The present invention relates to compositions which are particularly useful in application in pit latrines.

In many parts of the world it is not possible for dwellings to be provided with piped sewerage systems. In developed and developing countries the cost of piped sewerage systems is prohibitive, and in many places piped systems are technologically not feasible. Therefore, it is necessary to dispose of excreta on site. One of the most common methods of achieving this is the pit latrine. In its simplest form this is merely a hole in the ground which receives the sewerage. The sewerage itself comprises of a liquid component formed by the liquid fraction of excreta and sullage (i.e. domestic waste water) and a solid component.

In a simple pit latrine some of the liquid component is disposed of by seepage into the ground surrounding the pit so that the sewerage tends to become thicker and more compacted.

When a pit latrine becomes full it is sometimes possible to dig a new pit and use that pit, whilst the contents of the first are left to decompose into harmless and inoffensive material. However, in most cases it is not possible to construct a second pit, for example because there is insufficient space, or due to the cost of dismantling and reassembling the superstructure of the latrine. These problems are particularly acute in urban areas. Therefore, it is usually necessary to empty the pit latrine at regular intervals. It is undesirable to use manual methods due to the health hazards involved.

Where a new pit cannot / is not constructed this can lead to defecation in the open. Such practices can lead to rapid spread of disease through airborne, insect driven and / or ground water driven mechanisms.

There is a need to address the potential health issues associated with pit latrine management.

A further problem with pit latrines when they are not used because they are full or because the sanitary condition and smell is so bad that people prefer open defecation. In populated areas this causes rapid spread of virus and bacteria, causing illnesses.

It is an object of the present invention to address the potential health issues associated with pit latrine management.

According to a first aspect of the invention there is provided a composition suitable for treating a pit latrine comprising:- a) a bacterial spore,

b) a wetting agent,

c) an absorbent material. Whilst the invention is predominantly directed to pit latrines it is envisaged that the composition could find utility in other applications. Preferred, non-exhaustive examples of such alternative applications include in and around a squat toilet (especially where mains water is not available); as a means to quickly cover and absorb pet accidents; as a means to tackle / cover accidental discharges of one or more bodily fluids, especially in places where the discharge needs to be quickly addressed (absorbed and disinfected) such as in nurseries, hospitals etc.

It has been found that with the use of the formulation of the invention the potential health issues associated with pit latrine management are vastly reduced. Without wishing to be limited by theory it is postulated that this arises as the formulation when applied to pit latrines, brings about rapid decomposition of the contents thereof. Additionally it has been found that the pathogen content of the pit latrine contents is rapidly reduced.

Without wishing to be bound by theory it is postulated that the disinfection is achieved through dehydration of the pit latrine contents (faecal matter). It has been found that with the use of the composition of the invention the filling level of pit latrines is kept low and thus the life of the pit latrine is extended (with the reduction of the issues outlined above). It is thought that this is achieved by the use of the microorganisms that generates enzymes that degrade organic matter. These features have the effect that a higher proportion of people to use pit latrines (treated using a composition of the invention) with associated improvements in malodour and hygiene.

Preferably the bacterial spore is present in an amount of 0.01 to 5wt%, most preferably about 0.25wt%.

Optionally the bacterial spore is present as a bacteria/enzyme complex. This includes at least one enzyme and at least one microorganism which is capable of producing a hydrolytic enzyme.

Exemplary enzymes include cellulases, amylases, proteases and lipases, among which cellulases and lipases are preferred. These enzymes, as well as commercially available enzymatic preparations comprising the same are known to the art and are available from a variety of commercial suppliers. Alternatively, enzymatic preparations comprising these proteases may be produced by conventional methods, i.e., by extraction from a microorganism which is known to produce the desired enzyme under controlled laboratory conditions or in the alternative, in a bioreactor.

Cellulase is a term generally used to described the group of enzymes which hydrolyze cellulose. As is known, cellulose is a major constituent of paper products, and the use of cellulose is also becoming increasingly known to be an additive to certain foodstuffs. Thus, they are expected to constitute an appreciable portion of a waste stream. Cellulases include one or more subcategories of enzymes which hydrolyze cellulose which subcategories include endocellulases, exocellulases, beta-l,3-glucanases and beta- glucosidases. In the compositions and processes according to the present invention, any of these cellulases may be used alone or in combination but are used preferably in combination. Preferred cellulases for use include those which are derived from microorganisms of the genus Trichoderama, Chrysosporium, Aspergillus, Penicillium, Fusarium, Thielavia, Sporotrichium, Cellulominas, Ruminococcus, and Clostridium. Cellulases are also known to be produced by genetically engineered microorganisms of the genus Bacillus. Particularly preferred microorganisms useful as a source of the cellulase constituent include Aspergillus niger, Aspergillus aculeatus, Bacillus subtilis, Trichoderma longibrachiatum, and Bacillus lentus. Commercial sources for these cellulases are well known. Examples include those marketed under the tradenames MAXICEL available from the Geo. A. Jeffreys Co., Inc. (Salem Va.) as well as CELLUCLAST 250 1 and CELLUCLAST 100 1 available from Novo Nordisk, Inc., (New York, N.Y.). The activity of the cellulase enzymes may be expressed in units of cellulase units per gram; desirably the cellulase constituent exhibits an activity of at least about 100,000 CU units/gram, and still more desirably the constituent exhibits an activity of at least 200,000 CU units/gram, at a pH of 7.0. These CU units/gram may also be interchangeably expressed as CMC units determined by known viscosity measurement techniques, which techniques are known and recognized in the art. The cellulase enzyme further desirably exhibits activity in the pH range of about 4.0 to about 9.5, but preferably exhibits activity at a pH in the range of about 5.5 to about 7.5.

Cellulases may also be prepared from certain fungi, which are known to produce cellulase, and such cellulases harvested from fungi may also be used in the compositions of the invention.

Lipases which find use in the compositions of the invention are any which are found effective in the reduction of fats and oils. Fats which are particularly susceptible to decomposition by lipases find their origin in animal or plants. Such fats are generally deposited as food residues which are introduced into a drain and drain conduit as they are expected to constitute an appreciable portion of a waste stream. Fats and oils, particularly those which are solidified in a non-fluid form are also known to be an extremely difficult deposit to remove due to the hydrophilic nature of fats which resist dissolution in water.

In the compositions according to the invention, any lipase which is effective in the degradation of fats or oils which find their origin in animals or plants may be used. Useful lipases may be derived from a variety of sources including microorganisms of the genus Aspergillus, Rhizomucor and Candida. Particularly preferred microorganisms include those which include Aspergillus niger, Aspergillus oryzae, Rhizomucor miehei, Candida rugosa. Lipases which also may be used include those which may be derived from animal sources such as from animal pancreatic tissues as from forestomachs of certain livestock including calves, kids and lambs. The activity of the lipases may be expressed in various units, including the units of fatty acid (butyric acid) liberated from tributyrin, at pH 7.0 and 30 degrees centigrade. Such units are also interchangeably referred to as lipase units or "LU". Desirably, the lipases exhibit at least about 1,000 units butyric acid units liberated from tributyrin at pH 7.0 and 30 degrees centigrade, per gram, more desirably at least about 5,000 units/gram, and most desirably at least about 10,000 units/gram under these temperature and pH conditions.

The pH ranges wherein the lipases exhibit useful activity is between about 5.0-about 13.5, but more desirably is between about 7.0-12.0.

Various commercially available lipase containing preparations are available, such as LIPIDASE from available from the Geo. A. Jeffreys Co., Inc. (Salem Va.) and which is described to be a lipase from a fungal origin, which exhibits an enzyme activity level of at least about 10,000 butyric acid units liberated from tributyrin at pH 7.0 and 30 degrees centigrade, per gram, and which is useful in the pH range of 5.0-13.5.

Lipases may also be prepared from certain fungi, which are known to produce lipases, and such lipases harvested from fungi may also be used in the compositions of the invention.

Any proteases which are effective in breaking down proteins, particularly animal proteins may be used in the compositions according to the invention. Useful proteases may be derived from a variety of sources, including microorganisms such as those of genus Aspergillus, and Bacillus. Particularly, proteases derived from microorganisms Aspergillus niger, Aspergillus oryzae, Bacillus licheniformis, and Bacillus subtilis are advantageously used.

The activity of proteases may be described as protease units/gram which is established by known methods. Desirably the protease constituent exhibits an activity of at least about 100,000 protease units/gram, and still more desirably constituent exhibits an activity of at least 250,000 protease units/gram, but most desirably constituent exhibits an activity of at least 400,000 protease units/gram. These protease units/gram are interchangeably referred to as "CDU units/gram", or casein digestion units/gram which are known to the art and are determinable by well known techniques. The proteases further desirably exhibit activity in the pH range of about 3.5 to about 13.0, but preferably exhibit activity at a pH in the range of about 7.0 to about 10.5. Various commercially available lipase containing preparations are available, such as ALKAPRO from available from the Geo. A. Jeffreys Co., Inc. (Salem Va.) and which is described to be an alkaline serine type protease from a bacterial origin, which exhibits an enzyme activity level of at least about 400,000 protease units/gram and which is useful in the pH range of 3.5 to about 13.0, and which exhibits optimal activity at a pH in the range of about 7.0 to about 10.5.

Proteases which may be derived from other sources other than those elucidated above may also be used. Amylases which find use include those which a re effective in the breakdown of starches into sugars. Such useful amylases include those which are referred to as alpha-amylases, beta- amylases, iso-amylases, pullulanases, maltogentic amylases, amyloglucosidases, and glucoamylases as well as other amylase enzymes not particularly elucidated here. These include endo-active, and exo-active amylases. Useful amylases may be obtained from a wide variety of sources, including microorganisms of the genus: Aspergillus, Rhizopus, and Bacillus. By way of non-limiting example, specific microorganisms include: Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae, Rhizopus niveus. Bacillus subtilis. Bacillus amyloliquefaciens. Bacillus stearothermophilus, Bacillus licheniformis especially containing a Bacillus stearothermophilus gene for alpha-Amylase, Bacillus subtilis containing a Bacillus megaterium gene for alpha-Amylase, as well as Bacillus acidopullulyticus. Other sources include for example, barley malt, certain animal pancreatic tissue as well as others not elucidated here but which are nonetheless known to the art.

The activity of amylases may be described in units of bacterial amylase units per gram according to known methods such as those disclosed in the Food Chemicals Codex. Useful amylase containing preparations contain at least about 10,000 bacterial amylase units per gram (BAU/g), more desirably at least about 18,000 BAU/g, and most desirably at least about 22,000 BAU/g. Such BAU units may be determined by known techniques.

Useful amylase containing preparations are available from a variety of commercial sources including for example, a product marketed as IC 24,000 by the Geo. A. Jeffireys a nd Co., Inc. (Salem Va.) and which is described to be an amylase/carbohydrase preparation from a bacterial origin, which exhibits an enzyme activity level of at least about 24,000 bacterial amylase units per gram.

Optionally but desirably may further comprise amounts of further secondary enzymes including but not limited to: pectinase, carbohydrase, beta-glucanase, hemicellulase and xylanase. The addition of such further seconda ry enzymes such as pectinase aids in the decomposition of fruit containing wastes, carbohydrases effective in breaking down non- starch polysaccharides, beta-glucanase aids in the breakdown of vegetable gums, and xylanase assists in the decomposition of various types of polymeric gums and natural polymers.

I n addition to the enzymes described above, desirably further includes one or more microorganisms or other source for the enzymes described as present. Most desirably, an amount of at least one microorganism which is suited for the generation of at least one enzyme selected from: amylases, proteases, lipases or cellulases. More desirably includes amounts of at least one or more microorganisms suited for the generation of two or more enzymes selected from: amylases, proteases, lipases or cellulases, but most desirably includes amounts of at least one or more microorganisms suited for the generation of each enzyme of the group: amylases, proteases, lipases and cellulases. Various sources for such enzymes, particularly those described above in conjunction with the description of the respective enzymes may be utilized. As enzyme producing microorganisms, fungi a nd bacteria are especially preferred for use as the source of enzymes. Preferably the wetting agent is present in an amount of 0.01 to 5wt , most preferably about lwt%.

Preferably the wetting agent comprises a non-ionic alkoxylated alcohol surfactant, such as a Cio-Cis ethoxylated Alcohol.

Other surfactants such as one or more (other) nonionic; anionic, cationic and / or zwitterionic surfactants may be present. Preferably the absorbent material is present in an amount of 90 to 99wt%, most preferably about 97wt%.

Preferably the absorbent material comprises a material selected from wheat bran, rice husk, coconut shell powder, coco peat, sawdust, clay (such as bentonite), sugar cane powder, palm kern powder or an admixture thereof.

Optionally the composition comprises a fragrance. Ideally the fragrance acts with malodour counteraction performance. Alternatively / additionally most fragrance are known to include one or more essential oils; many of these oils display insect repellent properties.

Insect repellency is another way to reduce spread of pathogen (virus/bacteria) transmission. Exemplary fragrance materials may be based on natural and synthetic fragrances and most commonly are mixtures or blends of a plurality of such fragrances, optionally in conjunction with a carrier such as an organic solvent or a mixture of organic solvents in which the fragrances are dissolved, suspended or dispersed. Such may be natural fragrances, e.g., natural extracts of plants, fruits, roots, stems, leaves, wood extracts, e.g. terpineols, resins, balsams, animal raw materials, e.g., civet and beaver, as well as typical synthetic perfume compounds which are frequently products of the ester, ether, aldehyde, ketone, alcohol and hydrocarbon type, e.g., benzyl acetate, linalyl acetate, citral, citronellal, methyl cedryl ketone, eugenol, isoeugenol, geraniol, linalool, and Typically it is preferred to use mixtures of different perfume compounds which, together, produce an agreeable fragrance. Other suitable perfume oils are essential oils of relatively low volatility which are mostly used as aroma components. Examples are sage oil, camomile oil, clove oil, melissa oil, mint oil, cinnamon leaf oil, lime-blossom oil, juniper berry oil, vetiver oil, olibanum oil, galbanum oil, labolanum oil and lavendin oil.

In a preferred embodiment the fragrance itself displays anti-microbial properties. Preferred example of an anti-microbial fragrances are dislcosed in, for example, EP1776944, the contents of which are incorporated by reference. A preferred example of such an anti- microbial fragrance has the following composition.

Dihydrofamesol - 50wt

Neroridol - 30wt%

Dodecanal - 5wt%

Nookatone - 5wt%

2-Methyl decanal - 5wt%

Trans-2-Undecanal - 5wt% When present in a treatment composition, in accordance with certain of the preferred embodiments, the fragrance constituent may be present in any effective amount such that it can be discerned by a consumer of the composition, however is advantageously present in amounts of up to about 10%wt, preferably up to about 5%wt, preferably up to about 2%wt., e.g. in amounts of from about 0.00001 wt. to about 5%wt or 1.25%wt. of the composition.

According to a second aspect of the invention there is provided a method of treating a pit latrine comprising the application of a composition according to the first aspect of the invention thereto.

Preferably the composition is sprinkled around the pit latrine and then moved (e.g. brushed) into the pit latrine. This method of application has been found to be particularly beneficial: in this way it has been noticed that the composition becomes applied to the surface of the contents of the pit latrine. This has the effect of dissuading insects from coming to the pit latrine (with clear reduction of insecticidal carried infections). Moreover it has been found that after application the composition is absorbed into the pit: as this happens the composition (and particular elements thereof such as the microbiologically active fragrance) provides a malodour treatment / disinfection function as it is absorbed deeper into the pit contents.

Preferably the composition is applied daily to provide a layer of protection (associated with insecticidal activity causing spreading of bacteria / viruses).

Examples Frafirance Microbiology Test

The fragrance was tested similarly according to ASTM E2315 - 03 "Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure". The test Aromahygiene ¹⁸ fragrances,

Aromahygiene ^® is a registered trademark of Takasago International Corporation (USA), were diluted in ethanol and added to deionized water at various concentrations. Total amount of ethanol in the test solution was no more than 2% which did not affect activity. Microorganisms (Escherichia coli

ATCC 10536, Staphylococcus epidermidis ATCC 12228) were grown overnight in an enriched nutrient broth and sampled to determine the bacterial number. They were added to the test solutions, mixed using a vortex mixer and incubated at room temperature. Samples were re-moved at various fixed time intervals and diluted into D/E Neutralization broth (Difco, Detroit, Ml, USA). The treated samples were then spiral plated onto solid media and incubated at 37°C for 24 hours. Colony-forming units were then counted and normalized as log cfu/ml. Activity was calculated as the difference between initial counts versus final counts at that time interval. Table 1

£. co// ^'

Logio

Reduction

60 seconds

Lemon Burst (∑ g 0.2% 1 .07

Lemon Burst g 1 % 2.21

Table 2

E. coli E. coli E. coli

Log,o Logio Logio

Reduction Reduction Reduction

10 minutes 30 minutes 1 hour

Lemon Burst (ξ. 0.2% 1 .69 1 .84 >5

Lemon Burst (∑ § 1 % 2.98 3.28 >5

Table 3

S. epidermidis S. epidermidis S. epidermidis

Log ₁₀ Logio Logio

Reduction Reduction Reduction

10 minutes 30 minutes 4 hours

Lemon Burst (ξ. 0.2% 1 .07 1 .31 1 .54

Lemon Burst (∑ § 1 % 1 .13 1 .63 2.14

Malodour Testing Protocol

• Malodor

- Kitchen Malodor (proprietary composition from Takasago International Corporation (USA))

- Sweat Malodor (proprietary composition from Takasago International Corporation (USA))

• Protocol Using Smelling Jar

- Place 7.5cm filter paper in disposable plastic petri dish

- An aliquot of warm soap solution is placed onto the filter paper. Malodor is applied on top.

- Place petri dish in 3-digit random coded glass jar

- Cap the jar and allow to equilibrate for 30 minutes prior to evaluation

- The Malodor reference jar (no fragrance) will be presented to the panel before coded sample jars Jar Specifications:

2 litre wide-mouth glass jar

Plastic lid to fit with 5/8" hole drilled at 6 o'clock and ¼" hole drilled at 12 o'clock

Disposable plastic pipette to fit in the ¾" hole(23 ml capacity, 7.3 ml bulb draw), cork to fit 5/8" hole alodor alodor Reduction (%) Significance* (Pass/Fail)

Sweat 57% - CITRUS FRESH Pass

59% - CITRUS CLEAN

Kitchen 74% - CITRUS FRESH Pass

66% - CITRUS CLEAN