Field of the Invention

The present invention relates to a composition that is used to deliver hygiene on surfaces. Particularly, the present invention relates to a composition that delivers immediate kill of germs as well as over extended period of time which is evident from being able to solve the really difficult problem of biofilm removal.

Background of the Invention

Making hard surfaces hygienic is important for people inhabiting homes and using other common spaces like offices, restaurants, hotels and public places like bus and train stations and airports. This generally involves using a disinfectant composition that is diluted with water before use. Horizontal surfaces are disinfected by applying the diluted composition. They may then be left to air dry or are wiped dry with a clean cloth or mop. Vertical surfaces may be similarly disinfected by spraying the composition on to the surface followed optionally by wiping. Hard surfaces in such places include utensils which may be made of steel, ceramic, glass etc. Other important hard surfaces in such places include floors, walls, windows, kitchen platforms, and furniture among others. Such places also, many a time, remain with an unpleasant stain after the step of cleaning, in spite of use of products having very good cleaning and disinfecting agents included therein.

Toilets and bathrooms are places which especially require very frequent cleaning and disinfection as they are places for excrement and personal cleansing. Toilet cleaners include harsher chemicals like bleaches and strong antimicrobial actives which are not often liked by many consumers.

Washing fabrics, which have soft surfaces, also known as laundering or simply as laundry is a daily chore for most people. Typically, a fabric is washed by contacting it with a detergent composition either in neat or diluted form, rinsing the fabric one or more times with water; and finally, drying it. After the wash step involving a detergent composition, the fabric is rinsed free of the composition using copious amounts of water, two or three time. In order to provide various benefits like softening, antimicrobial, odour control, lingering perfume impact, whitening etc., fabric conditioning compositions may be added in the rinse stage, usually during the last rinse stage. In spite of using highly efficient antimicrobial agents including chlorine based bleaches, the problem often encountered in disinfecting any of the surfaces discussed in the preceding paragraphs is that while these products are good for providing instant kill of germs, they are not very effective in ensuring long term hygiene by way of inhibiting or killing the growth of germs that remain after the initial cleaning or of germs that may colonize the surfaces thereafter. In surfaces like difficult to reach corners that are not cleaned too often, stains accumulate which are also difficult to remove unless harsh chemicals like chlorine based bleaches are used. Biofilm formation is a phenomenon where tough stain are formed as a consequence of proteins or carbohydrates that stick on to the surface often including therein bacteria that help the biofilm adhere very strongly on to such surfaces.

Biofilms are microbial aggregates which include heterogeneous populations of bacteria, fungi, and algae. They are ubiquitous in nature and represent populations of cells that have functional interdependencies upon one another which collectively offer microbial activities that are not possible by any of the individual component species. The biofilm matrix surrounding bacteria and fungi makes them tolerant to harsh conditions and resistant to antimicrobial treatments. The formation of microbial biofilm presents a challenge to the establishment and maintenance of hygienic conditions in public health, the home and in industry. In indoor environments, biofilms are formed on surfaces which include kitchen surfaces, drainages and sinks, dishwash sponges, refrigerators, washing machines, toilets, damp surfaces and ceilings and poses a threat to human health. Biofilms can thus form on hard as well as on soft surfaces. The occurrence of many biofilm-based infections and their multiple antimicrobial resistance is a major concern to human health. It is critically important to design or screen anti-biofilm molecules or compositions that can effectively minimize and eradicate biofilm related infections.

Sometimes very aggressive mechanical scrubbing may be required in addition to harsh chemicals to remove such tough biofilms.

Thus, it is relatively easier to provide cleaning benefit (removal of dirt and oily/ particulate soil from surfaces). It gets progressively more difficult to ensure sufficient disinfection of germs (bacteria, virus and fungi) freely presently on surfaces. It is even more difficult to disrupt biofilms and kill the germs present therein.

There is thus, a need in the art, to provide for a solution to the more difficult problems listed above like immediate kill of germs, and removal of tough biofilms. The above problems are even more difficult to solve if the compounds and ingredients used are not considered harsh i.e are mild on skin and have acceptable sensory properties like having no offensive chlorine smell. The present inventors have now surprisingly found that a novel combination of conventional antimicrobial agents like cationic surfactants and bacterial spores solves at least a part of the problems mentioned above.

US6165965 (2000, Spartan) discloses an aqueous disinfectant and hard surface cleaning composition comprising an effective disinfecting amount of a quaternary ammonium compound; an effective amount of a spore forming microbial composition; and an effective water dispersing amount of a surfactant.

While the above publication discloses a composition which is effective in providing hygiene benefits, the present inventors have found that the novel composition invented herein is superior to the above, not only in hygiene benefits (both immediate as well as long term kill) but is vastly superior in biofilm removal.

It is therefore an object of the present invention to provide for a hygiene composition for cleaning surfaces that provides immediate as well as long lasting hygiene benefits against growth of germs.

It is another object of the present invention to achieve the above benefits using ingredients that are mild on the skin and have other pleasant sensorials.

It is yet another object of the present invention to provide for a hygiene composition that is able to remove biofilms from hard surfaces like those in homes, toilets and on utensils as well as on soft surfaces like those on clothes and fabrics.

Summary of the Invention

The first aspect of the present invention relates to a hygiene composition comprising

(i) 0.005 to 0.5 wt% a cationic surfactant; and

(ii) 0.00005 to 1 wt% of a combination of bacterial spores comprising Bacillus amyloliquefaciens, Bacillus megaterium, and Bacillus subtilis', and one or both of Bacillus pumilus and Bacillus thuringiensis wherein the cationic surfactant is selected from one or more of alkyl dimethyl benzylammonium chloride (ADBAC), bis (C8-C18) alkyl di methyl quaternary ammonium chloride preferably didecyl dimethyl ammonium chloride (DDAC), cetyl trimethyl ammonium chloride (CTAC), cetyl pyridinium chloride (CPC), cetrimonium bromide, benzethonium chloride (BZE); and dimethyldioctadecylammonium chloride.

According to another aspect of the present invention there is provided a method of ensuring immediate kill of germs as well as biofilm disruption comprising the step of contacting the desired surface with a composition of the first aspect preferably diluted with water.

Detailed Description of the Invention

For the avoidance of doubt, any feature of one aspect of the present invention may be utilised in any other aspect of the invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of” or "composed of". Thus, the term "comprising" is meant not to be limiting to any subsequently stated elements, but rather to optionally also encompass nonspecified elements of major or minor functional importance. In other words, the listed steps or options need not be exhaustive. Whenever the words "including" or "having" are used, these terms are meant to be equivalent to "comprising" as defined above. 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. Except in the 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 "x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "x to y", it is understood that all ranges combining the different endpoints are also contemplated. Unless specified otherwise, amounts as used herein are expressed in percentage by weight based on total weight of the composition and is abbreviated as “wt%”.

The composition of the invention includes a cationic surfactant selected from one or more of alkyl dimethyl benzylammonium chloride (ADBAC) which is also known as benzalkonium chloride (BKC), bis (C8-C18) alkyl di methyl quaternary ammonium chloride preferably didecyl dimethyl ammonium chloride (DDAC), cetyl trimethyl ammonium chloride (CTAC), cetyl pyridinium chloride (CPC), cetrimonium bromide, benzethonium chloride (BZE); and dimethyldioctadecylammonium chloride. Preferred aspect of the invention provides for the cationic surfactant to be chosen from one or more of BKC or DDAC, preferably BKC. They are available as a single quaternary ammonium compound, as well as mixtures of two or more different quaternary ammonium compounds e.g. under trademarks BARDAC™, BARQUAT® and HYAMINE® (all by Lonza); and BTC® (by Stepan). Quaternary ammonium compounds available as a single quaternary ammonium compound include didecyl dimethyl ammonium chloride (available as BARDAC™ 2250 R and BTC® 1010, both 50% active; and BARDAC™ 2280 R and BTC® 1010-80, both 80% active), alkyl dimethyl benzyl ammonium chloride (available as BARQUAT® MB-50, BARQUAT® MX-50, BARQUAT® QJ-50, HYAMINE® 3500, BTC® 50, BTC® 50E, BTC® 65, BTC® 776, BTC® 824, BTC® 835; each 50% active; and the same is available as BARQUAT® MB-80, BARQUAT® MX-80, HYAMINE® 3500-80, BTC® 8248, BTC® 8358; each 80% active

The cationic surfactant is included in at least 0.005%, preferably at least 0.01%, more preferably at least 0.05% and most preferably at least 0.1% by weight of the composition. The cationic surfactant is included in at most 0.5%, more preferably at most 0.4% by weight of the composition. Useful weight% ranges of the cationic surfactant for inclusion in the composition of the invention are 0.01 to 0.5%, preferably 0.05 to 0.5%, and most preferably 0.1 to 0.4% by weight of the composition. It is observed that using concentrations of the cationic surfactant higher than the claimed range adversely affects the viability of the bacterial spores which are included in the composition of the invention

The composition of the invention includes a combination of bacterial spores which is selected from a combination of Bacillus amyloliquefaciens, Bacillus megaterium, and Bacillus subtilis and one or both of Bacillus pumilus and Bacillus thuringiensis. By a combination of bacterial spores, as per this invention, is meant that the spores of the bacteria may be included in the composition.

A preferred aspect of the invention comprises a combination of bacterial spores which includes Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus subtilis and Bacillus pumilus. A further preferred aspect of the invention comprises a combination of bacterial spores which includes Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus subtilis, Bacillus pumilus and Bacillus thuringiensis.

It is preferred that each of the bacterial spores are present in 1 to 70%, more preferably in 5 to 40% of the total number of the the bacterial spores. An even more preferred aspect of the invention relates to the bacterial spores Bacillus amyloliquefaciens, Bacillus megaterium, and Bacillus subtilis being included in 5 to 40% of the total number of the bacterial spores. An especially preferred aspect of the invention relates to the bacterial spores Bacillus pumilus and Bacillus thuringiensis each being included in 1 to 30%, preferably 5 to 20%, most preferably 5 to 15% of the total number of the bacterial spores.

The combination of bacterial spores is included in 0.00005 to 1%, preferably 0.0001 to 1 %, more preferably 0.0005 to 1 %, further more preferably 0.005 to 1% by weight of the composition. The bacterial spores for inclusion in the composition of the invention can be in powder form having a count of around 10 ¹¹ cfu/g, but there could be other methods of growing and introducing the spores in the composition which are well known to a person skilled in the art.

The composition of the invention is preferably applied through use of diluents. Most preferred diluent is an aqueous base which preferably comprises 1 to 99% water. It is particularly prefererd that the pH of the composition is in the range of 5 to 7, preferably in the range of 5 to 6.

General purpose or toilet cleaners

The composition of the invention may be formulated as a general purpose cleaner which is usually used to clean floors (in which case it may be known as a floor cleaner) or used to clean toilets (in which case it may be known as a toilet cleaner). It may also be used to clean other hard surfaces like furniture, table tops, kitchen platforms and other surfaces in homes, offices, restaurants and other public places.

The composition of the invention delivered as general purpose cleaner may further comprise a nonionic surfactant. Typically these can be included for the purpose of stabilising the compositions. Suitable nonionic surfactants include addition products of ethylene oxide and/or propylene oxide with fatty alcohols, fatty acids and fatty amines.

Preferably the nonionic surfactant has an HLB of from about 7 to about 20, more preferably from 10 to 18, e.g. 12 to 16. Genapol™ C200 (Clariant) based on coco chain and 20 EG groups is an example of a suitable nonionic surfactant.

If present, the nonionic surfactant is present in an amount from 0.05 to 20%, more preferably 0.1 to 15 % based on the total weight of the composition.

Ingredients like amphoteric surfactant and sequestrant may be included in the floor cleaner and toilet cleaner composition of the present invention. General purpose cleaners and toilet cleaners may be diluted before use. When diluted, they may be diluted with water in a weight ratio in the range of 1 :10 to 1 :1000. The general purpose or toilet cleaners are usually delivered in liquid, gel, foam or cream format.

Fabric conditioning composition

The composition of the invention may also be delivered to provide the benefits of the invention to clothes or fabric that are laundered. It is preferably delivered through a fabric conditioning composition which additionally comprises 1 to 50% of a fabric softening active. Delivery through a fabric conditioning composition is preferred since it is included in the rinse stage (preferably the last rinse stage) of the laundering process. At this stage the fabric softening active ensures high levels of deposition of the bacteria on the fabric thereby ensuring long lasting hygiene benefit.

The fabric conditioner composition is also referred to as a fabric softener. Fabric conditioners comprise active materials which soften or condition fabric. Examples of suitable fabric softening actives include: quaternary ammonium compounds, silicone polymers, polysaccharides, clays, amines, fatty esters, dispersible polyolefins, polymer latexes and mixtures thereof. The fabric softening active is preferably selected from a quaternary ammonium compound, or silicone polymer and mixtures thereof, more preferably a quaternary ammonium compound.

The fabric softening compounds may preferably be cationic or non-ionic. Preferably, the fabric softening compounds of the present invention are cationic.

Fabric conditioning compositions for use in accordance with the invention may be dilute or concentrated. Dilute products typically contain up to about 6 wt.% of the composition softening compounds, generally about 1 to 5 wt.%, whereas concentrated products may contain up to about 50 wt. % of the composition softening compounds, preferably from about 5 to about 50 wt.%, more preferably from 6 to 25 wt.%. Overall, the products of the invention may contain from 1 to 50 wt. %, preferably from 2 to 25 wt. % of the composition softening compounds, more preferably 2 to 20 wt. % of softening compounds.

The preferred softening compounds for use in fabric conditioner compositions of the invention are quaternary ammonium compounds (QAC). The QAC preferably comprises at least one chain derived from fatty acids, more preferably at least two chains derived from a fatty acids. Generally fatty acids are defined as aliphatic monocarboxylic acids having a chain of 4 to 28 carbons. Preferably the fatty acid chains are palm or tallow fatty acids. Preferably the fatty acid chains of the QAC comprise from 10 to 50 wt. % of saturated C18 chains and from 5 to 40 wt. % of monounsaturated C18 chains by weight of total fatty acid chains. In a further preferred embodiment, the fatty acid chains of the QAC comprise from 20 to 40 wt. %, preferably from 25 to 35 wt. % of saturated C18 chains and from 10 to 35 wt. %, preferably from 15 to 30 wt. % of monounsaturated C18 chains, by weight of total fatty acid chains.

The preferred quaternary ammonium fabric softening compounds for use in compositions of the present invention are so called "ester quats". Particularly preferred materials are the ester-linked triethanolamine (TEA) quaternary ammonium compounds comprising a mixture of mono-, di- and tri-ester linked components.

The composition of the invention delivered as fabric conditioning composition may further comprise a nonionic surfactant. Typically these can be included for the purpose of stabilising the compositions. Suitable nonionic surfactants are those described above as suitable for use in general purpose or toilet cleaning compositions. If present, the nonionic surfactant is present in an amount from 0.01 to 10 wt.%, more preferably 0.1 to 5 wt.%, based on the total weight of the composition. Thus, the fabric conditioning composition of the invention preferably comprises 0.1 to 5 wt% of a non-ionic surfactant preferably an fatty alcohol ethoxylate.

Co-softeners may be used in the fabric conditioning composition. When employed, they are typically present at from 0.1 to 20 wt.% and particularly at from 0.5 to 10 wt.%, based on the total weight of the composition. Preferred co-softeners include fatty esters, and fatty N-oxides. Fatty esters that may be employed include fatty monoesters, such as glycerol monostearate, fatty sugar esters, such as those disclosed WO 01/46361 (Unilever).

The compositions of the present invention may comprise a fatty complexing agent. Especially suitable fatty complexing agents include fatty alcohols and fatty acids. Of these, fatty alcohols are most preferred. Preferably the composition of the present invention comprises 0.5 to 20 wt.% perfume materials by weight of the composition, more preferably 1 to 15 wt.% perfume materials, most preferably 2 to 10 wt. % perfume materials.

The fabric conditioner composition is preferably in an aqueous form. The compositions preferably comprise from 75 to 95 wt.% water

The fabric conditioning composition may be used to treat fabrics either in a hand washing or a machine washing process. Preferably the fabric conditioner is used in the rinse stage of the washing process. Preferably the clothes are treated with a 10 to 100 ml dose of fabric conditioner for a 4 to 7 kg load of clothes. More preferably, 10 to 80 ml for a 4 to 7 kg load of clothes.

The composition of the invention may also be delivered as a rim block. By a rim block is meant a shaped solid composition that is hung on to the rim of the toilet usually in a plastic cage like container so that every time the toilet is flushed, a quantity of water passes over the solid composition, eroding a part thereof before flushing into the toilet bowl. The toilet rim block preferably additionally includes a perfume to provide a lingering pleasant odour after every toilet flush.

Dishwash compositions

The composition of the invention may be useful for upkeep of kitchen items e.g.. utensils and dishes which may be cleaned using a liquid dishwash compositions. The composition is also useful for cleaning and disinfecting filters in dishwashing machines. The various ingredients other than the essential ingredients claimed in the present invention which may be included are anionic surfactants, non-ionic surfactants, and hydrotropes. Additionally, other ingredients like amphoteric surfactants and organic acids, may be included. Preferably, liquid dishwash composition further comprises water in an amount ranging from 5 to 99 wt%, more preferably from 15 to 80 wt%, further more preferably from 30 to 65 wt%, by weight of the composition. Preferably, liquid dishwash composition further comprises one or more sequestrants which may be included in 0.1 to 5 w% by weight of the composition. A preferred sequestrant is a phosphonic acid or a salt thereof. The most preferred sequestrant is 1 -Hydroxyethylidene-1 ,1 - diphosphonic acid (HEDP). It is preferable that the sequestrant is added to the formulation in acid form. Optionally, liquid dishwash composition further comprises an enzyme with or without a suitable enzyme stabilizer. Preferably, viscosity of the composition may suitably range from about 200 to about 10,000 mPa.s at 25°C at a shear rate of 21 sec ¹ . The liquid dishwash composition preferably has pH in the range from 2.0 to 8.0, more preferably from 3.5 to 7.0. The liquid dishwash composition may further comprise one or more polymers which may be included in 0.001 to 10 wt%, more preferably from 0.1 to 6 wt%, still further more preferably from 1 to 3 wt%.

Preferably, the composition may be used as is, i.e. neat, or it may be diluted before use. The extent of dilution is generally dependent on market choice. In some markets a more concentrated product is desired while in others a more dilute product is preferred. When the composition is a liquid dishwash compositions it is typically diluted with water in a weight ratio in the range of 1 :1 to 1 :10. The liquid dishwash composition may optionally comprise ingredients, such as fragrance, colorant, foam boosting agents, and odor absorbing materials.

It is especially preferred that the composition of the invention is substantially free of an antimicrobial agent other than the cationic surfactant. By substantially free of an antimicrobial agent is meant that the agent is not intentionally included in the composition at a concentration to provide a functional benefit. Preferably such an agent may at most be present in less than 0.1%, more preferably less than 0.05%, furthermore preferably present in less than 0.01% by weight of the composition, and ideally absent from the composition. It is especially preferred that the composition is substantially free of a bleach like an oxygen-based bleach or a chlorine-based bleach.

Another aspect of the present method relates to a method of disrupting biofilm on a surface comprising the step of contacting the desired surface with a composition of the invention preferably diluted with water.

Yet another aspect of the present invention relates to a method of removing stains on a surface comprising the step of contacting the desired surface preferably made of ceramic, with a composition of the invention preferably diluted with water.

The invention will now be illustrated with the help of the following non-limiting examples.

Examples

Examples A, 1-3: Effect of combination of bacterial spores on biofilm disruption:

Combination of bacterial spores as shown in Table -1 was prepared and its effect on biofilm disruption was determined using the protocol as given below:

A suspension of Pseudomonas strain (PA-01) culture is prepared in media. 1ml of this suspension is pipetted into the multi-well plate. This plate is incubated for 48 hours for the development of biofilm. After the incubation time, choice of formulation (1 ml) is added onto the pre-formed biofilms. Plate is incubated again for 48-72 hours. After incubation period, each well is treated with crystal violet dye (0.1 %) and is washed with distilled water 3-4 times. Plate is dried for a few hours and observed for the intensity of violet stain. The biofilm is extracted by adding 0.1 ml of 30% acetic acid to the wells, transferred to microplates and the absorbance read at 550 nm. Water is used as blank. The percentage of biofilm disruption is calculated over the control.

The % biofilm disruption as measured using the protocol as detailed above is also given in Table -1.

Table - 1

In the table above the following abbreviations are used for the various bacterial spores: Ba: Bacillus amyloliquefaciens Bm: Bacillus megaterium Bs: Bacillus subtilis

Bl: Bacillus licheniformis

Bp: Bacillus pumilus

Bt: Bacillus thuringiensis

Refers to the spores included in the following amounts: Ba, Bm, and Bs at 5 to 40%, and Bp and Bt at 5 to 15% of the total number of the bacterial spores included.

The data in the above table indicates that combination of bacterial spores as per the invention (Examples 1 to 3) gives vastly improved biofilm disruption as compared to a combination outside the invention (Example A)

Examples A, 3, 4: Experiments were conducted for understanding biofilm disruption better using various combination of bacterial spores. Several experiments on the following combination of bacterial spores as shown in Table - 2 below were conducted and the general observation on quantity and quality of biofilm disruption is summarized in the table.

Table - 2

+ : indicates poor biofilm disruption

++: indicates acceptable biofilm disruption

+++: indicates good biofilm disruption.

Stability of combination of bacterial spores at various concentrations of cationic surfactants: 500 ml of a combination of bacterial spores as in Example - 4 was kept at different temperature conditions (0°C, 25°C, and 37°C). The samples were stored and the probiotic spore count was measured at regular time intervals (4 weeks and 13 weeks). The data is summarized in the Table

- 3A - 3C below:

Table - 3A: Storage in the presence of 0.1 % BKC. All spore count is in log scale. Initial spore count is 8 log.

Table - 3B: Storage in the presence of 0.5% BKC. All spore count is in log scale. Initial spore count is 8 log.

Table - 3C: Storage in the presence of 0.75% BKC. All spore count is in log scale. Initial spore count is 8 log.

The data in the tables 3A to 3C above indicates that the spores are stable at a BKC concentration of up to 0.5 wt% while it is not viable above that concentration.

Examples B,C, 5-7: Effect of various compositions on immediate hygiene and biofilm disruption: Various compositions as shown in Table -4 were prepared and immediate hygiene as described below was measured as well as the biofilm disruption. All the compositions comprised a mixture of bacterial spores as per example -3. In addition, a certain amount of cationic surfactant benzalkonium chloride was included at a concentration as shown in Table -4 . Additionally, a second surfactant was included at the concentration given in the table. The results are summarized in Table - 4:

Measurement of immediate hygiene

1.0 ml of the interfering substance (Bovine Serum albumin) was pipetted out into a tube. 1.0 ml of the test suspension of 1.5 x 10 ⁷ cfu/ml to 5.0 x 10 ⁷ cfu/ml was added to the starting culture inoculum. 8.0 ml of one of the product test solutions/formulation was added. A stopwatch was started at the beginning of the addition till the required contact time. The tube was mixed and placed in a temperature controlled water bath at 20 °C for the chosen contact time. Just before the end of contact time, it was mixed again.

At the end of contact time, 1.0 ml sample of the test mixture was taken and transferred into a tube containing 9.0 ml neutralizer. It was mixed and placed in a water bath controlled at (20 ± 1)°C. After a neutralization time of 5 min ± 10 s, the sample was mixed and immediately 1.0 ml of the neutralized test mixture was taken (containing neutralizer, product test solution, interfering substance, and test suspension) in duplicate and inoculated using the pour plate or spread plate technique.

For spread plate technique, 0.25 ml of each sample was pipetted out into 4 separate Petri dishes and 15 ml to 20 ml of melted media was added, and cooled to (45 ± 1) °C.

Table - 4 The data in the table above indicates that compositions as per the invention (Examples 5 -7) provide both immediate hygiene as was as biofilm disruption while compositions outside the invention (Examples B and C) do not.