, the water content of the bars and the totalfatty mat ter content were determined and found to be inthe range 17-19% and 65-70% by weight respectively.

The bars were wrapped individually in glycine paper and/crpolyethylene bags and stored at 3 0°C. The residual enzymeactivity of bars was measured at intervals during storageusing a 5101 Scalar Aautoanalyser. 30 35

In each case, part of the bar was dissolved to form a weakaqueous solution. The enzyme activity was determined byphotometric détection of a complex formed from reaction ofthe enzyme in the solution with a protease substrate.

The results are presented below.

Enzyme activity %

Composition Storage time (deys) 14 27 41 i) enzyme containing soap bar withsodium formate(2%), borax(2%),propylene glycol(2%), sodiumsulphate (1%). enzyme : - a) Savinase* 8GU/mg(moisture 17.5%, TFM1 68.7%) 82 83 79 100 100 100 C3470 1 0056 b) Durazym" 8GU/mg (moisture 17.4%, TFM1 65.4%) ii) enzyme containing soap bar with5 sodium formate(2%), borax (2%), propylene çilycol(2%), sucrose(5%), sodium sulphate (1%).enzyme : -

a) Savinase 8GU/mg 89 9 2 85 (moisture 17.4%, TFM 64.1%) b) Durazym 8GU/mg 100 ICO ] CO (moisture 17.0 %, TFM 65.4%) iii) enzyme containing soap bar with15 borax(2%), propylene glycol{2%), sucrose (5%), sodium sulphate (1%)enzyme : -

a) Savinase 8GU/mg (moisture 18.7%, TFM1 64.8%) 87 84 7 5 20 b) Durazym 8GU/mg 100 100 ICO (moisture 18.8%, TFM1 65.6%)

Savinase 8.0L (ex Novo/Nordisk A/S)

Durazym 16.0L (ex Novo/Nordisk A/S), a mutantsubtilisin protease containing a glutamic residue et 25 position 195 and an alanine residue et position 222. 1 Total fatty matter.

The results show a bar containing the mutant subtilisinenzyme Durazym is much more stable, that is résiduelenzyme activity of the bar after storage is much higher,than a bar containing the enzyme Savinase. 30 1 0056 ίο

Example II

In this exemple she variation of residual enzyme; activity(enzyme stability) with level of the stabiliser System wasexamined.

Soap bars of compositions A and B were prepared accordingto the procedure; outlined in Example I.

Composition A % by weight B Citric acid 1 0.5 Sodium sulphate 1 0.5 Sodium Formate 2 1 Glycerol 2 1 Borax 2 1 Durazym** 5 GU/mg 0 . 3 0.3 of the bar composition Soap base (80% tallow, 20% coconut oil) Durazym 16.OL (ex Novo/Nordisk A/S)

The bars were wrapped individually in glycine paper andstored at 37°C and a relative humidity of 70. Theresidual enzyme activity of bars was measured periodicalLyduring storage.

The results are presented below. 100 56 11

Residual Enzyme Activity/% Composition A B Storage Time/Days 0 100 100 7 - 92 21 - 82 3 5 - 80 42 - 78 4 9 - 7 6 50 84 - 57 89 - 64 82 - 7 0 - 68 71 80 - 91 - 66 92 84 - 105 - 59 10 6 78 - 17 7 76 - The résulta show that even when the amount of the stabilising System is reduced the enzyme activity remain at an acceptable level. 1 0056 12

Example III

In this example the residual enzyme activity (enzymestability) of bars prepared using slurries of the mutant,subtilisin enzyme Durazym and Savinase were compared.

Soap bars of compositions C and D were prepared accordirigto the procedure; outlined in Example I.

Composition

Citric acidSodium sulphateSodium FormateGlycerolBorax

Enzyme

Durazym slurry*Savinase slurry 1 1 2 2 2 % by weight 1 2 2 2 0.3 0.3

Soap base (80% tallow, 2 0% coconut. oil)

Durazym 16.05L (Novo/Nordisk A/S)

The bars were wrapped individually in glycine paper andstored at 37°C and a relative humidity of 70%. Theresidual enzyme activity of bars was measured periodicallyduring storage.

The results are presented below.

Residual Enzyme Activity/ Composition C D Storage Time/Days 0 100 100 7 - 80 14 100 60 3 5 98 40 7 0 89 37 C347Ü 1 0056 13

The résulta show a bar containing the mutant subtilisinenzyme Durazym has a higher enzyme stability than acorresponding bar containing the enzyme Savinase.

Example IV

In this example, the cleaning performance of barscontaining Savinase and Durazym liquid enzymes werecompared. IC Soap bars of compositions E and F were prepared accordir.çr to the procedure outlined in Example I.

Composition % by weight 15 Coconut fatty acid

Sodium SulphateSodium, FormateGlycerolBorax 20 Enzyme Durazym Liquid* (5 GU/mg of the barcomposition)

Savinase Liquid**’ (5 GU/mg of the bar 25 composition) 1 1 1 2 2 0.12 1 1 1 2 2 0.12 "Durazym 16. OL (ex Novo/Nordisk A/S)‘"Savinase 16.OL (ex Novo/Nordisk A/S) 30 After manufacture, but before testing, the; bars were stored at 4°C to prevent enzyme dégradation. B3rs werestored for various lengths of time before testing.Thereafter, bars of compositions E and F were used. to wc.shstandard test cloths using a standardised procedure basée; 35 on a handwashinçj procedure commonly used with laundry bars. 1 0056 14

The test cloth was immersed in a quantity of deionisedwater. The wetted test cloth was squeezed out, reimmersedand squeezed out a second time, and then treated wi.th abai:. The bar was rubbed by hand on the cloth. The: barwas then set as.ide and the treated test cloth was immersedin deionised water, test cloth to liquor ratio of 2:.8g/Lwhich is équivalent to standard wash conditions, andrubbed 30 tirnes. The test cloth was rinsed in clean waterand dr?.ed.

The réflectance, at 460nm, of the test cloth was measuredbefore and after washing using a Micromatch RéflectanceSpectrometer. ΔΚ is the change in réflectance.

The procedure outlined above was repeated four times foreach bar composition at each storage time, and the resultspresented below represent an average value. AR460

E F

Storage Time/Days 0 17.24 19.45 6 16.87 17.07 8 16.80 16.73 10 16.77 16.38 12 16.74 16.04 2 0 16.60 14.68 3 0 16.43 12.97

The results show the increased stability of Durazymcontaining bars, gives rise to an overall performancebenefit after a storage time of only 6-8 days.

In the following examples soap bars of compositions G, H,I and J were ali prepared according to the procedureoutlined in Example !.. 1005«

Example V

In l.his examp Le the residual enzyme activity (enzyme stabidify) of a bar prepared wi th coconut. fatty acid, but in the absence of sodium formate, was examined. :ι.ο

Composition G % by weight

Coconut. fatty acid J

Sodium Sulphate 1

Sodium rormatt?

Glycero! 2

Borax 2’

Durazyrn ψ (5 GU/mg of the 0.3 bat. composition)

Soap base (80% tallow, 20 t coconut oi-1 ) ψ Durazyrn 16.0L (ex NOVO/Nordisk A/S)

20 The bar was wrapped in glycine paper and stored at 37 °C and a relative humidity of 70.. The residual enzymeactivity of the bar was measured periodically duringstorage. 25 The resuits are presented

Composition

St. orage Time/Days0 3 0 I 3 2CI4 08 0 below.

Residual Enzyme Activity/% 100 100 100 83 81

Example VI

In this example residual enzyme activity (enzyme stability) of a bar prepared with phosphoric acid v/as examined. 10056 16

Composition H % by weight

Phosphoric Acid 0.15

Sodium Sulphate 1

Sodium Formate 2

Propylene Glycol 2

Borax 2

Durazym Φ (5 GU/mg of the 0.3 bar composition)

Soap base (80% tallow, 20% coconut oil) ψ Durazym 16.0L (ex NOVO/Nordisk A/S)

The bar was wrapped in glycine paper and stored at 28°Cand a relative humidity of 70. The residual activity ofthe bar was measured periodically during storage.

The results are presented below.

CompositionStorage Time/Days 0 14 27 41

Residual Enzyme Activity/% 10 0100100100

Exemple VU. ,T.n this exemple the residual enzyme activity (enzyme stabil.it y) of bers prepared wi th and without Sodium

Formate was examined. t 0056

Composition I J % by weight

Ci trio acid 1 1

Glycerol 6 6

Sodium F'ormate! 2

Borax 2 2

Durazyiri =)= (5 GU/mg of the 0.3 0.3 lia r coinpos i t ion

Soap base (80% tallow, 2 0%; coconut oi 1 i 4: Durazym 16.0L (ex NOVO/Nordisk A/S)

The bars were wrapped individually in glycine paper and20 stored at 37°C and a relative humidity of 70. The residual enzyme activity of the bars were measuredperiodieally during storage.

The résulta aie presented below.2 5

Composation Residual Enzyme Ac:tivity/% Storage Tiine/Pays I J u 10 0 100 L 0 100 100 3 0 2 4 100 100 3 0 100 100 7 1 89 7 2 - 92 85 3 t- .j .J 107 82 - C347 0 î 1)056 18

The results demonstrate that even in the absence of sodiumformate good resrdual enzyme activity is maintained.

Claims (9)

1. Λ A 70 1 0 0 5 6 J1 a i ms L. A detergent composition in shaped solid form comprising a detergent. acti ve, water, and a mutantsubtilisin enzyme in which the amino acid sequencebas been altered at positions 195 and 222 bysubstitution with another amino acid.
2. 2. A detergent composition according to Claim 1 further LO comprising a stabilising System for tb.e enzyme, the stabilising system comprising a boron compound and a poiyol.
3. 3. A detergent composition according to Claim 2 wherein 15 the stabilising System further comprises at least one component selected f rom 1) an organic acid, an alkali métal sait of anorganic acid, an inorganic acid and mixtures 20 therecl:; and ii) an alkali métal sait of an inorganic acid, notbeing a boron compound. 25
4. 4. A detergent composition according to Claim 1 wherein the enzyme contains a glutamic acid residue atposition 195 and an alanine residue at position 222.
5. 5. A detergent composition according to Claim 1 wherein50 the composition contains the enzyme in an amount such that il corresponds with a proteolytic activity ofG1. I to 100 GU/mg based on l.he composition.
6. 6. A detergent composition according to Claim 155 comprising 25 to 80% by weight of soap or a mixture of soap and synthetic detergent active, 10 to 30% by 1 0 056 20 weight of water and 0.1 to 3 0% by weight of fi.Ller.
7. 7 . A detergent composition according to Claim 3 whereinthe boron compound is borax; the polyol is selectedfrom propylene glycol, glycerol and sorbitol; theorganic acid is selected from formic acid, succinicacid, adipic acid, glutaric acid, citric acid, soapacids and mixtures thereof; the alkali métal sait ofthe organic acid is selected from sodium formate,sodium citrate and sodium propionate; the inorganicacid is phosphoric acid; and the alkali métal sait ofthe inorganic acid is selected from sodium sulphate,sodium chloride, sodium carbonate, sodium bicarbonateand sodium phosphate.
8. 8. A detergent composition according to Claim 3 whereinthe boron compound is présent in an aiaount from 0.1to 10% by weight; the polyol is présent in an amountfrom 0.1 to 15% by weight; the organic acid, saitthereof, or inorganic acid is présent in an amountfrom 0.1 to 10% by weight; and the alkali métal saitof the inorganic acid is présent in an amount from0.1 to 5% by weight of the composition.
9. 9. Use of a detergent composition according to anypreceding cLaim to treat a stained fabric.