DETERGENT COMPOSITIONS COMPRISING BIOSURFACTANT AND LIPASE
Technical Field
This invention relates to detergent compositions comprising biosurfactant and lipase.
Background
A general description of biosurfactants is published by Rahman in Biotechnology 7 (2): 360-370, 2008 ISSN 1682-296X "Production, Characterisation and application of Biosurfactants - review".
Enzymes have been used in detergent formulations as a cleaning aid for many years. They may be derived from bacterial of other sources. The most commonly employed enzymes are proteases, amylases, mannanases, lipases and cellulases. They are often derived from fungal or yeast cultures.
Lipases are used in surfactant containing detergent formulations to aid the cleaning of oily soils from fabrics. Despite their isolation and characterisation some decades ago, these enzymes have been difficult to formulate in conventional surfactant formulations because there is a competition between the enzyme and the surfactant for the target substrate oil. Surfactants will win this competition for the surface and will out-compete or displace enzymes from the oily surface and therefore reduce the enzyme performance on those soils. Thus, the practical impact of lipases in detergent cleaning products is limited, especially when compared to the impact of other cleaning enzymes, such as proteases and amylases.

The move to more sustainable chemistries reinforces a desire to reduce the surfactant level in the wash. As a bio alternative, enzymes represent a weight efficient choice to maintain performance on oily soil removal as surfactant levels are lowered. The use of biosurfactants has been proposed in many prior art documents.
The following documents relate to combinations of biosurfactants and enzymes produced from bacteria.
JP51 68489A describes a method of making a biosurfactant using a lipase enzyme. The biosurfactant that is present in combination with the lipase does not have any acid moieties. The presence of a disaccharide moiety is optional.
"Lipase and biosurfactant production for utilisation in bioremediation of vegetable oils and hydrocarbon". Martins VG et al (2008) Quimica Nova No 3 1 vol 8, 1942-1947.
"Isolation and characterisation of a lipid degrading bacterium and its application to lipid containing wastewater treatment". Matsumiya Y. et al (2007) Journal of Bioscience and Bioengineering No 103, Vol 4, 325-330.
US20061061 20 describes a mixture of microorganism, biosurfactant and a plastic degrading enzyme for the bioremediation of man-made materials. The biosurfactant may be derived from bacterial or other sources; the preferred enzyme used in the examples is a cutinase of bacterial origin. It may be co expressed with amylase and hydrophobin. The compositions are not used for cleaning.
The following documents relate to combinations of biosurfactants and enzymes not specifically produced from bacteria, for cleaning.

CN1 0 1126052 describes a biosurfactant containing cleaning composition that also contains a protease. The origin of the protease is a pineapple plant.
US541 7879 (Unilever) describes synergistic dual surfactant laundry composition containing sophorolipid (from yeast), cellobiose lipid (from fungus) or rhamnolipid (from bacteria) glycolipid biosurfactant. Examples using these biosurfactants did not comprise any enzyme. In column 12 lines 24 to 25, it is mentioned as possible to combine the biosurfactants with an undisclosed amount of enzyme of undisclosed origin.
US2004171 5 12A (Igarashi Keisuke ; Hirata Yoshihiko ; Furuta Taro) discloses low-foaming detergent compositions comprising a biosurfactant (sophorolipid from yeast) which can replace a conventional low foaming block polymer nonionic surfactant. According to the general disclosure, the biosurfactant may be used with an undisclosed type of enzyme selected from amylase, protease, cellulose, lipase, pullulanase, isopullulanase, isoamylase, catalase, peroxidase, or the like. The enzyme can be added by selecting appropriately in light of its substrate specificity. For example, protease may be selected for a protein stain, and amylase may be selected for a starch stain. Examples use the sophorolipids for dishwashing (hard surface cleaning) in combination with Savinase 6.0T a protease from Novo Nordisk and Duramyl 60T a starch lytic enzyme (amylase) from Novo Nordisk. Duramyl is produced from Bacillus Licheniformis and Savinase is produced from Bacillus Clausii/lentus, both bacterial sources. These are not taught to be generically preferred sources in this document.
US20091 88055A (Stepan Co) discloses compositions comprising sulfonated estolides and other derivatives of fatty acids. Table 20 provides prophetic examples of these surfactants in combination with other surfactants, including rhamnolipids. Enzymes are not included in these examples. Elsewhere in the document, it is said that the cleaning performance on greasy soils is

synergistically improved with the estolides by using lipases. Suitable lipase enzymes include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, as disclosed in British Patent 1,372,034. Suitable lipases include those that show a positive immunological cross-reaction with the antibody of the lipase, produced by the microorganism Pseudomonas fluorescens IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P "Amano," hereafter referred to as "Amano-P". Further suitable lipases are lipases such as M 1 Lipase. RTM and Lipomax.RTM (Gist-Brocades). Highly preferred lipases are the D96L lipolytic enzyme variant of the native lipase derived from Humicola lanuginosa (a fungus) as described in U.S. 6,017,871 issued Jan. 25, 2000 (P&G). Preferably, the Humicola lanuginosa strain DSM 4106 is used. This enzyme is incorporated into the composition in accordance with the present technology at a level of from 50 LU to 8500 LU per litre wash solution. Preferably, the variant D96L is present at a level of from 100 LU to 7500 LU per litre of wash solution. More preferably at a level of from 150 LU to 5000 LU per litre of wash solution.
US2006080785A (Nero) describes carpet cleaning by applying a cleaning composition having biosurfactants and enzymes to the carpet; and bonnet cleaning the material. The enzymes are derived from Sea Kelp.
One document suggests combinations of biosurfactants and enzymes derived from bacteria for cleaning.
US200407271 3A (Unilever) discloses an article for use in an enzymatic fabric cleaning process, said article containing one or more types of harmless microorganisms capable of excreting enzymes useful in said fabric cleaning process. In one embodiment, the microorganism may be a bacterium, although fungal microorganisms are also exemplified. The examples all express bleaching

enzymes. Although not used in the examples the document speculates that it is especially useful if, in addition to enzymes, the micro-organisms are also capable of producing other chemical entities that contribute to the cleaning process, e.g. biosurfactants, for example lipopolysaccharides. No wash liquor or concentrate comprising a mixture of biosurfactants derived from bacteria together with enzymes derived from bacteria is actually disclosed in this document. We are confident that the concentration of biosurfactant would have been much less than 0.5 g/L.
Summary of the Invention
According to the present invention there is a cleaning composition comprising an effective amount of surfactant system and an enzyme system characterised in that the surfactant system comprises at least 1 wt% (based on the cleaning composition) of a biosurfactant, which is a glycolipid surfactant comprising at least 20 mol% of glycolipid having both disaccharide and acid moieties and at least one lipase enzyme of bacterial origin.
Surprising synergistic benefits on cleaning on stains and soils have been found when lipases, derived from bacteria, are combined with biological surfactants (biosurfactants) having both disaccharide and acid moieties.
The combination may be used in any biological formulation. Lipases are a key enzyme for insertion into detergent compositions, especially laundry detergents, but also compositions designed to clean hard surfaces such as dishwashing compositions, that clean everyday dirt and stains effectively at reduced surfactant levels to enable concentration of the formulation.
We tested three types of biosurfactant: (fungal, bacterial and yeast) in combination with two types of lipase enzyme (fungal and bacterial). The bacterial

enzymes consistently outperformed the fungal ones with the biosurfactants. The best result comes from a combination of bacterially derived enzyme with bacterially derived biosurfactant comprising at least 80mol% of biosurfactant having disaccharide and acid moieties (di-Rhamnolipid).
According to a second aspect of the present invention there is a process for cleaning a substrate comprising the steps of immersing the substrate in water adding a composition according to any preceding claim to the water to form a wash liquor and washing the substrate characterised in that the wash cycle time is less than 60 minutes, preferably less than 30 minutes and the water temperature is less than 35 °C at all times.
Detailed Description of the Invention
Bacterial Lipase
Suitable lipases include those of bacterial origin. Chemically modified or protein engineered mutants are included. Examples of useful bacterial lipases include lipases from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), B. stearothermophilus (JP 64/744992) orB. pumilus (WO 91/1 6422).
Bacterial genes encoding bacterial lipase enzymes can be transferred to preferred expression production hosts, which are not limited to bacterial and includes for example other microbial hosts. The term bacterial lipase includes lipase produced from such expression hosts but originating from bacteria.

The enzyme may be a phospholipase classified as EC 3.1 .1.4 and/or EC 3.1 .1.32. As used herein, the term phospholipase is an enzyme, which has activity towards phospholipids. Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1 ) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol. Phospholipases are enzymes that participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases Ai and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in lysophospholipid. Phospholipase C and phospholipase D (phosphodiesterases) release diacyl glycerol or phosphatidic acid respectively.
The term phospholipase includes enzymes with phospholipase activity, e.g., phospholipase A (Ai or A 2), phospholipase B activity, phospholipase C activity or phospholipase D activity. The term "phospholipase A" used herein in connection with an enzyme of the invention is intended to cover an enzyme with Phospholipase Ai and/or Phospholipase A2 activity. The phospholipase activity may be provided by enzymes having other activities as well, such as, e.g., a lipase with phospholipase activity. The phospholipase activity may, e.g., be from a lipase with phospholipase side activity. In other embodiments of the invention the phospholipase enzyme activity is provided by an enzyme having essentially only phospholipase activity and wherein the phospholipase enzyme activity is not a side activity.
Preferably, the phospholipase is of bacterial origin Bacillus, e.g., B. megaterium, B. subtilis; Citrobacter, e.g., C. freundii; Enterobacter, e.g., E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g., K. pneumoniae; Proteus, e.g., P. vulgaris; Providencia, e.g.,

P. stuartii; Salmonella, e.g. S. typhi murium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella, e.g., S. flexneri;
The composition may further comprise Enzymes that are not of bacterial origin. Particularly protease, amylase and cellulase, although non-bacterial lipases could also be included.
Biosurfactants
These are derived from microbial sources including bacteria, yeasts and fungi. The term Biosurfactant in this patent specification does not include surfactants derived from plant material such as Alkyl polyglucosides (APG).
a) Bacterially derived Biosurfactants
These are, for example, the Rhamnolipids typically from Pseudomonas sp. Information about other bacterially derived biosurfactants is available from "Mapping of Patents in Bioemulsifiers and biosurfactants - review, published in the Journal of Scientific and Industrial Research Vol 65, 2006, P91 . Within the definition of bacterially produced biosurfactants, we include those where a bacterial gene is cloned and subsequently expressed from another organism as a manufacturing technique. For example, Rhamnolipids have been produced from E. coli in this way.
b) Biosurfactants from non-bacterial sources
Biosurfactants from non-bacterial microbial sources include those derived from fungi and yeasts, e.g. sophorolipids from Candida sp and Torulopsis sp. Candida apicola, Candida bombicola, Candida lipolytica, Candida bogoriensis. See: Environmental applications for biosurfactants - Environmental Pollution, Volume 133, 2005, Pages 183-198 Catherine N. Mulligan. See also, Towards commercial

production of microbial surfactants - Trends in Biotechnology, Volume 24, 2006, Pages 509-515 : Soumen Mukherjee, Palashpriya Das, Ramkrishna Sen.
Mannosylerythritol Lipids are typically from Pseudozyma (formerly Candida) Antarctica. Cellobiose lipids are typically from Ustilago maydis. Trehalose Lipids typically from Rhodococcus sp.
Further information is given in Production, Characterisation and Applications of Biosurfactants Review- Biotechnology - Volume 7,2008, page 370: Pattanathu, Rahman and Gakpe.
The detergent composition may comprise other ingredients commonly found in laundry liquids. Especially polyester substantive soil release polymers, hydrotropes, opacifiers, colorants, perfumes, other enzymes, other surfactants, microcapsules of ingredients such as perfume or care additives, softeners, polymers for anti redeposition of soil, bleach, bleach activators and bleach catalysts, antioxidants, pH control agents and buffers, thickeners, external structurants for rheology modification, visual cues, either with or without functional ingredients embedded therein and other ingredients known to those skilled in the art. The composition is preferably a liquid and is advantageously packaged in either a multidose bottle or in a unit dose soluble pouch
The invention will now be further described with reference to the following non-limiting examples.
Examples
Example 1
In this example, various Enzyme / biosurfactant compositions were tested to
determine their ability to remove a coloured beef stain from cotton cloth.

Wash solutions were prepared by dispersing lipase at a concentration of 4mg protein per litre together with detergent surfactant at the required concentration in phosphate buffered saline (PBS) adjusted to pH 8 and 12° FH water hardness. 10 mis of the wash solution were mixed in 25 ml plastic vials at 37 °C with agitation at 200 rpm in an orbital incubator for 30 minutes. Swatches (approximately 1 cm2) of cotton cloth stained with Sudan Red coloured Beef fat were then added and the vials returned to the shaking incubator. Swatches were removed at timed intervals, rinsed in cold water and dried at 37 °C. The residual colour was monitored using a Macbeth Colour Eye, and compared with untreated stained cloths. Results are shown in Table 1 for 30 minutes and Table 2 for 4 hours.
Bacterial enzyme is "Lipomax", a bacterially derived Lipase variant M21 L of the lipase of Pseudomonas alcaligenes as described in WO 94/25578 to Gist-Brocades (M. M.M.J. Cox, H.B.M. Lenting, L.J.S.M. Mulleners and J.M. van der Laan).
Fungal enzyme is "Lipolase", derived from Humicola languginosa as described in EP 0 258 068 and available from NovoZymes A/S.
Details of the surfactants were as follows:
SL = Sophorolipid: a biosurfactant of fungal origin comprising disaccharide
moieties and at least 20% acid moieties.
AC = Accell: a biosurfactant derived from a yeast.
RL = Rhamnolipid: a biosurfactant of bacterial origin comprising acid moieties and
wherein R2 comprises disaccharide moieties. RL is approximately 70 mol% di
Rhamnolipid and 30 mol% mono Rhamnolipid. Only the di Rhamnolipid has the
required disaccharide moiety.

Table 1-30 min

Biosurfactant No Enzyme Bacterial lipase Fungal enzyme
0.25g/l SL 2.83 8.42 4.29
0.25g/l AC 0.96 2.39 1.25
0.25 g/l RL 3.35 5.40 3.29
0.5 g/l SL 8.98 11.20 8.44
0.5 g/l AC 1.05 1.95 1.00
0.5 g/l RL 1.28 10.00 0.82
Table 2-4 hours

Biosurfactant No Enzyme Bacterial lipase Fungal enzyme
0.25g/l SL 5.52 12.98 8.61
0.25g/l AC 3.67 9.15 3.19
0.25 g/l RL 3.12 8.01 3.36
0.5 g/l SL 12.23 13.59 11.29
0.5 g/l AC 2.22 8.40 3.52
0.5 g/l RL 1.38 12.01 2.34
The bacterial lipase enzyme consistently outperforms the fungal lipase enzyme across all stain types. For the Sophorolipids at higher concentration and long wash times the presence of the fungal lipase enzyme provides no benefit over using the surfactant without a lipase.
The enzymes were all dosed at the same level by determining the amount of active enzyme protein in each of the samples by use of a standard BCA protein Assay kit (ex Pierce) following the manufacturer's protocol.

Example 2
In this example, various enzyme/Biosurfactant compositions were examined to determine their ability to remove a coloured beef stain.
The same experimentation was carried out as in Example 1 except the rhamnolipid material was separated into its mono-rhamnolipid and di-rhamnolipid components. The di rhamnolipid having two rhamnose sugars on the acyl group. We use the notation R1 for the mono rhamnolipid and R2 for the di-rhamnolipid material. The cleaning results for 1 hour and 4 hours are given in Tables 3 and 4.
Table 3 - 1 hour

Biosurfactant No Enzyme Bacterial lipase Fungal lipase
0.5 g/l SL 6.34 10.28 9.72
0.5 g/l RL 1.15 8.88 1.04
0.5 g/l R1 9.85 11.31 12.25
0.5 g/l R2 0.80 8.87 1.05
Table 4-4 hours

Biosurfactant No Enzyme Bacterial lipase Fungal lipase
0.5 g/l SL 10.25 12.54 11.17
0.5 g/l RL 1.1 8 10.68 1.89
0.5 g/l R1 14.52 12.43 14.1 9
0.5 g/l R2 1.14 11.42 2.85

Claims
1. A cleaning composition comprising an effective amount of surfactant system
and an enzyme system characterised in that the surfactant system comprises at least 1 wt% (based on the cleaning composition) of a biosurfactant, which is a glycolipid surfactant, comprising at least 20 mol% of glycolipid having both disaccharide and acid moieties and at least one lipase enzyme of bacterial origin.
2. A composition according to claim 1 in which the biosurfactant is selected from
Rhamnolipids, Sophorolipids and mixtures thereof
3. A composition according to claim 1 in which the biosurfactant is of bacterial
origin.
4. A composition according to any preceding claim in which the biosurfactant is
a rhamnolipid.
5. A composition according to claim 4 in which the rhamnolipid comprises two or
more rhamnose units on the acyl chain and is at least 60% di-rhamnolipid.
6. A process for cleaning a substrate comprising the steps of immersing the
substrate in water adding a composition according to any preceding claim to the water to form a wash liquor and washing the substrate characterised in that the wash cycle time is less than 60 minutes, preferably less than 30 minutes and the water temperature is less than 35 °C at all times.