FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
AN ANTIMICROBIAL COMPOSITION
HINDUSTAN UNILEVER LIMITED, a company incorporated under
the Indian Companies Act, 1913 and having its registered office
at 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed.

Technical Field
The invention relates to transparent/translucent liquid antimicrobial composition.
Background of the invention
Hygiene has been and continues to be one of the most important requirements among consumers. Consumers all over the world use different types of antimicrobial compositions for disinfecting various surfaces like hard surfaces in the homes, factories and offices e.g. kitchen tops, floors and furniture; soft and porous surfaces like clothes, carpets and upholstery; and personal surfaces like skin and hair.
Many micro organisms like bacteria and virus are found on such surfaces some of which are pathogenic such as Escherichia coli, also referred to as E. coli, and Staphylococcus aureus, also referred to as S. aureus. Consumers like to keep surfaces free of such germs so that they can live without fear of contacting debilitating diseases. For cleaning hard surfaces the most commonly known disinfectants in house hold care are hypochlorite salts. However hypochlorite causes bad smell, bleaches clothes that come in contact with it and is unpleasant to skin which is mostly caused by its high alkalinity. Surfactant based detergent compositions are often used to clean soft surfaces like fabrics and such compositions often include bleaches like peroxides. For personal hygiene, soap is often used for disinfecting skin.
Many of the above compositions are considered harsh and consumers are on the look out for milder solutions to maintain their hygiene. The other problem with such compositions is that a lot of water is required to rinse away the surfactant and/or the bleach present in those compositions.
One class of products which overcomes the above problems is class of liquid disinfectants. This is a class of products where the compositions are predominantly water or a mix of water and solvents with a small amount of disinfectants which may synthetic or naturally derived.

Such products comprising naturally derived disinfectants e.g. essential oils are especially preferred since using the right combination not only provides the high level of disinfection desired but also give the consumers the satisfaction of having used natural products which they believe have fewer side effects on them as they handle the product and/or minimally impact the environment.
Such natural actives like essential oils are insoluble in water and have to be formulated in water with the aid of emulsifiers which lead to a composition which is opaque or turbid and kinetically unstable. Providing such a composition in a transparent or translucent form is highly appealing to the consumer.
In order to facilitate ease of packing and transportation, these products are often manufactured at much higher concentration of antimicrobial actives than are actually required in use. The consumers are instructed to dilute the product with water before actual use. In addition to the providing the consumers with a transparent or translucent composition when packed it is important to provide the consumer with a visual cue that the product has been sufficiently diluted for actual use. Such a cue could be change of the formulation from a transparent/translucent form to the formation of a turbid suspension or cloudiness on dilution to the desired level. Formation of such turbid suspension on dilution, referred to as blooming, is often achieved using high concentration of essential oils e.g. pine oil at concentrations higher than 3% is known to bloom when diluted with water. However, inclusion of such high concentrations of essential oils are sometimes undesirable as they have certain other negative connotations of off-odour, incompatibility with other ingredients, and instability of emulsions/ microemulsions Therefore, there is a need in the art to provide compositions where the antimicrobial agents like essential oils are used at low concentrations which not only cause blooming on dilution with water but ensures the desired anti-microbial action at such low concentrations.

The present inventors have been working on solving this problem of providing such a disinfectant composition that is transparent when packed and forms a turbid suspension when diluted with water and have come up with a novel solution in the form of the present invention.
The present invention provides a solution to the above problem by way of incorporating the antimicrobial agents in low concentration in a specific microemulsion composition that comprises selective amount of a surfactant, an specified class of organic solvents and a clouding agent selected from fatty acid, or alkyl ester of fatty acid that ensures solubility of the antimicrobial agents oils in the composition thus ensuring a transparent appearance when stored and transported and gives a turbid/cloudy appearance when diluted with water, to provide a visual cue to the consumer about the delivery of the antimicrobial agents. Further, the turbidity is ensured in short time span after the composition is diluted with water.
Compositions comprising some of the above components with such essential oils in either a microemulsion or emulsion format have been reported viz. US5591708, US2005107278, US2009281012, US2006084589 and US5486307. However, the compositions reported in these publications do not have all of the constituents of the present invention thereby unable to provide either the transparent appearance or the turbid appearance on dilution with water.
GB 2320927 discloses a blooming type, germicidal hard surface cleaning and disinfecting concentrate composition which comprises (a) 0.1 to 10%wt of a terpene containing solvent which desirably includes both pine oil and d-limonene; (b) 0.1 to 12%wt. at least one non-ionic surfactant constituent which desirably includes at least one non-ionic surfactant which exhibits a cloud point in water of less than 20 °C; (c) 0.1 to 5% at least one amphoteric surfactant selected from imidazoline and amino propionate surfactants; at least one germicidal cationic surfactant; and water.

The blooming in the above patent publication is achieved due to the high concentration of the essential oil viz. pine oil constituent.
It is thus an object of the present invention to provide for an anti-microbial composition that has a clear transparent / translucent appearance and becomes turbid on dilution with water.
It is another object of the present invention to provide for an anti-microbial composition that has a clear transparent/ translucent appearance and quickly changes to have a turbid appearance when diluted with water.
It is yet another object of the present invention to provide for an anti-microbial composition that comprises natural anti-microbial actives at low concentration that has a clear transparent/translucent appearance and becomes turbid on dilution with water.
Summary of the invention
The first aspect of the present invention provides for a transparent/ translucent liquid antimicrobial composition in microemulsion format capable of forming turbid suspension on dilution with water comprising (i) 0.05% to 2% of an antimicrobial active selected from the class comprising
terpenes, essential oils, or cationic oils having a solubility in water of less than
2000 ppm at 25°C; (ii) 0.05 to 10% of a surfactant; (iii) 1 to 50 % of an organic solvent selected from linear or branched alcohol having
a Log P (Experimental octanol / water partition coefficient) value in the range of
-1.5 to 3.5; (iv) a clouding agent selected from the class comprising C8 to C22 fatty acid or
alkyl esters of fatty acids. (v) 40 to 99% water.

The second aspect of the present invention provides for use of a composition of the first aspect of the present invention for disinfecting external surfaces of human or animal body, for disinfecting soft and porous substrates like fabric or for disinfecting hard surfaces.
The third aspect of the present invention provides for a method of disinfecting a surface comprising the steps of
(i) applying a composition as claimed in the first aspect of the present
invention on to a desired surface followed by; (ii) removing the composition from said surface after a predetermined time period.
Detailed description of the invention
These and other aspects, features and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description and the appended claims. For the avoidance of doubt, any feature of one aspect of the present invention may be utilized in any other aspect of the invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of or "composed of." In other words, the listed steps or options need not be exhaustive. It is noted that the examples given in the description below are intended to clarify the invention and are not intended to limit the invention to those examples per se. Similarly, all percentages are weight/weight percentages unless otherwise indicated.
Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this description indicating amounts of material or conditions of reaction, physical properties of materials and/or use are to be understood as modified by the word "about". Unless specified otherwise, numerical ranges expressed in the format "from x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "from x to y", it is understood that all ranges combining the different endpoints are also contemplated.

The composition of the invention may be formulated in any product primarily for disinfecting surfaces but may also be formulated in products that also provide other benefits like cleansing, odor control or general aesthetics. The composition of the present invention is generally in the form of a liquid but may be thickened to a gel. Non-limiting examples of compositions include disinfectant liquids, shampoos, conditioners, shower gels, and deodorant liquids,
The invention provides for a transparent/translucent liquid antimicrobial composition in microemulsion format capable of forming turbid suspension on dilution with water comprising a selective antimicrobial active, a surfactant, a selective organic solvent, a selective clouding agent and water.
The microemulsion composition of the invention is preferably transparent. This format of the composition is especially preferred since the transparent form makes for an appealing visual impact on consumers. In a preferred aspect, the dispersed phase droplets in the microemulsion are in the size range of 6 to 50 nm, more preferably in the size range of 6 to 15 nm.
The antimicrobial agent is selected from the class comprising terpenes, essential oils, or cationic oils having a solubility in water of less than 2000 ppm at 25°C. Examples of aromatic essential oils suitable for use in the present invention include amyl salicylate, carvacrol, cymene, e.g. p-cymene, dihydroeugenol, eugenol, hexyl eugenol, hexyl salicylate, isoeugenol, methyl eugenol, methyl isoeugenol, methyl salicylate, tert butyl cresol, thymol, and vanillin. Examples of non-aromatic essential oils of terpenoid compounds include cedrene, cineole, citral (including geranial and neral), citronellal, citronellol, eucalyptol (also known as 1,8 cineole) paradihydrolinalool, dihydromyrcenol (DH myrcenol), farnesol, geranio!, hexyl cinnamaldehyde, hydroxycitronallol, hydroxycitronellal, isocitral, limonene, preferably d-limonene, linalool, longifolene, menthol, nerol, nerolidiol, pinene, e.g. α-pinene, phellendrene, terpinene, e.g. a-terpinene and v-terpinene, terpineol, e.g. y-terpineol and terpin-4-ol, and tetrahydromyrcenol (THM).

Preferred cationic oils include Quaternary Ammonium Cationic Vegetble Oil and Charged aminopolydimethylsilane of the chemical formula (CH3)3-Si-[Si(CH3)2-O]-[Si(CH3)-((CH2)3-NH-(CH2)2-NH2)-O]2-Si-(CH3)3. Preferably the solubility of the antimicrobial active is less than 2000 ppm at 25°C. More preferred antimicrobial actives are terpineol, thymol, eugenol, borneol or limonene and mixtures thereof. Furthermore preferred antimicrobial actives are terpineol, thymol or eugenol. An especially preferred composition comprises thymol and terpineol. An even further preferred composition comprises thymol, terpineol and eugenol. The antimicrobial active is present in 0.05 to 2%, more preferably 0.1 to 2% by weight of the microemulsion composition.
The microemulsion composition of the invention comprises 0.05 to 10%, preferably 0.05 to 8 %, more preferably 0.5 to 5% surfactant by weight of the composition. The surfactant is preferably of the cationic, anionic, or zwitterionic class. Cationic or anionic surfactant is more preferred, most preferred is of the cationic class. When anionic surfactant is present it is preferably chosen from alkali or alkaline earth metal salts of alkyl sulphonic acid, fatty acid, or alkyl ether sulphate. When zwitterionic surfactant is present it is preferably chosen from betaines, sulphobetains, hydroxyl sulphobentains, or amino carboxylates. When a cationic surfactant is present, it is preferably benzalkonium chloride, alkyl pyridinium chloride or quaternary ammonium gemini surfactants.
The microemulsion composition of the invention comprises 1 to 50% of an organic solvent selected from linear or branched alcohol having a Log P value in the range of -1.5 to 3. Log P is a property defined in the article Hiroshi Chuman, Atsushi Mori and Hideji Tanaka, "Prediction of the 1-Octanol/H20 Partition Coefficient, Log P, by Ab Initio MO Calculations: Hydrogen-Bonding Effect of Organic Solutes on Log P", Analytical Sciences, September 2002, Vol. 18, 1015-1020. Log P is basically the 1-octanol/water partition coefficient and is experimentally determined by the shake-flask method. Preferred alcohols have a carbon chain length of 1 to 7.

Examples of such organic solvents are ethyl alcohol, isopropyl alcohol, octanol, or propyl alcohol. The useful alcohols and their log P values are given in Table below.

Alcohol log P at 25°C Vapour Pressure mm Hg @ 25°C
Ethyl alcohol -0.32 83
Isopropyl alcohol 0.42 44
Octyl alcohol 3.13 0.07
Preferred organic solvents have a log P value from -0.1 to 3. Most preferred alcohols are ethyl alcohol or isopropyl alcohol.
The solvent used in the microemulsion composition of the invention preferably has a vapor pressure greater than or equal to 0.05 mm Hg at 25°C. The organic solvent is preferably present in 1 to 45%, more preferably 1 to 30 % by weight of the composition.
A selective clouding or blooming agent is included in the microemulsion composition of the invention. The clouding agent has to be carefully selected such that the microemulsion stability and concomitant transparency are not compromised while ensuring the desired quick precipitation when diluted with water. Without wishing to be bound by theory it is believed that the solvent and the clouding agent interact synergistically with the surfactant across the various boundaries of the multi-component phase equilibria to ensure the desired microemulsion stability in the storage stage while ensuring clouding and turbidity on dilution. All of the above desirable properties are achieved while ensuring that the antimicrobial efficacy of the composition is not compromised even at the low concentration of the antimicrobial active present.

The clouding agent is selected from the class comprising C8 to C22 fatty acid or alkyl esters of fatty acids. Preferred clouding agents have carbon chain length 8 to 22 more preferably 8 to 16 Most preferred fatty acids/ fatty acid esters are caprylic acid, lauric acid or methyl/ethyl caprylate The clouding agent is preferably present in 0.5 to 10 %, more preferably in 0.5 to 5% by weight of the composition. Clouding agent preferably has a melting point greater than 25°C. The clouding agent preferably has a solubility in water of less than 2000 ppm in water at 25°C.
The composition of the invention preferably comprises 40 to 99%, preferably 50 to 80%, further more preferably 50 to 70 % water by weight of the composition.
It is an optional aspect of the invention to include an electrolyte in the composition of the invention. Suitable electrolyte may be chosen from a chloride, sulphate, acetate, or carbonante of an alkali, alkaline earth or transition metal. Of these, multivalent electrolyes are especially preferred. More useful electrolytes include chloride, sulphate or acetate of an alkaline earth metal. Examples of electrolytes useful in including in the composition of the invention are calcium chloride, sodium chloride, magnesium chloride, zinc sulphate, aluminium chloride or zinc acetate. The more preferred electrolytes are calcium chloride, magnesium chloride, zinc sulphate or zinc acetate while the most preferred electrolytes are zinc sulphate or zinc acetate. Electrolyte is preferably present in 0.005 to 10%, more preferably 0.05 to 10 %, further more preferably 0.05 to 5 % by weight of the composition. Without wishing to be bound by theory it is believed that the inclusion of the electrolyte in the microemulsion composition of the invention allows for meeting all of the objects of the invention while minimizing the amount of solvent to be used.
The microemulsion composition of the invention is usually to be used for disinfecting a surface by first diluting the composition in water. The preferred weight ratio of composition: water for the dilution step is in the range of 1:1 to 1:200, more preferably 1:5 to 1:50 and optimally 1:15 to 1:30 and ideally about 1:20.

By the term transparent or translucent microemulsion composition is meant that the composition has a turbidity value of 0 to 200 NTU. The turbidity was measured using a Merck Turbiquant 1500T turbidity meter. On diluting with water, it is preferred that the turbidity increases to greater than 500 NTU in less than 5 seconds, preferably less than 2 seconds, more preferably less than 1 second, most preferably immediately after the dilution step.
The composition of the invention preferably has a pH ranging from 3 to 8, more preferably from 3.5 to 8 at 25QC.
According to another aspect of the invention there is provided a method of disinfecting a surface comprising the steps of (i) applying the composition of the invention to a desired surface followed by (ii) removing the composition from the surface after a predetermined time period. The composition is preferably diluted with water before applying on the desired surface. The dilution ratio is preferably in the range of 1:1 to 1:200, more preferably 1:5 to 1:50 and optimally 1:15 to 1:30 and ideally 1:20. Suitable surfaces for disinfecting include external surfaces of human or animal body and soft and porous substrates like fabric and hard surfaces. More suitable surfaces include animate surfaces like the externa! surfaces of animal or human body e.g. skin and hair or inanimate surfaces like hard surfaces e.g. floors, furniture, kitchen tops, utensils and soft/porous surfaces e.g. clothes/ fabric, carpets or upholstery. The composition/ diluted composition is then removed from the disinfected surface. By removing is meant that it is wiped off from the surface using a suitable materia! or implement e.g. a sponge, fabric or a wipe. Alternately the composition/ diluted composition is rinsed off from the surface using desired amount of water. The predetermined time for removing the composition from the surface may vary according to the amount of cleanliness desired and the type of surface but may vary from 5 to 3000 seconds, preferably 15 to 120 seconds.

According to another aspect of the invention there is provided use of the composition of the invention for disinfecting external surfaces of human or animal body, for disinfecting soft and porous substrates like fabric or for disinfecting hard surfaces. The use is preferably non-therapeutic.
The invention will now be illustrated with the help of the following non-limiting Examples
Examples
Examples 1 to 7: Comparison of the antibacterial efficacy and desired turbidity of compositions as per the invention as compared to a composition out side the invention.
The following compositions, as shown in Table -1 were prepared. The number presented are in % by weight of the composition.

Table-1:
Example 1 2 3 4 5 6 7
Terpineol 1 1 1 1 1 1 1
Thymol 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Eugenol 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Benzalkonium Chloride 3 3 3 3 3 3 3
Isopropyl alcohol 7 18 23 30 23 16 23
Caprylic acid 0 2.5 2.5 0 0.5 0 1.0
Methyl caprylate 0 0 0 2.5 2.5 0 1.5
Octanol 0 0 0 0 0 2.5 0
Buffer of 5.25 PH 0 0 @ 0 0 0 -
Water To 100 To 100 To 100 To
100 To 100 To 100 To
100
PH 6.8 3.7 4.5 6.5 4.2 6.4 4.1

@The buffer used was a mixture of citric acid (1M) and sodium citrate (1M) at a ratio of<=37:13.
The composition was tested for the following desired properties.
Efficacy of antibacterial kill:
The compositions were tested for antibacterial kill using the following protocol.
Time/Contact Kill assay
Organism; E, coli,
Test System: Distilled water (Dw) 8ml
Contact Time: 30 sec
Neutralizer: D/E broth (9ml)
Method: Pour plating (CY agar)
Principle: To check the antibacterial activity of a antiseptic solution in suspension
at specific time points (invitro).
Procedure:
Prepare a suspension of 0.8OD 620nm of mid-log phase E. coli culture (~108 cells/mi)
Dilute the composition to 9 ml of a suitable concentration and mix well.
Add 1ml of 0.8 OD 620nm culture suspension into the system (10ml), mix well and
allow contact for 30 sec.
At the time point (30 sec) take 1ml from the above system and neutralize by adding
into 9 ml D/E broth, vortex.
Plate different dilutions in CY agar.
The compositions having greater than 5 log kill of bacterial were considered as acceptable.
Turbidity on dilution:
The compositions were tested to check if the desired turbidity was attained on diluting
the composition. The test procedure used was as follows.

200 ml deionized water was taken and desired composition was added in the sequence of 1 ml in a stirred condition. After each addition of the composition, turbidity was measured by taking a well mixed sample. All the measurements were performed as quickly as possible to avoid further segregation and coalescence of droplets. Temperature was kept constant at about 25°C.
The data on the efficacy and turbidity of the various compositions 1 to 7 is summarized in Table - 2. When the efficacy passed the criterion of 5 log kill of bacteria the sample is evaluated as 'Yes'. If the turbidity passed the criterion of >800 NTU, the sample was evaluated as 'Yes', else as 'No'.

Table - 2
Example 1 2 3 4 5 6 7
Efficacy Yes Yes Yes Yes Yes Yes Yes
Turbidity No Yes Yes Yes Yes Yes Yes
The data in Table - 2 indicates that samples as per the invention (Examples 2 to 7) provide for the desired antimicrobial efficacy and turbidity on dilution while the composition outside the invention (Example - 1) does not provide all the desired functionalities.

Examples 8 to 10: Other useful compositions as per the invention
Compositions as per the invention were prepared as shown in Table - 3. Table - 3
Example Example- 8, wt% Example- 9, wt% Example- 10,
wt%
Terpineol 1 0 1
Thymol 0.5 0 0.5
Eugenol 0.1 0 0.1
Limonene 0 1.0 0
Benzalkonium chloride 1.5 3.0 0
Isopropyl alcohol 20 25 20
C12E07 1.5 0 0
SLES 0 0 1.5
Caprylic acid 2.5 2.5 2.5
Water To 100 To 100 To 100
The compositions of Table -3 exhibited the desired anti-bacterial efficacy and turbidity on dilution.
The invention thus provides for an anti-microbial composition comprising natural actives that has a clear transparent / translucent appearance and becomes turbid on dilution with water, in very quick time.

Claims
1. A transparent/transluscent liquid antimicrobial composition in microemulsion
format capable of forming turbid suspension on dilution with water comprising
(i) 0.05 to 2% of an antimicrobial active selected from the class comprising
terpenes, essential oils, or cationic oils having a solubility in water of
less than 2000 ppm at 25°C; (ii) 0.05 to 10% of a surfactant; (iii) 1 to 50% of an organic solvent selected from linear or branched alcohol
having a Log P (Experimental octanol/ water partition coefficient) value
in the range of -1.5 to 3.5 ; (iv) a clouding agent selected from the class comprising C8 to C22 fatty acid
or alkyl esters of fatty acids; and (v) 40 to 99% water.
2. A composition as claimed in claim 1 wherein said surfactant is either a cationic, anionic, or zwitterionic surfactant.
3. A composition as claimed in claim 2 wherein said surfactant is cationic.
4. A composition as claimed in claim 2 wherein said cationic surfactant is benzalkonium chloride or alkyl pyridinium chloride or quaternary ammonium gemini surfactants.
5. A composition as claimed in any one of the preceding claims wherein said antimicrobial active is terpineol, thymol, eugenol, borneol, or limonene.

6. A composition as claimed in any one of the preceding claims wherein said solvent has a vapour pressure greater than or equal to 0.05 mm Hg @ 25°C.
7. A composition as claimed in anyone of the preceding claims wherein said solvent is a linear or branched alcohol having 1 to 7 carbon atoms.
8. A composition as claimed in claim 7 wherein said organic solvent is ethyl alcohol or isopropyl alcohol.
9. A composition as claimed in any one of the preceding claims comprising 0.01 to 10% electrolyte.
10. A composition as claimed In claim 9 wherein said electrolyte is chosen from chloride, sulphate, acetate, or carbonate of an alkali, alkaline earth or transition metal.
11. A composition as claimed in claims 9 or 10 wherein said electrolyte is a multivalent electrolyte.
12. A composition as claimed in any one of the preceding claims having a pH ranging from 3-8 at 25°C.

13. A method of disinfecting a surface comprising the steps of
(i) applying a composition as claimed in any one of the preceding
claims to a desired surface followed by; (ii) removing the composition from said surface after a predetermined
time period.
14. A method as claimed in claim 13 wherein said composition is diluted with water
in a ratio of 1:5 to 1:50 before applying on said substrate.