WO2015091294A1

WO2015091294A1 - Mono-rhamnolipid based compositions.

Info

Publication number: WO2015091294A1
Application number: PCT/EP2014/077650
Authority: WO
Inventors: Mayri Alejandra DIAZ DE RIENZO; Paul Simon Stevenson
Original assignee: Unilever Plc; Unilever N.V.; Conopco, Inc., D/B/A Unilever
Priority date: 2013-12-18
Filing date: 2014-12-12
Publication date: 2015-06-25

Abstract

A bacteriostatic composition comprising a rhamnolipid, the rhamnolipid comprising at least 50 wt % mono-rhamnolipid.

Description

MONO-RHAMNOLIPID BASED COMPOSITIONS.

The present invention relates to an improved bacteriostatic composition

comprising a mono-rhamnolipid.

CN103215207A discloses bacteriostatic rhamnolipid. Despite the prior art there remains a need for improved bacteriostatic rhamnolipid compositions.

Accordingly, and in a first aspect, the present invention provides a bacteriostatic composition comprising a rhamnolipid, said rhamnolipid comprising at least 50 wt % of mono-rhamnolipid.

Mono-rhamnolipids have a single rhamnose sugar ring. The lUPAC 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. The lUPAC 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 the case of rhamnolipids, throughout this patent specification, the prefixes mono- and di- are used to indicate respectively mono-rhamnolipids (having a single rhamnose sugar ring) and di-rhamnolipids (having two rhamnose sugar rings). If abbreviations are used R1 is mono-rhamnolipid and R2 is di- rhamnolipid. The mono-rhamnolipid may be L-rhamnosyl- -hydroxydecanoyl- - hydroxydecanoate (RhaC-ioC-io with a formula of C26H₄8Og) produced by P.

aeruginosa_.

A typical di-rhamnolipid is L-rhamnosyl-L-rhamnosyl- -hydroxydecanoyl- - hydroxydecanoate (Rha2CioCio with a formula of C32H58O13).

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 US5767090: Example 1 , some enzymes can convert mono-rhamnolipid to di-rhamnolipid.

The following rhamnolipids are sources of mono- and di- rhamnolipids

encompassed within the invention (C12:1 , C14:1 indicates fatty acyl chains with double bonds): - Rhamnolipids produced by P. aeruginosa (mono-rhamnolipids):

Rha-Cs-C-io, Rha-C-io-Ce, Rha-C-io-C-ιο, Rha-C-io-C-12, Rha-Cio-Ci2:i j Rha- C12-C10, Rha-Ci2:i-Cio

Rhamnolipids produced by P. chlororaphis (mono-rhamnolipids only):

Rha-C-io-Ce, Rha-Cio"CiOi

Rha- C"i2:i"C"i2j Rha-C"i₄-C"i0i Rha-Ci_4:i-Cio.

- Mono-rhamnolipids may also be produced from P.putida by introduction of genes rhIA and rhIB from Psuedomonas aeruginosa [Cha et al. in Bioresour Technol. 2008. 99(7):2192-9 ]

- Rhamnolipids produced by P. aeruginosa (di-rhamnolipids):

Rha-Rha-C₈-Cio, Rha-Rha-C₈-Ci_2:i , Rha-Rha-Ci₀-C₈, Rha-Rha-Ci₀-Ci₀,

Rha-Rha-Cio-Ci2:i , Rha-Rha-Ci₀-Ci₂, Rha-Rha-Ci₂-Ci₀, Rha-Rha-Ci_2:i- C"i2i Rha-Rha-Cio"Ci_4:i - Rhamnolipids produced by Burkholdera pseudomallei (di-rhamnolipids only):

Rhamnolipids produced by Burkholdera (Pseudomonas) plantarii (di- rhamnolipids only):

Rha-Rha-C ₄-C ₄.

- Rhamnolipids produced by P. aeruginosa which are initially unidentified as either mono- or di-rhamnolipids:

Ce-Ce, C-8-C-10, C-10-C-8, C8-Ci2:1 , Ci2:1-C8, CiO"Cio, Ci2"Cio, Ci2:1-C-|0, C-|2" Ci2, Ci2:1-Ci2, C-|₄-C-|0, Ci_4: Cio, C14-C14.

Preferably, the rhamnolipid comprises at least 50 wt % mono-rhamnolipid, more preferably 60 wt %, even more preferably 75 wt % and most preferably at least 90 wt % mono-rhamnolipid.

The composition according to the invention can be used as bacteriostatic raw material such that the user e.g. consumer dilutes in a further composition or the composition may be a consumer product the application of which is intended to provide bacteriostatic effect to a substrate or even as a preservative within the consumer composition.

The composition may comprise further surfactants.

Preferably the or each further surfactant comprises a synthetic anionic surfactant. 'Anionic surfactants' are defined herein as amphiphilic molecules comprising one or more functional groups that exhibit a net anionic charge when in aqueous solution at the normal wash pH of between 4 and 1 1 .

Preferably the alkali metal salts of organic sulphur reaction products having in their molecular structure an alkyl moiety containing from about 6 to 24 carbon atoms, more greater than 12 carbon atoms and preferably also a moiety selected from the group consisting of sulphonic and sulphuric acid ester moieties.

Additionally or alternatively, the anionic surfactant preferably has low levels of ethoxylation, preferably comprising 1 -12 ethylene oxide units per molecule, more preferably 1 -3 and even more preferably 1 . The units of ethylene oxide may be an average.

Although any anionic surfactant hereinafter described can be used, such as primary alkyl sulphates (PAS) e.g. sodium lauryl sulphate (SLS) and e.g. alkyl ether sulphate such as sodium lauryl ether sulphate(SLES), soaps, fatty acid ester sulphonates, fatty acid sulphates or sulphonates; alkyl benzene sulphonates (LAS), sulphosuccinate esters, olefin sulphonates, paraffin sulphonates and organic phosphates; fatty alcohol sulphates; alkyl phenol ether sulphate; fatty acyl isethionate products which products comprise fatty acyl isethionate and free fatty acid and/or fatty acid salt; alkyl sulphonates such as sodium alkane sulphonate. Preferred anionic surfactants are the alkali (ammonium or triethylammonium for example) and alkaline earth metal salts of the above. The source oil / alcohol can be plant or animal derived for example coconut or palm or tallow etc. The further surfactant may also comprise a non-ionic, cationic and/or amphoteric and mixtures thereof.

Preferably the rhamnolipid is present at 1 wt % - 95 wt % of the total surfactant (rhamnolipid and further surfactant) in the composition. More preferably the rhamnolipid is present at 10 - 70 wt % of the total surfactant.

Preferably the further surfactant is present in the fabric or hard surface washing compositions at a level of from 3 to 85% by weight, preferably from 3 to 60% by weight, more preferably from 3 to 40% by weight, most preferably from 3 to 35% by weight. Preferably the further surfactant is present in personal (human skin and hair) wash compositions at a level of 5 to 60%, preferably 10 to 40% surfactant, while cosmetic compositions need not comprise any surfactant, but preferably comprise 1 % to 30% by wt., more preferably 1 to 15% by wt. surfactant.

Preferably, the composition is a home care and personal product.

Preferred home care products include laundry detergent/cleaning compositions, laundry conditioning compositions, and hard surface cleaners such as hand and machine dish washing compositions, kitchen and bathroom cleaners. Hard surfaces include kitchen and bathroom surfaces, cutlery, crockery etc.

Personal care compositions include shampoos, hair conditioners, deodorants, skin cleansing compositions and oral care products such as toothpastes and mouthwashes.

The following are non limiting embodiments of the invention, included by way of example only. EXAMPLE 1

The following data illustrates the bacteriostat efficacy of a composition comprising a mono-rhamnolipid. Materials and Methods

Microorganisms and Culture conditions

P. aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 9144, were maintained in nutrient broth plus 20% glycerol at -20°C. Bacterial growth from a nutrient agar slant incubated for 24h at 30°C was used to obtain a bacterial suspension with an optical density at 570nm adjusted to give 108 cfu/mL. Rhamnolipid Characteristics

Rhamnolipid containing 10% (w/v) mono-rhamnolipid (C26H₄8O9, MW: 504, Critical Micelle Concentration: 20 mg/L M at neutral pH) and 10% (w/v) dirhamnolipid (C₃2H58O ₃, MW: 650, CMC: 1 .5x10-4 30 mg/L at neutral pH) was obtained separated from a sample obtained from Jeneil Biosurfactant Co. (Saukville, Wisconsin).

The Mono- and di-rhamnolipid was separated from the sample obtained from Jeneil using the protocol below and the individual R1/ R2 fractions obtained were used to produce the 10% rhamnolipid solution mentioned above

A quantified amount of JBR425 was acidified to pH 3 using 12M HCI 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 μιτι 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 μΙ. Table 1 - Analysis of JBR425 via HPLC/MS

Growth of "static" biofilms on Oxoplates®.

P. aeruginosa ATCC 15442 was grown overnight as previously described and diluted 100-fold with TSB 50% following which a 100μΙ_ sample of each diluted culture was dispensed (eight replicates) to fill a 96 well Oxoplate OP96C®. After 48h each well was rinsed twice with PBS 1 XS, and the active different treatments were added to each well during 30min at 30°C. OxoPlate OP96C (PreSens, Regensburg, Germany) contains oxygen-sensitive particles PSLi-Pt-1 (Opto- Sense, Worth, Germany), which consist of small polystyrene particles. The sensor has a thickness of about 10μηη and is fixed at the bottom of each well of a 96-flat bottom-well plate (Greiner, Frickenhausen, Germany). The oxygen concentration in each well was followed for 21 h at 20min intervals. Fluorescence of each well was measured in dual kinetic mode (BMG Labtech GmbH, Germany). Filter pair 1 (544/650 nm) detects fluorescence of the indicator dye. The second filter pair (544/590 nm) measures fluorescence of the reference dye. All experiments were repeated on independent days. Oxygen concentration as percentage air saturation was calculated for each well by using the following equation:

Effect of different biosurfactants on oxygen consumption by Biofilms of Pseudomonas aeruginosa ATCC 15442.

Biofilms of Pseudomonas aeruginosa ATCC 15442 were formed on an Oxoplate® after 48h at 30°C, once the biofilms were rinsed with PBS 1 X twice they were incubated for half an hour under different treatments as follows:

Biosurfactants used to determinate the oxygen consumption of biofilms of

Pseudomonas aeruginosa ATCC 15442.

TREATMENT CONCENTRATION (v/v)

Phosphate Buffered Saline Solution 1X - Made up in the following manner

Preparation of PBS 1X

NaCI - (8gpl) final concentration at 1 X = 137mM

KCI - (0.2gpl) final concentration at 1 X = 2.7mM

Na₂HPO₄ - (1 .44gpl) final concentration at 1 X = 10mM

KH₂PO - (0.24gpl) final concentration at 1X = 1 .8mM To prepare 1 L of PBS 1 X dissolve the reagents listed above in 800 ml_ of H₂O. Adjust the pH to 7.4 with HCI, and then add H₂O to 1 L. Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1 .05 kg/cm²) on liquid cycle. Mono-Rhamnolipids 0.04% were produced by dilution of R1 (mono - rhamnolipid) Stock solution. Di-Rhamnolipids 0.04% were produced by dilution of R2 (Di rhamnolipid) stock solution.

A minimum number of cells were required to consume a threshold amount of oxygen before they were detected in the system. All the results were beyond this threshold (high inoculum density), in consequence consumption of oxygen was detected immediately and the growth medium was essentially free of oxygen after 1 h, in presence of all treatments used. As described above, oxygen consumption was quantified for mono- and di-rhamnolipids at 0.04% v/v. The results are shown in Table 2 below.

Table 2 Oxygen Consumption for mono- and di-rhamnolipids.

Time (Mins)

0 30 60 90 120 150 180

P02 (%)

Air

Saturation

PBS 43.31053 5.046361 5.74107 5.494804 5.615807 5.167131 5.269409

DiRhl 48.68312 71.48805 80.82274 87.44776 91 .24868 96.53764 96.62697

MonoRhl 65.60897 98.40783 100.0019 96.96805 87.2077 53.74983 10.85155

Time (Mins)

210 240 270 300 330 360

PBS 5.305869 4.780303 4.710532 4.556791 4.31215 4.189293

DiRhl 99.2276 99.38459 99.7057 99.00303 101.3567 101 .9028

MonoRhl 6.821 1 12 6.787163 6.368684 6.028308 5.86853 5.841625 The results of Table 2 are shown in Fig. 1 , in which the curves also give insight into the kinetics of bacterial growth inhibition. The cells with mono-rhamnolipid treatment showed a delay of oxygen consumption during the first 2h of active growth, however the oxygen level in each well declined gradually to 0% and stayed at this level, behaviour typical of bacteriostatic compounds. On the other hand, the cells treated with di-rhamnolipid displayed a strikingly dissimilar pattern, because of the initial effect after addition of the treatment when the oxygen concentration increased. This increase is attributable to the enhanced diffusion of atmospheric oxygen into the wells after cell death indicating bactericidal, and not bacteriostatic action.

Claims

A bacteriostatic connposition comprising a rhamnolipid, the rhamnolipid comprising at least 50 wt % mono-rhamnolipid.

Composition according to claim 1 wherein the rhamnolipid comprises at least 60 wt% mono-rhamnolipid.

Composition according to claim 1 wherein the rhamnolipid comprises at least 75 wt% mono-rhamnolipid.

Composition according to claim 1 wherein the rhamnolipid comprises at least 90 wt% mono-rhamnolipid.

Composition according to any preceding claim including a further surfactant comprising a synthetic anionic surfactant.

Composition according to any preceding claim wherein the rhamnolipid is present at 1 wt % - 95 wt % of the total surfactant, preferably 10 - 70 wt %.

Composition according to any of claims 1 to 6 which is a homecare or personal care composition.

Composition according to claim 7 which is a personal care compositon, preferably selected from a shampoo, conditioner, deodorant, skin cleansing composition, antiperspirant.

Composition according to claim 7 which is a homecare composition preferably selected from a laundry composition and hard surface cleaner. Use of a composition according to any of claims 1 to 9 for bacteriostatic prevention or disruption of microbial growth.