This invention relates to cleaning compositions comprising mono-rhamnolipids in combination with enzymes.
Rhamnolipids are a class of glycolipid. They are constructed of rhamnose combined with beta-hydroxy fatty acids. Rhamnose is a sugar. Fatty acids are ubiquitous in animals and plants. The carboxyl end of the fatty acid end is connected to the rhamnose. Rhamnolipids are compounds of only three common elements; carbon, hydrogen, and oxygen. They are a crystalline acid. Rhamnolipids may be produced by strains of the bacteria Pseudomonas aeruginosa. There are two major groups of rhamnolipids; mono-rhamnolipids and di-rhamnolipids.
Mono-rhamnolipids have a single rhamnose sugar ring. A typical mono-rhamnolipid produced by P. aeruginosa is L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RhaC10C10). It may be referred to as Rha-C10-C10, with a formula of C26H48O9. Mono-rhamnolipids have a single rhamnose sugar ring. The IUPAC Name is 3-[3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxydecanoyloxy]decanoic acid.
Di-rhamnolipids have two rhamnose sugar rings. A typical di-rhamnolipid is L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha2C10C10). It may be referred to as Rha-Rha-C10-C10, with a formula of C32H58O13. The IUPAC name is 3-[3-[4,5-dihydroxy-6-methyl-3-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid.
In practice a variety of other minor components with different alkyl chain length combinations, depending upon carbon source and bacterial strain, exist in combination with the above more common rhamnolipids. The ratio of mono-rhamnolipid and di-rhamnolipid may be controlled by the production method. Some bacteria only produce mono-rhamnolipid, see
In various publications mono-rhamnolipids have the notation Rha-, which may be abbreviated as Rh or RL2. Similarly di-rhamnolipids have the notation Rha-Rha or Rh-Rh- or RL1. For historical reasons "rhamnolipid 2" is a mono-rhamnolipid and "rhamnolipid 1" is a di-rhamnolipid. This leads to some ambiguity in the usage or "RL1" and "RL2" in the literature. Throughout this patent specification, we use the terms mono- and di-rhamnolipid in order to avoid this possible confusion. However, if abbreviations are used R1 is mono-rhamnolipid and R2 is di-rhamnolipid. For more information on the confusion of terminology in the prior art see the introduction to
The following rhamnolipids have been detected as produced by the following bacteria: (C12:1, C14:1 indicates fatty acyl chains with double bonds).
Rhamnolipids produced by P. aeruginosa (mono-rhamnolipids):
Rhamnolipids produced by P. aeruginosa (di-rhamnolipids):
Rhamnolipids produced by P. aeruginosa (unidentified as either mono- or di-rhamnolipids):
Rhamnolipids produced by P. chlororaphis (mono-rhamnolipids only):
Rhamnolipids produced by Burkholdera pseudomallei (di-rhamnolipids only):
Rhamnolipids produced by Burkholdera (Pseudomonas) plantarii (di-rhamnolipids only):
Because rhamnolipids are produced in various chemical formulas, each with a different HLB, it is known that rhamnolipids can be produced or mixed to have a range of foaming properties. Rhamnolipids are an anionic surfactant with both hydrophilic end and a lipophilic end. When their concentration increases to a certain level it is known that the rhamnolipids join together inside a liquid in a micelle.
It has been suggested that rhamnolipids with two shorter fatty acids are more active in reducing surface tension and as an emulsifier. Those rare rhamnolipids with a single fatty acid chain are not as effective.
The bacterium Pseudomonas aeruginosa is found naturally in soils, in water, and on plants. Metabolically, P. aeruginosa is chemoheterotrophic, generally aerobic, utilizing a wide range of organic compounds for sources of carbon and nitrogen.
There are over 100 strains of P. aeruginosa on file at the American Type Culture Collection (ATCC). There are also a number of strains that are only available to manufacturers of commercial Rhamnolipids. Additionally there are probably thousands of strains isolated by various research institutions around the world. Some work has gone into typing them into groups. Each strain has different characteristics including how much rhamnolipid is produced, which types of rhamnolipids are produced, what it metabolizes, and conditions in which it grows. Only a small percentage of the strains have been extensively studied.
Through evaluation and selection, strains of P. aeruginosa can be isolated to produce rhamnolipids at higher concentrations and more efficiently. Strains can also be selected to produce less byproduct and to metabolize different feedstock or pollutants. This production is greatly affected by the environment in which the bacterium is grown.
Various documents have proposed to use Rhamnolipids in detergent compositions.
The ability of microbes to co-generate enzyme and biosurfactants is disclosed in "
According to the present invention there is provided a detergent composition with a novel ratio of mono to di rhamnolipids in combination with lipase. The amount of mono-rhamnolipid present is more than the amount of di-rhamnolipid present (if any). Preferably, at least 80 wt%, more preferably at least 90 wt% or even 100 wt% of the rhamnolipid in the composition is mono-Rhamnolipid.
The lipase is preferably derived from either fungal or bacterial sources. By bacterial sources, we include expression from other microbes, such as yeast, of genes that have been cloned from bacteria.
The rhamnolipid is preferably present in an amount of from 0.5 to 40 wt%. The lipase is preferably present in an amount of from 0.0001 to 5 wt%.
The detergent composition is preferably unbuilt. That is zeolite, phosphate or silicate builders are absent.
The detergent composition is preferably a liquid detergent composition and if citric acid builder is present, it is limited to a maximum level of 2 wt%. The composition is especially useful as a laundry detergent and may be used with advantage for washing in water with a low water hardness of less than 15°F. A process whereby the laundry and the composition are washed in presoftened water is particularly advantageously used with the compositions of the invention.
The compositions are used to remove fatty soils from laundry, especially from cotton cloths. Removal of soils from cotton is of increasing concern because many of the sophisticated soil removal and soil release technologies included in modern laundry detergents work best on polyester cloths. Accordingly, it is advantageous to combine the detergent system of the present invention with a polyester soil release polymer.
Using mono-rhamnolipids and lipase in a detergent composition according to the invention leads to enhanced cleaning benefits and possibly synergies with synthetic anionic surfactants, for example C12-14 alkyl benzene sulphonate synthetic anionic surfactant. This surfactant is commonly employed in laundry detergent compositions and is typically used with a nonionic surfactant, such as the ethoxylated nonionic surfactant used in
A preferred fatty soil is beef fat.
A large proportion of biosurfactants are generated by the action of bacteria on renewable feedstocks and are increasingly becoming more and more viable options as sustainable replacements of current synthetic surfactants. Within the current portfolio of biosurfactants that are currently commercialised, Rhamnolipids, formed by the degradation of oils and fats by Pseudomonas Aeg, show poor cleaning benefits when used at concentrations of components generated by the bacterial breakdown process. However, when the mono and di rhamnolipid components of the expressed rhamnolipids are extracted and the mono-rhamnolipid is used with lipase superior cleaning results. Moreover, by producing blends and mixing with synthetic anionic surfactants further enhancement in detergency may be achieved.
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.
Suitable lipases for use in the compositions of the invention include those of bacterial, fungal or yeast origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in
Other examples are lipase variants such as those described in
Further examples of suitable lipases are described and referenced but not limited to those in
Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™, Lipex™, Novozym 525L (Novozymes A/S).
The composition may comprise a cutinase. classified in EC 3.1.1.74. The cutinase used according to the invention may be of any origin. Preferably, cutinases are of microbial origin, in particular of bacterial, of fungal or of yeast origin.
Cutinases (Esterases) are enzymes that are able to degrade cutin. In a preferred embodiment, the cutinase is derived from a strain of Aspergillus, in particular Aspergillus oryzae, a strain of Alternaria, in particular Alternaria brassiciola, a strain of Fusarium, in particular Fusarium solani, Fusarium solani pisi, Fusarium roseum culmorum, or Fusarium roseum sambucium, a strain of Helminthosporum, in particular Helminthosporum sativum, a strain of Humicola, in particular Humicola insolens, a strain of Pseudomonas, in particular Pseudomonas mendocina, or Pseudomonas putida, a strain of Rhizoctonia, in particular Rhizoctonia solani, a strain of Streptomyces, in particular Streptomyces scabies, or a strain of Ulocladium, in particular Ulocladium consortiale. In a most preferred embodiment the cutinase is derived from a strain of Humicola insolens, in particular the strain Humicola insolens DSM 1800. Humicola insolens cutinase is described in
Other suitable esterases are those described in
Preferred commercial cutinases include NOVOZYM™ 51032 (available from Novozymes A/S, Denmark).
The composition may also comprise 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 which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases A1 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 (A1 or A2), 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 A1 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.
The phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian), e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom. Preferably the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g., A. niger, Dictyostelium, e.g., D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g., R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer, Sclerotinia, e.g., S. libertiana; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g., W. sclerotiorum; 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. typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella, e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber, Yersinia, e.g., Y. enterocolitica. Thus, the phospholipase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum. The phospholipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae.
Preferred phospholipases are derived from a strain of Humicola, especially Humicola lanuginosa. The phospholipase may be a variant, such as one of the variants disclosed in
In another preferred embodiment the phospholipase is derived from a strain of Fusarium, especially Fusarium oxysporum. The phospholipase may be the one concerned in
In a preferred embodiment of the invention the phospholipase is a phospholipase A1 (EC. 3.1.1.32). In another preferred embodiment of the invention the phospholipase is a phospholipase A2 (EC.3.1.1.4.).
Examples of commercial phospholipases include LECITASE™ and LECITASE™ ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S, Denmark).
The composition may further comprise other enzymes enhancing the detergency of the composition such as softening agents, an amylase (e.g. Fungamyl(R) from Novo Nordisk A/S, Denmark), a lipase (e.g. Novocor(R) AD from Novo Nordisk A/S, Denmark), a cellulase (e.g. Celluzyme(R), Carezyme(R), and/or Celluclast(R), all from Novo Nordisk A/S, Denmark), a xylanase (e.g. Biofeed(R) PLUS or Shearzyme(TM) from Novo Nordisk A/S, Denmark), a beta-glucanase (e.g. Viscozyme(R) or Ultraflo(TM) from Novo Nordisk A/S, Denmark), a pectinase (e.g. Pectinex(TM) Ultra from Novo Nordisk A/S, Denmark), a peroxidase (e.g. Guardzyme(TM) from Novo Nordisk A/S, Denmark), a laccase (e.g. obtained from Myceliophthora or Polyporus), an enhancing agent for the peroxidase/laccase (e.g. PPT or methylsyringic acid methylsyringate or derivatives thereof) and/or a buffer (e.g. citric acid).
The invention will be further described with reference to the following non-limiting examples.
Various Lipase and Rhamnolipid compositions were examined to determine their ability to remove a coloured beef stain.
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 mls 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.
Bacterial enzyme is "Lipomax", a bacterially derived Lipase variant M21 L of the lipase of Pseudomonas alcaligenes as described in
Fungal enzyme is "Lipolase", derived from Humicola languginosa as described in
Details of the surfactants were as follows:
In addition to the commercially supplied Rhamnolipid, two further samples were made up by separating it into R1 and R2 rich fractions. These fractions were also used in combination with the lipases. The cleaning data for 1 hour and 4 hours is given in Tables 1 and 2.
For our analysis of this Rhamnolipid in Table 3, a known amount of JBR425 was acidified to pH 3 using 12M HCl and placed in a refrigerator overnight. The supernatant was then extracted three times using a 2:1 mixture of Chloroform and Ethanol. The solvent was then removed by rotary evaporation and the isolated rhamnolipid mixture was then re-dissolved in methanol.
The process of separating and characterising the mixture was carried out using an HPLC connected to an Ion Trap Electrospray ionisation Mass Spectrometer. The mode of ionisation was in negative mode with a scanning range of 50-1200Da. The column used to separate was a Phenomenex luna C18 250 x 4.6mm 5 µm column. The mobile phase: water (mobile phase A) and acetonitrile (mobile phase B) were used to separate via a gradient of 60:40 (A:B) changing to 30:70 (A:B) over 30 minutes. The system was then held for 5 minutes before returning to the start conditions all at a flow rate of 0.5ml/min. The injection volume was 10 µl.
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.