WO1996034936A1

WO1996034936A1 - Detergent compositions

Info

Publication number: WO1996034936A1
Application number: PCT/EP1996/001743
Authority: WO
Inventors: Christophe Andre; Lucien Charmoille; Gerhard Konieczny-Janda
Original assignee: Genencor International, Inc.
Priority date: 1995-05-05
Filing date: 1996-04-26
Publication date: 1996-11-07
Also published as: EP0823938A1; ZA963285B; BE1009312A3; AU5761996A; CA2220089A1; AR001827A1

Abstract

The invention relates to detergent compositions containing a lipase and at least one surfactant. More particularly, it relates to a detergent composition containing a lipase produced by a strain of Pseudomonas. The invention also relates to the utilization of such detergent compositions and to a surface-active composition usable in a detergent composition.

Description

Detergent compositions

The invention relates to detergent compositions, and more particularly detergent compositions containing a lipase and at least one surfactant, and the use of these detergent compositions.

Detergent compositions containing a lipase and one or more surfactant (s) are known. Such compositions have, in particular, been described in European patent application 0 430 315, European patent application 0 341 999, international patent application WO 94/03578, international patent application WO 94/25556 and international patent application WO 91/00910. In particular, international patent application WO 94/25556 describes a detergent composition which can be used for washing dishes. This detergent composition contains various enzymes (protease, amylase), a silicate, a polyacrylate copolymer, fillers, bleaching agents, stabilizing agents and surfactants. The surfactants used, which can be nonionic, anionic or amphoteric, must be compatible with the enzymes contained in the detergent composition. Among the nonionic surfactants, mention may be made of condensation products of one mole of an alcohol or of a fatty acid containing from 10 to 20 carbon atoms with 4 to 40 moles of ethylene oxide. Among the anionic surfactants, mention may be made of soluble alkyl sulfates and alkyl sulfonates containing from 8 to 18 carbon atoms. Although lipases have been mentioned in this patent application, no composition containing a lipase is exemplified.

Although the detergent compositions containing a lipase and a surfactant, described in the patent applications cited above, have utility in certain aspects, they seem to have insufficient efficacy. Indeed, either the lipases contained in these compositions are not very stable, or their washing performance is poor. It seems in fact that certain surfactants decrease, or at least do not contribute to, the stability and / or the washing performance of the enzymes, and in particular the lipases, present in these detergent compositions.

The incorporation of a lipase, produced by a strain of Pseudomonas, in a detergent composition, seems to increase the effectiveness of these compositions during washing.

However, to our knowledge, it seems that no detergent composition containing a lipase, derived from a strain of Pseudomonas, and a surfactant is known.

Consequently, there is currently a need for such a detergent composition containing a lipase which can be used in the detergency field, which is very stable and also very active in a wide range of pH and temperature. It is therefore necessary, on the one hand, to identify the surfactants, or mixtures of surfactants, which contribute to, or at least do not decrease, the stability of the lipases present in the detergent compositions, and this in particular for the lipases produced by Pseudomonas strains. This is why it is necessary to supply and prepare detergent compositions containing such surfactants, or such mixtures of surfactants, and a lipase, and more particularly a lipase produced by a strain of Pseudomonas.

In addition, there is also a need for a detergent composition, containing a lipase, which is particularly effective during washing on oily stains and this from the first washing cycles. In addition, there is also a need for a detergent composition, containing a lipase, which is particularly effective during washing at low temperatures, that is to say temperatures up to 5 ° C.

It is also necessary, on the other hand, to identify the surfactants, or mixtures of surfactants, which contribute to, or at least do not decrease, the washing performance of the lipases present in the detergent compositions, and this in particular for lipases produced by Pseudomonas strains. Therefore, it is necessary to provide and prepare detergent compositions containing such surfactants, or such mixtures of surfactants, and a lipase, and more particularly a lipase produced by a strain of Pseudomonas.

The object of the present invention is to identify the surfactants, and the mixtures thereof, which included with a lipase in a detergent composition, make it possible to provide a very stable detergent composition. More particularly, the object of the present invention is to identify the surfactants, and mixtures thereof, which, included with a lipase produced by a strain of Pseudomonas, in a detergent composition, make it possible to provide a very stable detergent composition.

The present invention aims to identify surfactants, and mixtures thereof, which included with a lipase in a detergent composition, make it possible to provide a detergent composition having a high washing performance. More particularly, the present invention aims to identify the surfactants, and mixtures thereof, which, included with a lipase produced by a strain of Pseudomonas, in a detergent composition, make it possible to provide a detergent composition having high washing performance.

The present invention aims to identify surfactants, and mixtures thereof, which, included with a lipase in a detergent composition, make it possible to provide a detergent composition active in a wide range of temperature, active in a wide range of alkaline pH, and effective on oily stains from the first washing cycle.

More particularly, the present invention aims to identify surfactants, and mixtures thereof, which, included with a lipase produced by a strain of Pseudomonas in a detergent composition, make it possible to provide a detergent composition which is active in a wide range temperature range, active in a wide range of alkaline pH, and effective from the first washing cycle, and particularly effective on oily stains from the first washing cycles.

The object of the present invention is to identify the surfactants, and the mixtures thereof, which, included with a lipase in a detergent composition, make it possible to provide a detergent composition which is particularly effective during washing at low temperatures, c that is to say temperatures up to 5 ° C.

The present invention also aims to prepare and provide a new detergent composition, containing a lipase and at least one surfactant, which is very stable in the presence of surfactants. More particularly, the present invention also aims to identify and provide a new detergent composition, containing a lipase, derived from a strain of Pseudomonas, and at least one surfactant, which is very stable in the presence of surfactants. The present invention also aims to prepare and provide a new detergent composition, containing a lipase and at least one surfactant, and which has a high washing performance. More particularly, the present invention aims to prepare and provide a detergent composition, containing a lipase derived from a strain of Pseudomonas and at least one surfactant, which has a high washing performance.

To this end, the present invention relates to the identification of surfactants which are particularly effective when used with a lipase in a detergent composition. More particularly, the present invention relates to the identification of surfactants which are particularly effective when used with a lipase produced by a strain of Pseudomonas in a detergent composition.

Thus, the surfactant compositions according to the present invention, which have given excellent results for this purpose, consist of a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxy¬ lation of 9; and of an anionic surfactant chosen from the alcoholic sodium ethoxy sulphate salts containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2. The detergent compositions containing a lipase, and more particularly a lipase produced by a strain of Pseudomonas, and a surfactant composition as described above, have given excellent results.

The present invention also relates to the identification of surfactants which make it possible to prepare a stable detergent composition containing a lipase. And more particularly, the present invention relates to the identification of surfactants which make it possible to prepare a stable detergent composition containing a lipase produced by a strain of Pseudomonas.

Thus, the surfactants which have given good results are polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of between 3 and 9. The detergent compositions containing a lipase, and more particularly a lipase produced by a strain of Pseudomonas, and a surfactant as described above, have given excellent results.

The present invention also relates to the identification of surfactants which make it possible to prepare a detergent composition having a high washing performance and containing a lipase. And more particularly The present invention relates to the identification of surfactants which make it possible to prepare a detergent composition having a high washing performance and containing a lipase produced by a strain of Pseudomonas. Thus, the surfactants which have given good results, are the polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9. The surfactants which have also given good results, are a mixture comprising a surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9, and a surfactant chosen from sodium alcoholic ethoxy sulfate salts having from 12 to 14 carbon atoms and a degree of ethoxylation equal to 2. The detergent compositions containing a lipase, and more particularly a lipase produced by a strain of Pseudomonas, and a surfactant as described above, have given excellent results.

The present invention also relates to a detergent composition containing a lipase produced by a strain of Pseudomonas and at least one surfactant.

The detergent composition according to the invention contains at least one nonionic surfactant.

Generally, the detergent composition according to the invention contains at least one nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from approximately 10 to approximately 20 carbon atoms and having a degree of ethoxylation of between approximately 2 and approximately 40. The Polyethoxylated aliphatic alcohols are also known as ethoxylates (Ullman's Encyclopedia of Industrial Chemistry, 1994, 5th edition, Vol A25, pages 785-787) or ethoxylated fatty alcohols.

Usually, the detergent composition according to the invention contains at least one nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from approximately 10 to approximately 18 carbon atoms and having a degree of ethoxylation of between approximately 2 and approximately 20. De preferably, the detergent composition according to the invention contains at least one nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from approximately 12 to approximately 14 carbon atoms and having a degree of ethoxylation of between approximately 3 and approximately 11. From in a particularly preferred way, the detergent composition according to the invention contains at at least one nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of between about 3 and about 9. Good results have been obtained with a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about

14 carbon atoms and having a degree of ethoxylation of 3, such as the product sold under the trademark MARLIPAL 24/30 by HULS AG; polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 6, such as the product sold under the trademark MARLIPAL 24/60 by HULS AG; polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 7, such as the product sold under the trademark MARLIPAL 24/70 by HULS AG; and polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9 such as the product sold under the trademark MARLIPAL 24/90 by HULS AG.

Excellent results have been obtained with a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9. The detergent composition according to the invention may also contain at least an anionic surfactant.

Generally, the detergent composition according to the invention contains at least one anionic surfactant chosen from the alkyl sulfate salts containing from 8 to 24 carbon atoms and the alcoholic ethoxysulfate salts containing from 6 to 20 carbon atoms and having a degree of ethoxylation between about 2 and about 20.

The alkyl sulfate salts are also known as the salts of the sulfuric acid esters (Ullman's Encyclopedia of Industrial Chemistry, 1994, 5th edition, Vol A25, pages 779-780). The salts of alcoholic ethoxysulphates are also known by the name of ethersulphates, alcohol ether sulphate or salts of esters of sulfuric acid and ethers of oligooxyethylenes or ethoxylates (Ullman's Encyclopedia of Industrial Chemistry, 1994, 5th edition, Vol A25 , pages 781-782) or sulfates of ethoxylated fatty alcohols. Usually, the detergent composition according to the invention contains at least one anionic surfactant chosen from the alkyl sulfate salts containing from 12 to 22 carbon atoms and the salts of alcoholic ethoxysulfates containing from 8 to 18 carbon atoms and having a degree of ethoxylation of between approximately 2 and approximately 16. Preferably, the detergent composition according to the invention contains at least one anionic surfactant chosen from sodium alkyl sulphates containing from 14 to 20 carbon atoms and alcoholic ethoxysulphate salts (sodium salt) containing from 10 to 20 carbon atoms and having a degree of ethoxylation of between approximately 2 and approximately 8. In a particularly preferred manner, the detergent composition according to the invention contains at least one anionic surfactant chosen from sodium alkyl sulphates containing from 16 to 18 carbon atoms and the salts of alcoholic ethoxysulphates (sodium salts) containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2. Good results have been obtained with sodium alkylsulfate containing from 16 to 18 atoms carbon, sold under the brand MARLINAT T55 by HULS AG.

Very good results have been obtained with the alcoholic sodium ethoxysulfate salt containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2, sold under the trademark MARLINAT 242/70 by HULS AG. Excellent results have been obtained with a detergent composition containing a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9; and an anionic surfactant chosen from the alcoholic sodium ethoxysulfate salt containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2.

Lipases are classified in the international system under number E.C. 3.1.1.3 .; it is a glycerol ester hydrolase.

The lipases which are suitable for the invention can be of various origins. In general, the detergent composition according to the invention contains a microbial lipase. Usually, the detergent composition according to the invention contains a fungal or bacterial lipase.

Among the bacterial lipases, mention may be made of lipases derived from aerobic bacteria, such as in particular the strains of Bacillus, in particular a strain of Bacillus pumilus, the strains of Staphylococcus, the strains of Acinetobacter and the strains of Pseudomonas. Preferably, the detergent composition according to the invention contains a lipase derived from a strain of Pseudomonas, such as in particular strains of Pseudomonas wisconsinensis, mendocina, stutzeri, cepacia, putida, aeroginosa, fluorescens, fragi, nitroreducans, gladioli, plantari, glumae, pseudoalcaligenes, alcaligenes. Good results have been obtained with a detergent composition containing a lipase derived from a strain of Pseudomonas wisconsinensis. Excellent results have been obtained with the strain of Pseudomonas wisconsinensis T 92.677 / 1 or a derivative or mutant of this strain.

The strain of Pseudomonas wisconsinensis T 92.677 / 1 was deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS (LMG culture collection, University of Ghent, Laboratory of Microbiology - KL Ledeganckstraat 35, B-9000 Ghent, Belgium) in accordance with the Budapest Treaty under the number LMG P-15151 on October 12, 1994. Among the fungal lipases, mention may be made of lipases derived from fungi, such as in particular the strains of Aspergillus, the strains of Rhizopus, the strains of Fusarium, the strains of Mucor, Penicillium strains, Candida strains, Humicola strains and Thermomyces strains. Preferably, the detergent composition according to the invention contains a lipase derived from a strain of Aspergillus or from a strain of Humicola.

The lipase and surfactant contents in the detergent composition strongly depend on the intended use of this composition.

The detergent compositions according to the invention usually comprise at least 0.001% (w / w) of lipase. Generally, they comprise at least 0.003% (w / w) lipase. Preferably, they comprise at least 0.005% (w / w) of lipase. The abbreviation% (w / w) represents a percentage expressed in weight by weight.

The detergent compositions according to the invention usually comprise at most 5% (w / w) lipase. Generally, they comprise at most 4% (w / w) of lipase. Preferably, they comprise at most 3% (w / w) of lipase.

The detergent compositions according to the invention usually comprise at least 0.001% (w / w) of surfactants. Generally, they comprise at least 0.01% (w / w) of surfactants. Preferably, they comprise at least 0.1% (w / w) of surfactants. The detergent compositions according to the invention usually comprise at most 60% (w / w) of surfactants. Generally, they comprise at most 50% (w / w) of surfactants. Preferably, they comprise at most 40% (w / w) of surfactants. The preparation of the detergent compositions according to the invention can be carried out in any manner known per se.

The lipase can be incorporated into the detergent composition in solid form or in liquid form. In particular, it can be incorporated in the form of a suspension or a granule. The present invention also relates to the use of the detergent composition containing a lipase, as defined above. An example of use in detergents is described in particular in British patent application 1372034 and in European patent application 0 218 272. The multiple additives of the detergent compositions are known to those skilled in the art and are adapted according to the intended use. of the composition. Such additives are in particular enzymes, such as for example proteases, amylases and / or cellulases; bulking agents, such as sodium tripolyphosphate; bleaching agents, such as perborate; peroxide compounds; formulation additives; surfactants. Examples of such additives, added to detergent compositions, are in particular described in the British patent application 1372034, in the European patent application 0 218 272, in the European patent application 0 430 315, the European patent application 0 341,999, international patent application WO 94/03578, international patent application WO 94/25556 and international patent application WO 91/00910.

These additives must be compatible with the lipase and the surfactants contained in the detergent composition. In particular, they must be chemically inert with respect to lipase and surfactants at normal temperatures of use and storage of the detergent composition according to the invention. The nature of the additives and their content in the composition must also be chosen so as not to significantly affect the action of the lipase.

The detergent compositions according to the invention may also contain other substances chosen according to the special field of application of the detergent composition. Among these, mention may be made of optical brighteners, tarnish inhibitors, anti-epileptic agents dirt, disinfectants, corrosion inhibitors, perfumes, dyes, pH regulating agents.

The detergent compositions of the invention can be used, depending on their formulation, as washing powder, granule or liquid for washing clothes; as a stain remover for detaching or degreasing objects or detaching laundry prior to cleaning; and as a powder, granule or liquid for washing dishes.

The present invention is illustrated by the following examples. Example 1 Production of the lipase by the strain of Pseudomonas wisconsinensis T 92.677 / 1.

The Pseudomonas wisconsinensis T 92.677 / 1 strain, deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS (LMG culture collection) under the number LMG P-15151 on October 12, 1994, is cultured in 25 ml of LB medium at 30 °. C for 12 hours.

The LB medium contains 10 g / l of tryptone (DLFCO), 5 g / l of yeast extract and 10 g / l of NaCl. The pH is adjusted to 7.0 with 0.1N NaOH. The medium is sterilized at 123 ° C for 30 minutes. The culture is performed at 30 ^° C with orbital shaking at a rate of

200 revolutions per minute with an amplitude of about 2.54 cm.

Then, starting from this culture, a culture is carried out in 40 l of LB liquid medium contained in a fermenter with a total capacity of 72 1. The pH of the medium is adjusted to 7.0 with 0.1N NaOH, the medium is sterilized at 123 ° C for 30 minutes. The culture is carried out at 30 ° C. with axial stirring at the rate of 150 revolutions per minute and under aeration of 7 l of air per minute, the regulation of dissolved oxygen being fixed at 10% (v / v) by regu¬ agitation speed.

In this request, the abbreviation% (v / v) represents a percentage expressed in volume by volume. The abbreviation% (v / p) represents a percentage expressed by volume by weight. The abbreviation% (w / v) represents a percentage expressed by weight per volume. The abbreviation% (w / w) represents a percentage expressed in weight by weight.

After 16 hours of incubation, this culture is introduced into a 0.75 m ³ fermenter containing 0.4 m ³ of sterilized liquid medium D. Medium D contains K ₂ HPO ₄ 2.5 g / 1, KH ₂ PO ₄ 2.5 g / 1, MgSO ₄ .7H ₂ 0 1 g / 1, (NH4) ₂ Sθ4 2 g / 1, (NH ₂ ) ₂ CO 2 g / 1, CaCl ₂ 1 g / 1, soy flour 20 g / 1, yeast extract 2 g / 1, glucose 20 g / 1, antifoam

Mazuôl (MAZES CHEMICALS) 5 g / 1. The pH is adjusted to 7.4 (with normal phosphoric acid and normal caustic soda) before and after sterilization in the fermenter. Glucose is sterilized, apart, at pH

3.5 (pH adjusted with normal sulfuric acid) for 30 minutes at

123 ° C. The medium is sterilized in the fermenter for 30 minutes at 123 ° C. The culture in the fermenter is carried out at a temperature of 23 ° C, under a pressure of 0.15.10 ^ Pa (Pa = Pascal) (0.15 bar), with an aeration of 4 m ³ / h, with axial stirring 300 revolutions per minute, the dissolved oxygen regulation being fixed at 10% (v / v) by regulating the stirring speed.

After 24 hours of fermentation, the enzyme activity of the culture thus obtained is measured by the following technique. The hydrolysis of triolein is quantified by the neutralization of the fatty acids released under the action of lipase. This measurement is carried out using an automatic titration apparatus, which maintains the pH constant at a set value by the addition of 0.01 N NaOH.

A lipase unit (LU) is defined as the quantity of enzyme which catalyzes the release of one micromole of fatty acid per minute under the standard conditions of the test described below.

10 g of Triolein (ROTH 5423.1) and 10 g of gum arabic (FLUKA 51200) are mixed in 100 ml of distilled water. This mixture is emulsified using an ULTRA-TURRAX mixer at 13,500 rpm (axial stirring) 3 times for 5 minutes, keeping the mixture under nitrogen and in an ice bath.

A dilution buffer is prepared containing 2.34 g / l of NaCl,

2.94 g / 1 CaCl ₂ .2H ₂ 0 and 0.61 g / 1 Tris (2-amino-2-hydroxy-methyl-1, 3-propanediol). An automatic titration apparatus is used, equipped with a burette containing 0.01N NaOH, a temperature probe and a pH probe, and equipped with a thermostatically controlled reactor.

Into the reactor thermostatically controlled at 30 ° C., a magnetic stir bar, 10 ml of triolein emulsion and 20 ml of dilution buffer are introduced. The pH of the solution thus obtained is adjusted to 9.5 with

0.1N NaOH. Then, 0.5 ml of the test sample containing the lipase, the sample is optionally diluted so that it contains only a maximum of 5 LU. The pH is checked for the first two minutes with 0.01N NaOH. Then, the soda consumption is recorded between 2 and 4 minutes, while keeping the pH constant (volume of soda consumed between 2 and 4 minutes = VI in ml).

Then, the same test is carried out, replacing the sample containing the lipase with 0.5 ml of dilution buffer (volume of soda consumed between 2 and 4 minutes = V2 in ml).

A lipase unit (LU) is determined as follows: 1 LU / ml = (V1-V2) x possible dilution of the sample x 10

By this method, lipase activity is detected in the culture. Example 2 Preparation of a concentrated lipase solution.

The culture, obtained in Example 1, is cooled to a temperature of 15 ° C. The pH of the culture is adjusted to a pH of approximately 9 with concentrated 10N caustic soda.

Then, 1% (v / v) of Triton X-114 (UNION CARBIDE) is added to this culture. The mixture is stirred gently (axial agitation of 50 revolutions per minute) for 2 hours at 15 ° C. Next, 0.5% (v / w) of OPTIFLOC FC205 is added to the mixture.

(SOLVAY) in the form of a 10% (v / v) solution. The mixture is stirred gently (axial agitation of 50 revolutions per minute) for 1 hour at 15 ° C.

Then, to this mixture, maintained at 15 ° C, 4% (w / w) of CELATOM FW6 product (EAGLE-PICHER) is added. The mixture is stirred gently (axial agitation of 50 revolutions per minute) for 15 minutes at 15 ° C.

The mixture is passed through a WELDERS filter press fitted with plates carrying MARSYNTEX PPD2427 membranes, limiting the inlet pressure to 4.1 (r Pa (4 bar). A clear filtrate is recovered. Then, this filtrate is concentrated to using ultrafiltration cartridges

AMICON S120, having a cut-off characteristic of 10 kD.

Then 30% (w / w) NaCl (SOLVAY) is added to the ultrafiltrate. The mixture is stirred and heated at 40 ° C for 5 minutes. The ultrafiltrate obtained is transferred to a 10 1 separating ball. It is cooled to 4 ° C. Then, it is left to stand for 16 hours at 4 ° C. The upper phase is then recovered by eliminating the lower phase of the ball at decant.

20% (v / w) of n-butanol (JANSSEN 23.208.25) is added to the upper phase. The mixture is stirred.

The mixture is centrifuged for 10 minutes at 8500 rpm (BECKMAN J21, rotor JA10) at a temperature of 4 ° C. The centrifugation precipitate is kept.

The centrifugation precipitate is dissolved in 5 l of a buffer F solution containing 40% (v / v) of propylene glycol, 3 mM Tris, 12 mM CaCl ₂ and 24 mM NaCl. The mixture is centrifuged for 1 hour at 8500 rpm

(BECKMAN J21, rotor JA10) at a temperature of 4 ° C. The centrifugation supernatant is kept.

50% (v / v) acetone is added to the centrifugation supernatant. The acetone suspension is left to stand for 1 hour at 4 ° C. Then, the suspension is centrifuged for 10 minutes at 8500 rpm

Acetone is added to the centrifugation supernatant at 4 ° C until an acetone concentration of 80% (v / v) is obtained. The mixture is left to stand for 1 hour at 4 ° C.

Then, the mixture is centrifuged for 10 minutes at 8500 rpm (BECKMAN J21, rotor JA10) at a temperature of 4 ° C. The centrifugation precipitate is kept. This precipitate is suspended in 1 l of the buffer solution F. Then, the suspension is centrifuged, containing the precipitate, 1 hour at

8500 rpm (BECKMAN J21, rotor JA10) at a temperature of 4 ° C.

The centrifugation supernatant is stored, which forms a concentrated solution of lipase. Example 3

Purification of lipase.

To purify the concentrated lipase solution, as obtained in Example 2, the purification technique using hydrophobic interaction chromatography is used. By following the directions for use prescribed by the supplier

(PHARMACIA) about the interaction chromatography column hydrophobic, a PHARMACIA Phenyl-Sepharose Fast column is loaded

Flow (High Sub - ref 17-0973-05) with a dilute lipase solution. The concentrated lipase solution, as obtained in Example 2, is diluted with

4 volumes of buffer containing 3 mM Tris, 12 mM CaCl ₂ and 24 mM NaCl. As a balance buffer, a buffer containing 50% is used

(v / v) propylene glycol, 3 mM Tris, 12 mM CaCl ₂ and 24 mM NaCl. The flow rate is fixed at 5 ml per minute.

The lipase is recovered with the fraction eluted. Purity is around

45%. Then, the fraction, containing the lipase, is concentrated up to 50 ml by ultrafiltration using an AMICON cell, equipped with a membrane.

YM10.

Example 4

Preparation of a detergent composition containing lipase. A liquid detergent composition containing 1 mg / 1 of a lipase having an enzymatic activity of approximately 9600 LU / ml is prepared, and a mixture of additives.

Lipase is used as obtained in Example 3, which is diluted with water of hardness (water containing 1.98 mM calcium chloride, 0.69 mM magnesium chloride and sodium bicarbonate

2.5 mM), in order to obtain the desired concentration.

The mixture of additives used contains 0.5 g / l of LAS, Na

(MARLON A350, C10-C13 sodium alkylbenzene sulfonate having from 10 to

13 carbon atoms); 0.02 g / 1 soap; 0.6 g / 1 of FAEO (9EO) [product sold under the brand name MARLIPAL 24/90, polyethoxylated aliphatic alcohol having 12 to 14 carbon atoms and a degree of ethoxylation equal to

9 (ethoxylate) (number of EO (ethylene oxide) group equal to 9)];

0.06 g / l propanediol; 0.12 g / 1 ethanol; 0.12 g / 1 sodium citrate;

0.025 g / 1 of phosphonate [diethylene triamine penta (methylenephosphonic acid)].

The initial pH of the liquid detergent composition is adjusted to approximately

9.5 with NaOH (1 mole / 1).

Example 5

Preparation of a detergent composition. Example 4 is reproduced, with the exception of the formulation of the mixture of additives. In this example, the mixture of additives used contains 0.25 g / l of LAS, Na (MARLON A350); 0.02 g / 1 soap; 0.8 g / 1 of FAEO (9EO) (MARLIPAL 24/90); 0.25 g / 1 FAES (MARLINAT 242/70); 0.06 g / l propanediol; 0.12 g / 1 ethanol; 0.12 g / 1 sodium citrate; 0.025 g / 1 of phosphonate [diethylene triamine penta (methylene-phosphonic acid)]. Example 6 Use of a detergent composition.

A piece of white fabric (R. HOPPE Gmbh) is prepared based on cotton (65%) and polyester (35%) of 10 cm by 10 cm, impregnated with bacon fat and Sudan red. The impregnation of the fabric is carried out as follows.

A homogeneous solution of Sudan red 7B (SIGMA cat. No. F 1000) and bacon fat (LARU Gmbh) is prepared by adding 0.1% (w / w) of Sudan red to the bacon fat. The mixture is heated to 90 ° C. until the Sudan red is completely dissolved. A roll of fabric is immersed in the solution of Sudan red and bacon fat kept at 90 ° C and continuously moved into this solution at the speed of 0.5 m / minute. Then, the fabric is spun under constant linear pressure between two rollers.

The fabric thus impregnated contains 31.5% (w / w) of fat. The impregnated fabric is placed at -18 ° C for 22 hours until the fat has completely crystallized. The fabric is cut into 10 cm by 10 cm pieces. Then, the pieces of impregnated fabric are stored at -18 ° C in the dark.

Then, the fabric impregnated with bacon fat and Sudan red is washed with 200 ml of the liquid detergent composition, as obtained in Example 4, in a 250 ml reactor. Washing takes place at 10 ° C for 25 minutes with stirring at 40 revolutions per minute (back and forth stirring for 20 seconds). After washing, the fabric is rinsed three times with

200 ml of water with a hardness of 15, then dried at 22 ° C between two papers for 20 hours.

Next, the reflectance (RL) at 560 nm of the dried fabric is determined using a colorimeter (TRICOLOR LFM 3), the measured value (Y) being converted into a value L = 10 / Y which corresponds to the reflectance.

We repeat this test three times, i.e. we then perform three both the washing cycle with the liquid detergent composition containing the lipase and the rinsing cycle on the same tissue sample without impregnating it with Sudan red and bacon fat between each cycle. At each end of the cycle, the reflectance at 560 nm of the dried fabric is determined.

A strictly identical test is carried out with a detergent composition, as obtained in Example 4, but in which the lipase has not been added. At each end of the cycle, the reflectance (RO) at 560 nm of the dried fabric is determined.

Washing performance in% is defined as the difference between the reflectance (RL) obtained with washing in the presence of lipase and the reflectance (RO) obtained with washing in the absence of lipase, i.e. RL - RO expressed in%.

The results obtained are collated in Table 1.

Table 1

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 26.16 1 28.17

2 0 56.85 1 66.53

3 0 69.57 1 78.31

4 0 78.52 1 86.73

The results of this example show that the detergent composition of the invention is effective at a relatively low temperature and in particular at 10 ° C. Example 7 Use of a detergent composition.

The protocol described in Example 6 is followed using the detergent composition as obtained in Example 5.

The results obtained are collated in Table 2. Table 2

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 6.81 1 8.23

2 0 18.67 1 63.04

3 0 29.96 1 79.18

4 0 58.3 1 88.29

The results of this example show that the detergent composition of the invention is effective at a low temperature and in particular at 10 ° C. Example 8 Use of a detergent composition containing lipase.

Example 7 is repeated, with the exception of the washing temperature and the amount of lipase used.

In this example, the washing takes place at 30 ° C. and 2 mg / 1 of lipase having an enzymatic activity of 9600 LU / 1 is used.

The results obtained are collated in Table 3.

Table 3

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 41.12 2 64.22

2 0 52.86 2 76.68

3 0 62.2 2 91.23

4 0 72.82 2,100

The results of this example show that the detergent composition of the invention is effective from the first washing cycle. Example 9

Preparation of a detergent composition. An aqueous solution is prepared containing 290.8 mg / 1 of calcium chloride CaCl ₂ , 2 H2O; 140 mg / 1 of magnesium chloride MgCl ₂ , 2 H ₂ O and 210 mg / 1 of sodium carbonate NaHC03-

To this aqueous solution, a surfactant FAEO (3EO) [product sold under the brand MARLIPAL 24/30 by HULS AG (Mari, Germany), a polyethoxylated aliphatic alcohol having 12 to 14 carbon atoms and an equal degree of ethoxylation is added. to 3 (ethoxylate) (number of EO (ethylene oxide) group equal to 3)], at a concentration of 0.5 g / 1.

To this aqueous solution containing the surfactant FAEO (3EO), a lipase concentration equivalent to 20 LU / 1 is added. Lipase is used as obtained in Example 3, which is diluted with water of hardness 15 in order to obtain the desired concentration.

The pH of the composition obtained is adjusted to a pH of 9.5 with NaOH (1 mole / 1). The enzymatic activity of this detergent composition is measured immediately. The method for measuring enzyme activity is described in example 2.

Example 10

Preparation of a detergent composition.

Example 9 is reproduced, replacing the FAEO surfactant (3EO) with the FAEO surfactant (6EO) [product sold under the brand

MARLIPAL 24/60 by HULS AG (Mari, Germany), polyethoxylated aliphatic alcohol having 12 to 14 carbon atoms and a degree of ethoxylation equal to 6 (ethoxylate) (number of group EO (ethylene oxide) equal to

6)], at a concentration of 0.5 g / 1. Example 11

Preparation of a detergent composition.

Example 9 is reproduced, replacing the surfactant FAEO (3EO) with FAEO (7EO) [product sold under the brand MARLIPAL 24/70 by

HULS AG (Mari, Germany), polyethoxylated aliphatic alcohol having 12 to 14 carbon atoms and a degree of ethoxylation equal to 7 (ethoxylate) -

(number of group EO (ethylene oxide) equal to 7)], at a concentration of 0.5 g / 1.

Example 12

Preparation of a detergent composition. Example 9 is reproduced, replacing the FAEO surfactant (3EO) with the FAEO surfactant (9EO), [product sold under the brand

MARLIPAL 24/90 by HULS AG (Mari, Germany), polyethoxylated aliphatic alcohol having from 12 to 14 carbon atoms and a degree of ethoxylation equal to 9 (ethoxylate) (number of group EO (ethylene oxide) equal to 9 )] at a concentration of 0.3 g / 1.

Example 13

Preparation of a detergent composition.

Example 9 is reproduced, replacing the surfactant FAEO (3EO) by the surfactant LAS, Na [product sold under the brand MARLON A350 by HULS AG (Mari, Germany), sodium alkylbenzene sulfonate having 10 to 13 carbon atoms ], at a concentration of 0.5 g / 1.

Example 14

Preparation of a detergent composition.

Example 9 is reproduced, replacing the FAEO surfactant (3EO) by the FAES surfactant [product sold under the brand MARLINAT 242/70 by HULS AG (Mari, Germany), the alcoholic ethoxysulfate salt of sodium having 12 to 14 carbon atoms and a degree of ethoxylation equal to 2], at a concentration of 0.5 g / 1. Example 15

Preparation of a detergent composition. Example 9 is reproduced, replacing the surfactant FAEO (3EO) by the surfactant PAS, Na [product sold under the brand MARLINAT T55 by HULS AG (Mari, Germany), sodium alkyl sulfate having from 16 to 18 carbon atoms] , at a concentration of 0.5 g / 1. Example 16 Preparation of a detergent composition.

Example 9 is reproduced, replacing the FAEO surfactant (3EO) with a mixture of two surfactants:

- the FAES surfactant, at a concentration of 0.5 g / 1; and

- the surfactant FAEO (9EO), at a concentration of 0.5 g / 1. Example 17

Preparation of a detergent composition.

Example 9 is reproduced, replacing the FAEO surfactant (3EO) with a mixture of three surfactants:

- the surfactant LAS, Na, at a concentration of 0.25 g / 1; - the FAES surfactant, at a concentration of 0.25 g / 1; and

- the surfactant FAEO (9EO), at a concentration of 0.5 g / 1. Example 18

Stability of detergent compositions.

The stability of the detergent compositions as obtained in Examples 9 to 17 is tested.

The detergent compositions, as obtained in Examples 9 to 17, are incubated for 45 minutes at 35 ° C. Then, the enzymatic activity of the detergent compositions incubated is measured.

The results are expressed as% of the residual enzymatic activity based on the activity of the lipase theoretically added. The results are collated in Table 4. Table 4

Example Residual activity%

Composition before incubation after incubation

9 131 112

10 130 123

11,124,117

12,163,161

13 0.5 0.4

14 115 98

15 97 92

16 138 126

17 130 107

These results show that the detergent compositions containing one of the surfactants FAEO (3EO), FAEO (6EO), FAEO (7EO), FAEO (9EO), FAES and PAS, Na, or a mixture of these are stable; while the detergent compositions containing only the surfactant referenced LAS, Na are rapidly inactivated.

These results show that the detergent compositions containing one of the surfactants FAEO (3EO), FAEO (6EO), FAEO (7EO), FAEO (9EO), or a mixture of FAEO (9EO) and FAES are very stable.

Excellent results are obtained with the detergent composition containing the surfactant FAEO (9EO). Example 19 Preparation of a detergent composition.

An aqueous solution is prepared containing 290.8 mg / 1 of calcium chloride CaCl, 2 H ₂ O; 140 mg / 1 of magnesium chloride MgCl ₂ , 2 H ₂ O and 210 mg / 1 of sodium carbonate NaHCO ₃ .

To this aqueous solution are added 120 mg / 1 of sodium citrate, 60 mg / 1 of propylene glycol, 120 mg / 1 of ethanol, 25 mg / 1 of phosphonic acid (DEQUEST 2060S, MONSANTO) and 20 mg / 1 palm oil soap (EDENOR HPK). To the aqueous suspension thus obtained, two surfactants are added: the surfactant LAS, Na at a concentration of 0.5 g / 1 and the surfactant FAEO (6EO) at a concentration of 0.3 g / 1.

To this aqueous suspension containing the two surfactants mentioned, a lipase concentration equivalent to 20 LU / 1 is added. Lipase is used as obtained in Example 3, which is diluted with water of hardness 15 in order to obtain the desired concentration.

The pH of the detergent composition obtained is adjusted to a pH of 9.5 with NaOH (1 mole / 1).

The enzymatic activity of the detergent composition is measured immediately. The method for measuring the enzymatic activity is described in Example 2. Example 20 Preparation of a detergent composition.

Example 19 is repeated, replacing the FAEO surfactant (6EO) with the FAEO surfactant (7EO) at the same concentration of 0.3 g / l. Example 21

Preparation of a detergent composition.

Example 19 is repeated, replacing the FAEO surfactant (6EO) with the FAEO surfactant (9EO) at the same concentration of 0.3 g / l. Example 22 Preparation of a detergent composition.

Example 21 is repeated, replacing the surfactant LAS, Na with the surfactant FAES at a concentration of 0.5 g / l. Example 23

Preparation of a detergent composition. Example 19 is reproduced, replacing the mixture of two surfactants [LAS, Na and FAEO (6EO)] with a mixture of three surfactants: the surfactant LAS, Na at a concentration of 0.25 g / l, the FAES surfactant at a concentration of 0.25 g / 1 and the FAEO surfactant (9EO) at a concentration of 0.5 g / 1. Example 24

Preparation of a detergent composition.

Example 19 is repeated, replacing the FAEO surfactant (6EO) with the FAEO surfactant (3EO) at the same concentration of 0.3 g / l. Example 25 Stability of detergent compositions.

Following the procedure described in Example 18, the stability of the detergent compositions as obtained in Examples 19 to 23.

The detergent compositions, as obtained in Examples 19 to 24, are incubated for 45 minutes at 35 ° C.

Then, the enzymatic activity of the detergent compositions incubated is measured.

The results are expressed in% of the residual enzymatic activity based on the activity of the lipase theoretically added.

The results are collated in Table 5.

Table 5

Example Residual activity%

Composition before incubation after incubation

19 132 2

20 130 2

21,117 1

22 170 163

23 128 112

24 155 75

These results show that the detergent composition containing two surfactants: the surfactant FAES and the surfactant FAEO (9EO) is very stable.

These results also show that the detergent composition containing three surfactants: the surfactant LAS, Na, the surfactant FAES and the surfactant FAEO (9EO) is stable. Example 26

Preparation of a detergent composition.

A liquid detergent composition containing 0.6 mg / 1 of a lipase having an enzymatic activity of approximately 9600 LU / ml is prepared and a mixture of additives. Lipase is used as obtained in Example 3, which is diluted with water of hardness (water containing 1.98 mM calcium chloride, 0.69 mM magnesium chloride and 2.5 mM sodium bicarbonate) to obtain the desired concentration.

The mixture of additives used contains 0.5 g / l of LAS, Na; 0.02 g / 1 soap; 0.3 g / 1 of FAEO (3EO); 0.06 g / l propanediol; 0.12 g / 1 ethanol; 0.12 g / 1 sodium citrate; 0.025 g / 1 of phosphonate [diethylene triamine penta (methylenephosphonic acid)].

The initial pH of the liquid detergent composition is adjusted to approximately 9.5 with NaOH (1 mole / 1).

Example 27

Preparation of a detergent composition.

Example 26 is repeated, replacing the FAEO surfactant (3EO) with the FAEO surfactant (6EO) at the same concentration of 0.3 g / l. Example 28

Preparation of a detergent composition.

Example 26 is repeated, replacing the FAEO surfactant (3EO) with the FAEO surfactant (7EO) at the same concentration of 0.3 g / l.

Example 29 Preparation of a detergent composition.

Example 26 is repeated, replacing the FAEO surfactant (3EO) with the FAEO surfactant (9EO) at the same concentration of 0.3 g / l.

Example 30

Preparation of a detergent composition. Example 26 is repeated, replacing the surfactant FAEO (3EO) with the surfactant FAEO (9EO) at the same concentration of 0.3 g / 1 and the surfactant LAS, Na with the surfactant FAES at the concentration of 0.5 g / 1.

Example 31

Preparation of a detergent composition. Example 26 is reproduced, using the surfactant

FAEO (3EO) at a concentration of 0.15 g / 1 and adding the surfactant

FAEO (9EO) at a concentration of 0.15 g / 1.

Example 32

Preparation of a detergent composition. Example 31 is repeated, replacing the FAEO surfactant (9EO) with the FAEO surfactant (7EO) at the same concentration of 0.15 g / l.

Example 33

Use of a detergent composition.

The protocol described in Example 6 is reproduced, with the exception of the washing temperature which is 35 ° C. in this example, and not 10 ° C., and with the exception of the duration of the washing which is 45 minutes in this example, not 25 minutes. The detergent composition as obtained in Example 26 is used.

The results are collated in Table 6.

Table 6

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 65.16 0.6 51.76

2 0 76.42 0.6 69.60

3 0 80.72 0.6 75.29

4 0 83.83 0.6 79.03

Example 34

Use of a detergent composition.

Example 33 is reproduced using the detergent composition as obtained in Example 27.

The results are collated in Table 7.

Table 7

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 67.01 0.6 70.11

2 0 80.26 0.6 81.51

3 0 87.21 0.6 89.41

4 0 93.49 0.6 95.62

Example 35

Use of a detergent composition.

Example 33 is reproduced using the detergent composition as obtained in Example 28.

The results are collated in Table 8.

Table 8

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 60.46 0.6 64.15

2 0 79.57 0.6 80.64

3 0 86.29 0.6 88.62

4 0 92.35 0.6 95.56 Example 36

Use of a detergent composition.

Example 33 is reproduced using the detergent composition as obtained in Example 29.

The results are collated in Table 9.

Table 9

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 19.13 0.6 23.07

2 0 32.12 0.6 50.44

3 0 44.72 0.6 68.52

4 0 56.64 0.6 80.95

Example 37

Use of a detergent composition.

Example 33 is reproduced using the detergent composition as obtained in Example 30.

The results are collated in Table 10.

Table 10

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 7.34 0.6 7.51

2 0 8.92 0.6 12.13

3 0 10.16 0.6 29.27

4 0 11.17 0.6 65.23

These results also show that the detergent composition containing two surfactants: the surfactant LAS, Na and the surfactant FAEO (9EO) has a good washing performance. Example 38 Use of a detergent composition.

Example 33 is repeated using the detergent composition as obtained in Example 31.

The results are collated in Table 11.

Table 11

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 67.01 0.6 69.19

2 0 80.62 0.6 80.99

3 0 87.47 0.6 88.25

4 0 93.64 0.6 94.33

These results also show that the detergent composition containing two surfactants: the surfactant FAES and the surfactant FAEO (9EO) has a good washing performance. Example 39 Use of a detergent composition.

Example 33 is reproduced using the detergent composition as obtained in Example 32.

The results are collated in Table 12.

Table 12

Amount of lipase Washing performance

Number of washings implemented mg / 1%

1 0 78.00 0.6 73.69

2 0 84.90 0.6 82.09

3 0 91.17 0.6 88.35

4 0 96.05 0.6 93.30

Claims

R E V E N D I C A T I O N S

1 - Detergent composition containing a lipase produced by a strain of Pseudomonas and at least one surfactant.

2 - Detergent composition according to claim 1, characterized in that the surfactant is a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 10 to about 20 carbon atoms and having a degree of ethoxylation of between about 2 and about 40.

3 - Detergent composition according to claim 2, characterized in that the nonionic surfactant is chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9.

4 - Detergent composition according to claim 1, 2 or 3, caracté¬ ized in that the detergent composition contains an anionic surfactant chosen from the alkyl sulfate salts containing from 8 to 24 carbon atoms and the alcoholic ethoxysulfate salts containing from 6 with 20 carbon atoms and having a degree of ethoxylation of between about 2 and about 20.

5 - Detergent composition according to claim 4, characterized in that the anionic surfactant is chosen from sodium alkyl sulphates containing from 16 to 18 carbon atoms and the alcoholic sodium ethoxysulphate salts containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2.

6 - Detergent composition according to any one of claims 1 to 5, characterized in that the lipase comes from a strain of

Pseudomonas wisconsinensis or a derivative or mutant of this strain capable of producing lipase.

7 - Detergent composition according to claim 6, characterized in that the lipase comes from the strain of Pseudomonas wisconsinensis T 92.677 / 1 (LMG P-15151) or a derivative or mutant of this strain capable of producing a lipase. 8 - Detergent composition containing a lipase and at least one surfactant, characterized in that the detergent composition contains a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9 and an anionic surfactant chosen from the salts of alcoholic sodium ethoxysulfates containing 12 to 14 carbon atoms and having a degree of ethoxylation of 2.

9 - Detergent composition according to claim 8, characterized in that the lipase is a microbial lipase.

10 - Detergent composition according to claim 9, characterized in that the lipase comes from a strain of Pseudomonas.

11 - Detergent composition according to any one of claims 1 to 10, characterized in that the lipase comes from a strain of Pseudomonas wisconsinensis or a derivative or mutant of this strain capable of producing a lipase.

12 - Detergent composition according to claim 11, characterized in that the lipase comes from the strain of Pseudomonas wisconsinensis

T 92.677 / 1 (LMG P-15151) or a derivative or mutant of this strain capable of producing a lipase.

13 - Surfactant composition, characterized in that it contains a nonionic surfactant chosen from polyethoxylated aliphatic alcohols containing from about 12 to about 14 carbon atoms and having a degree of ethoxylation of 9 and an anionic surfactant chosen from the salts of alcoholic sodium ethoxysulfates containing from 12 to 14 carbon atoms and having a degree of ethoxylation of 2.