WO1991013142A1

WO1991013142A1 - Lipase containing liquid pre-spotter and use of such pre-spotter

Info

Publication number: WO1991013142A1
Application number: PCT/DK1991/000059
Authority: WO
Inventors: Erik Gormsen
Original assignee: Novo Nordisk A/S
Priority date: 1990-03-01
Filing date: 1991-02-28
Publication date: 1991-09-05
Also published as: DE69101219D1; JP2930719B2; WO1991013141A1; ES2051122T3; DE69101219T2; DK0517761T3; EP0517761A1; EP0517761B1; JPH05504368A

Abstract

The pre-spotter comprises a liquid mixture of an alcohol, water, a surfactant, and an effective amount of lipase. This pre-spotter is simple, and it exhibits an improved fat and oil removing effect and an improved lipase stability.

Description

LIPASE CONTAINING UQUID PRE-SPOTTER AND USE OF SUCH PRE-SPOTTER

The invention relates to a lipase containing liquid pre-spotter and to a use of such pre-spotter.

In relation to washing processes it is described that fat or oil stains, which are difficult to remove, can be treated with a lipase containing liquid pre- spotter, which is applied directly to the stain before the washing process. However, to the best of our knowledge no lipase containing pre-spotter is commercialized, probably due to lipase stability or performance problems. It is further described that the thus treated laundry is subsequently washed, usually at relatively low temperature, normally after a short period of time. It is one of the advantages by using a lipase containing pre-spotter that the subsequent washing process can be carried out at relatively low temperature, i.e. below around 60 °C. If no lipase containing pre-spotter is used, usually the washing process will have to be carried out at around 100^βC and at high alkalinity in order to have the fat or oil stains removed effectively, but even under these conditions, i.e. at around 100^βC and at high alkalinity, some obstinate fat or oil stains cannot be removed. Reference can be made to EP 177 183, which describes special lipase containing pre-spotters, in which the enzymes are encapsulated in reversed micelles. It has been found that the fat and oil removing effect of the prior art lipase containing pre-spotters and the lipase stability in the prior art lipase containing pre-spotters are open to improvement.

Thus the purpose of the invention is the provision of a simple lipase containing liquid pre-spotter, which exhibits an improved fat and oil removing effect and an improved lipase stability. The lipase containing liquid pre-spotter according to the invention comprises a liquid mixture of an alcohol and water with an alcohol content of above 10% by weight and a content of a surfactant of between 0.5 and 10% by weight, and an effective amount of lipase, whereby the balance is water. It is to be understood that the surfactant can be of the non-ionic, the anionic, the cationic and the zwitterionic type. The pre-spotter according to the invention is able to remove spots of oil of both mineral, vegetable and animal origin, at least partially. The spots of mineral oil are removed due to the surfactant, and the oil spots of vegetable and animal origin are removed due to the lipase. In this specification with claims the term alcohol comprises both monovalent alcohols like ethanol and polyvalent alcohols (polyols) like propylene glycol. If the alcohol content is below 10% by weight, the effect and the stability of the lipase tend to be unsatisfactory.

The term "an effective amount of lipase" means an amount, which is able to remove fat and oil stains effectively, when evaluated by swatch tests described later in this specification. It is intended that all lipases can be used in the pre-spotter according to the invention. Reference can be made to Research Disclosure June 1988, No. 290, 29056.

In a preferred embodiment of the pre-spotter according to the invention the surfactant is non-ionic. In this manner both a very high fat and oil removing effect and a very high lipase stability is obtained.

In a preferred embodiment of the pre-spotter according to the invention the pre-spotter further comprises a lipase stabilizer. Examples of suitable lipase stabilizers are CaCl2 (e.g. 0.1-0.5%) or a protease inhibitor (such as borate and formate).

In a preferred embodiment of the pre-spotter according to the invention the alcohol is a mono- or polyvalent alcohol with 2-3 carbon atoms, preferably ethanol, propylene glycol, glycerol or a mixture of two or more of these. It has been found that the fat and oil removing effect is most satisfactory with these alcohols.

In a preferred embodiment of the pre-spotter according to the invention the lipase activity is at least 10,000 LU/liter of liquid pre-spotter. It has been found that the time interval between application of pre-spotter and main wash is undue long in order to obtain an efficient fat and oil removal, if the lipase activity is less than 10,000 LU/liter of liquid pre-spotter. On the other hand, for economic reasons a suitable upper limit for the lipase activity is 5,000,000 LU/liter of liquid pre-spotter.

In a preferred embodiment of the pre-spotter according to the invention the lipase is Lipolase^®. It has been found that the fat and oil removal is very efficient in this embodiment.

In a preferred embodiment of the pre-spotter according to the invention the pre-spotter contains an antimicrobial agent. Examples of such agents are inorganic salts (such as NaCI), sugars (such as sucrose and glucose), organic acids (such as benzoic, sorbic, propionic, lactic and formic acids) which are generally effective in amounts of 0.01-2% at low pH (below 5). Other examples of stabilizing agents are antioxidants (such as sulphur dioxide), 1 ,2-benz-iso- thioazolin-3-one (BIT) and parabens. Some of these may also serve to improve enzyme stability.

In a preferred embodiment of the pre-spotter according to the invention the pH value is between 4 and 10, preferably between 5 and 9. In selecting the pH value for a specific pre-spotter, the stability optimum and the activity optimum of the lipase should be taken into consideration.

Also, the invention comprises a use of the pre-spotter according to the invention, and this use is characterized by the fact that the pre-spotter is applied locally to the fat or oil spots on the laundry, that the laundry is left alone for a certain time period, and that the laundry subsequently is exposed to a low temperature washing process, or that alternatively the pre-spotter is applied locally to the fat or oil spots on the laundry and that the thus treated laundry is treated by rinsing with water. It has hitherto been assumed that it was necessary to use surfactants during laundering in order to remove fat or oil from the laundry. However, as appears from the above, according to the invention it has been found that fat and oil can be removed effectively by means of the pre-spotter according to the invention, i.e. before the laundering because in this instance the fat or oil spot can be removed without any subsequent main wash, as during pre-spotting it is broken down to degradation products, which can be removed just by rinsing the treated laundry with tap water.

The certain time period alluded to above is normally at least 5 minutes, but it can be shorter in case the lipase activity in the pre-spotter is extraordinarily high.

From the brochure Novo Detergent Enzymes B 435b-GB July 1989 it appears that very difficult stains can be removed by introduction of a pre-spotter with a Lipolase^® containing detergent in a specific test, which utilized a heavy duty liquid detergent. This heavy duty liquid detergent contained around 28% surfactant. It has been found that during the pre-spotting not exclusively a fat hydrolysis takes place but additionally an ester synthesis based on the alcohol in the pre-spotter and the fatty acids liberated from the fat or oil stains by means of the lipolytically catalyzed hydrolysis. It is surprising that an ester synthesis takes place because of the high water activity. Also, the ester formed by the above indicated ester synthesis performs as a surfactant, improving the fat and oil removing effect. Thus, it is assumed that part of the improved fat and oil removing effect of the pre-spotter according to the invention has to be ascribed to this ester synthesis; however, applicant does not want to be bound to this hypothesis.

In the examples shown below the lipase activity is measured in LU lipase activity units. The LU activity determination method is based on hydrolysis of tributyrin in a pH-stat. 1 LU (lipase Unit) is the amount of enzyme which liberates 1 μmole titratable butyric acid per minute at 30° C and pH 7.0 with gum arabic as an emulsifier. Further details are given in Novo Nordisk analytical Method AF 95/5, available on request from Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.

In the examples shown below the following general procedure was used. Method: Embodiments of liquid pre-spotter is applied to the fat stain on a test swatch by means of a pipette. The amount of pre-spotter used depends on the size of the stain and on the kind of fabric; typically 0.5-3 ml of pre-spotter is satisfactory. After 5-30 minutes of incubation the swatches are washed in a Laboratory washing machine (Launder-o-meter or Terg-o-tometer) at low temperature (20-40° C). The detergent used in the washing process is a standard powder or liquid detergent.

Swatches: The swatches are made from coloured (e.g. blue or green) cotton fabric. The fabric is cut into test pieces with the dimensions 12x12 cm, and 75- 150 il of fatty material (e.g. lard, margarine, butter, or olive oil) is applied to the center of each swatch. The amount of fatty material applied depends on the type of fatty material used. The fatty material is heated to approx. 70 °C before application in order to facilitate the penetration of fatty material into the fabric. The stained fabric was aged at room temperature for 1 day before treatment.

Evaluation: After washing and drying the swatches are gently ironed. The swatches are visually inspected and compared to reference swatches which have been identically treated but without any lipase added to the pre-spotter formulation. In order to quantify the efficiency of the lipase, extraction of the residual fatty material can be carried out (Soxhlet, chloroform, 5 hours).

EXAMPLE 1

Effect of Lipolase* in pre-spotter formulations based on water, propylene glycol, and a non-ionic surfactant

Stain: Lard on green cotton sheet.

75 μ\ of lard was applied on each swatch. After stain application, the swatches were heat treated in an oven for 30 minutes at 75 °C in order to obtain a better distribution of the stain.

Pre- spotting: 1 ml of pre-spotter is applied with a pipette on the centre of the stains and subsequently the swatches are incubated for 15 minutes at room temperature.

The pre-spotter formulations contained Lipolase* 100L (100,000 LU/g), an alcohol ethoxylate (Berol 160, Berol Kemi AB, Sweden), and a mixture of water and propylene glycol in the later indicated concentrations.

For comparison, the same pre-spotter formulations, but without added lipase, were evaluated as well.

Washing: Immediately after pre-spotting the swatches were washed in a Terg- o-tometer (40^βC, 20 minutes, 18° dH, pH 9.5) with a simplified European powder detergent containing 1.75 g/l of sodium triphosphate, 0.40 g/l of sodium metasilicate, 2.00 g/l of sodium sulphate, 0.30 g/l of Berol 160 (alcohol ethoxylate from Berol Kemi AB, Sweden), and 0.50 g/l of Nansa S80/S (LAS from Albright & Wilson, UK). Evalua¬ tion: A trained test panel (13 persons) evaluated the stain removal by visual inspection. Def. of score: 0 Heavy stain (= untreated stain)

1 Clearly visible stain

2 Visible stain

3 Hardly visible stain

4 Mere trace of stain

5 Stain completely removed

Results: The mean score of the investigated pre-spotter formulation were as follows:

EXAMPLE 2

Effect of lipase dosage in pre-spotter formulations based on water, propylene olvcol. and a nonionic surfactant

The same kind of test material and the same pre-spotting, washing, and evaluation method as described in Example 1 was used; the only difference being that the dosage of Lipolase* 100 L was varied between 0 and 5% and that between 0 and 10% of an alcohol ethoxylate (Dobanol 25-7 from Shell) was included in the pre-spotter formulation.

Results (mean score of test panel evaluation):

EXAMPLE 3

15 Effect of different lipases in pre-spotter formulations based on water, propylene qlvcol, and a nonionic surfactant

The same experiments as described in Example 3 were carried out, the only difference being that four lipases of different origin (2 fungal, 1 bacterial and 1 from yeast) were investigated. The lipases were compared on an LU-activity 0 basis.

Results (mean score of test panel evaluation):

1) see Huge- Jensen et al. (1989), Lipids 24, 781-785

2) see EP 0214761

3) Sigma L1754

EXAMPLE 4

Storage stability of a pre-spotter formulation

The enzymatic stability of Lipolase* was investigated in a pre-spotter formulation:

% w/w Lipolase 100L 2

Deionized water 38

Propylene glycol 50

Berol 160 10 pH 6.4

The pre-spotter formulation was stored at 30° C in a closed bottle, and the activity was followed over a 2 months period.

Storage (days) Residual activity (% ) 0 100

4 111

19 111

63 112 EXAMPLE 5

Synthesis of esters vs hydrolysis under pre-spotting conditions

In order to investigate lipase catalyzed formation of esters vs hydrolysis the following experiment was carried out. Olive oil adsorbed to PVC particles was incubated in pre-spotting formulations containing a lipase. After 5 minutes of incubation the reaction was stopped and fatty matter was extracted and analyzed by chromatography. Conditions of incubation: 30°C, pH 7.0, 0.33 g olive oil adsorbed on 1.67 g PVC powder, 20 g pre-spotter, 1500 LU.

No surfactant was present in the above formulations of Example 5. Even so, the example is considered an illustration of the invention because a surfactant in regard to the ester synthesis vs hydrolysis aspect would only be an indifferent 5 or inactive component, as the only decisive factor for the ester synthesis vs hydrolysis aspect is the ratio between alcohol and water.

Claims

'-CLAIMS

1. Lipase containing liquid pre-spotter comprising a liquid mixture of an alcohol and water, with an alcohol content of above 10% by weight and a content of a surfactant of between 0.5 and 10% by weight, and an effective amount of

5 lipase, whereby the balance is water.

2. Pre-spotter according to Claim 1 , wherein the surfactant is non-ionic or anionic.

3. Pre-spotter according to Claims 1 to 2, which further comprises a lipase stabilizer.

10 4. Pre-spotter according to Claims 1 to 3, in which the alcohol is a mono- or polyvalent alcohol with 2-3 carbon atoms, preferably ethanol, propylene glycol, glycerol or a mixture of two or more of these.

5. Pre-spotter according to Claims 1 to 4, in which the lipase activity is at least 10,000 LU/liter of liquid pre-spotter.

15 6. Pre-spotter according to Claims 1 to 5, wherein the lipase is Lipolase*.

7. Pre-spotter according to Claims 1 to 6, wherein the pre-spotter contains an antimicrobial agent.

8. Pre-spotter according to Claims 1 to 7, wherein the pH value is between 4 and 10, preferably between 5 and 9.

9. Use of the pre-spotter according to Claims 1 to 8, characterized by the fact that the pre-spotter is applied locally to the fat or oil spots on the laundry, that the laundry is left alone for a certain time period, and that the laundry subsequently is exposed to a low temperature washing process, or that alternatively the pre-spotter is applied locally to the fat or oil spots on the laundry and that the thus treated laundry is treated by rinsing with water.