Probiotics for cat litter

Abstract

The present invention relates to probiotics in cat litter, primarily to a cat litter composition, obtained or obtainable by a method for producing a cat litter composition. It relates furthermore to above method for producing a cat litter composition and to the use of one or more probiotic strains, wherein one or the, several or all of the probiotic strain(s) is / are selected from the species Bacillus paralicheniformis and Priestia megaterium as component(s) for cat litter compositions and / or for reducing or avoiding malodors.

Description

[0001] Probiotics for cat litter

[0002] The present invention primarily relates to a cat litter composition. The present invention furthermore relates to a method for producing a cat litter composition as defined herein. Another aspect of the present invention relates to a cat litter composition as defined herein, obtained or obtainable by a method as defined herein. Finally, the present invention relates to the use of one or more probiotic strain(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium as component(s) for cat litter compositions and / or for reducing or avoiding malodors.

[0003] Further aspects of the present invention will arise from the description below, in particular from the examples, as well as from the attached patent claims.

[0004] One quarter of all households worldwide have a pet cat, with the trend rising, making the pet market a significant economic sector. It is well-known that pets make an important contribution to the health of the cat owner, which is why most owners are very concerned about optimizing their cohabitation for both pet and owner. This also includes a cat-friendly furnishing of their homes, whereby an essential part is the installation of a device where the cats excrements can be collected. These requirements are usually satisfied with the installation of a cat litter box. Thereby, for a comfortable human habitation, it is important that unpleasant odors of the cat litter box are minimized. Besides the regular cleaning, it is desirable to avoid or reduce the formation of unpleasant odors by the used cat litter in advance. Particularly the odor of the excreted urine represents a major issue.

[0005] Urine in general, but especially cat urine, contains a large amount of urea. While in human urine the urea concentration is approx. 500 mM, the urine concentration of a castrated male cat is approx. 900 mM, and that of an adult male cat even up to approx. 1600 mM. Besides other malodor causing substances like felinine, in particular urea is the main source of the malodor of cat urine. Over time, enzymatic degradation of urea occurs by urease-positive bacteria contained in the cat urine, resulting in the formation of ammonia and carbonic acid. Ammonia has a penetrating fishy odor. Over time, the concentration of ammonia increases, consequently leading to an increased intensity of malodor. Unfortunately, the human odor detection threshold of ammonia is about 5-20 ppm, which additionally results in a very early malodor perception.

[0006] Since this is a well-known problem, there are various approaches to resolve this issue - and / or to reduce the frequency of having to exchange the cat litter material - in the previous state of the art. One of the frequently used substances, which are added to cat litter forthe purpose of malodor reduction, is Methylisothiazolinone (MIT). However, this compound has several drawbacks, as it exhibits allergenic properties for humans, e.g. the causing of contact dermatitis. Furthermore, it is cytotoxic, corrosive, causes serious environmental hazard, and is only synthetically accessible.

[0007] The application of probiotic bacteria, more specifically spore forming bacteria of the genera Bacillus, e.g., for malodor reduction in pet housing or bedding is described in several patent applications.

[0008] In US 8999318 B2 it is disclosed that a composition including bacteria or bacterial strains of the species Baci / ius subti / is or Baci / ius licheniformis can be used in animal bedding to control odors from animal waste.

[0009] Furthermore, the use of probiotic strains in other fields like animal farming is well established. WO 2014151837 A1 discloses the use of a probiotic strain from the species Baci / ius subti / is for maintaining or improving the paw health of poultry. For that purpose, it discloses feeding the animals with said probiotic strain compositions or the application of the bacterial composition on the poultry litter. Another patent application in this context is e.g., US 20150037307 A1. US 6025187 A1 discloses bacterial complexes, which may be used in the digestion and decomposition of residues of biological origin in the form of biomass, and the transformation of these residues into non-polluting organic compounds. Furthermore, it discloses the applications of these bacterial complexes to the treatment of waste of biological origin such as excrement and liquid manures. In these applications, this waste is converted into compost or other stable, biodegradable, and non-polluting nitrogenous compounds. The used bacterial complexes, essentially containing both non-pathogenic Bacillus and non- pathogenic Lactobacillus, are capable of converting inorganic nitrogen (e.g., ammonia) into organic nitrogen in the form of bacterial proteins.

[0010] In US 5507250 A1 it is disclosed that the addition of urease negative bacteria to sodium smectite clay minerals in pet litter inhibits growth of urease positive bacteria for a period of several days, thereby retarding formation of ammonia and other obnoxious odors.

[0011] A method for making an animal litter for absorbing animal wastes comprising the steps of forming particles of an absorptive material and combining the particles of absorptive material with non-enteric bacteria is disclosed in US 5154594 A1 .

[0012] Although the use of probiotic bacteria in this context may be known in connection with selected specific applications, not every species and every probiotic strain can be considered. Numerous species have low stability on the litter or on carrier materials (e.g. mold formation), have low or even no efficacy, or may even amplify ammonia production. In addition, the concentration of bacteria in most applications is relatively high, which results in higher production costs. Another drawback in this field is that in some processes for the production of bacterial compositions, freeze-drying is used, which requires an enormous amount of energy and expensive and complex instrumentation. In addition, some inventions comprise the use of chemicals to improve the malodor reduction performance. However, many of these chemicals are environmentally unfriendly as well as harmful to human and animal health.

[0013] Thus, the primary object of the present invention was to provide a cat litter composition as natural and eco-friendly solution for reducing or avoiding malodor in pet housing. Furthermore, the object of the present invention was to substitute bioactive substances such as methylisothiazolinone with a natural biological solution and its mode of action at low dosages, to support stability of the used microorganism in an „unfriendly“ environment and to make it technically applicable for a variety of cat litter matrices. Another object of the present invention was to provide a method for production of a cat litter composition with the above mentioned advantageous properties. Further objects underlying the present invention follow from the description below and the present patent claims.

[0014] According to a first aspect of the present invention, the stated objects are achieved by a cat litter composition comprising or consisting of the following components:

[0015] (a) one or more litter material(s),

[0016] (b) one or more probiotic strain(s),

[0017] (c) one or more carrier materials) for the probiotic strain(s), and

[0018] (d) optionally one or more fragrance(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium, and preferably, wherein the litter material does not consist of zeolite, preferably wherein the cat litter composition comprises at least one additional litter material in case the cat litter composition comprises zeolite as litter material, more preferably wherein the cat litter composition does not comprise zeolite.

[0019] In the context of own studies it was surprisingly found, that if the cat litter composition as defined above is used in pet housing, a malodor prevention, more precisely ammonia prevention, for up to at least 10 days, in some of the cases even up to 20 days, was achieved. Furthermore, an efficacy at low dosages was observed and the use of various litter materials and carrier materials was optimized, providing high formulation flexibility. The application of probiotic strain(s) from the species Bacillus paralicheniformis and / or Priestia megaterium, preferably the deposited strains mentioned below is particularly advantageous, since they showed the highest stability on both carrier material and in the final cat litter composition. In more detail, the application of probiotic strains according to the invention is easy to process, easy to integrate and forms a stable product for up to six months with proven anti-malodor activity, even by storage at elevated temperatures. In the field of formulations with probiotics, it is a relatively cost-effective process of manufacturing. Furthermore, there are several advantages of the use of probiotic strain(s) from the species Bacillus paralicheniformis and / or Priestia megaterium, compared to the use of chemical agents like methylisothiazolinone. Exemplarily, by previous colonization of the cat litter with the probiotic strain(s), there should be a competitive exclusion of colonization of the litter with other undesired, smell-generating bacteria. Possibly, as a side effect, the influence on urea degradation by change of the milieu is that then, by enzymatic action, non-smelling derivatives are formed. Besides its mode of action, the use of probiotics according to the invention provides a sustainable and natural solution, since cytotoxicity and environmental hazards like corrosive action are excluded.

[0020] A preferred embodiment according to the invention is a composition as defined above, wherein one or the, several or all litter material(s) is / are selected from the group consisting of sand, clay, diatomite, sepiolite, bentonite, crystalline silica, silica gel, pine wood pellets, recycled newspaper, clumping sawdust, Brazilian cassava, corn, wheat, walnuts, barley, soy pulp, dried orange peel, grass seed, paper, tofu, chalk, and quartz.

[0021] It is advantageous that various kinds of litter materials may be used in connection with the present cat litter composition, as this allows addressing different preferences but also health problems like allergies of both customers and pets. A litter material is a material, which is able to absorb liquids, in this case in particular urine.

[0022] US 20120128620 A1 discloses an animal litter including zeolite, a sub-blend comprising organic proteins, amino acids, carbohydrates, surfactants, and oxidizers, and a blend of bacteria that functions as a biological inhibitor, wherein the zeolite and the sub-blend are coupled together by the application of the blend of bacteria. Underthe condition that animal waste comes into contact with the animal litter, the zeolite absorbs the liquid in the animal waste, the blend of bacteria destroys parasites and bacteria within the animal waste as well as constructs a physical barrier around the animal waste to inhibit the spread of the parasites and bacteria outside the physical barrier, and the sub-blend controls and neutralizes the odors emanating from the animal waste. In the mentioned patent application, bacteria, including the species Bacillus licheniformis, are used to kill pathogens and create a physical barrier to protect the health of both pets and owners. The bacteria in this case are not used to reduce malodor, but rather it is explicitly stated that the compounds of the sub-blends are responsible for the decomposition of the odor causing substances, such as ammonia. As mentioned above, in connection with the present invention, using zeolite as litter material is not preferred or, respectively, is not in accordance with the invention if used as sole litter material. A particularly preferred strain of Bacillus paralicheniformis has been deposited under the Budapest Treaty at the American Type Culture Collection (ATCC®) under the accession number PT A-127480 (Bacillus paralicheniformis BL-01) by BIO-CAT Microbials, 689 Canterbury Rd, Shakopee, MN 55379 on 07 December 2022. Therefore, another preferred embodiment according to the invention is a composition as defined above, wherein the or one of the probiotic strain(s) is Bacillus paralicheniformis BL-01 . The application of probiotic strain BL-01 is particularly advantageous. For example, it showed a particularly high stability in the cat litter composition and a high efficacy for ammonia prevention, even at low dosages, in particular when combined with the herein described preferred carrier material(s).

[0023] A particularly preferred strain of Priestia megaterium has been deposited under the Budapest Treaty at the American Type Culture Collection (ATCC®) under the accession number PT A-127646 (Priestia megaterium 1 -70) by BIO-CAT Microbials, 689 Canterbury Rd, Shakopee, MN 55379 on 15 September 2023. Therefore, another preferred embodiment according to the invention is a composition as defined above, wherein the or one of the probiotic strain(s) is Priestia megaterium 1 -70. The application of probiotic strain 1-70 is particularly advantageous. For example, it showed a particularly high stability in the cat litter composition and a high efficacy for ammonia prevention, even at low dosages, in particular when combined with the herein described preferred carrier material(s).

[0024] Another preferred embodiment according to the invention is a composition as defined above, wherein one or the, several or all carrier material(s) is / are selected from the group consisting of maltodextrin, calcium carbonate, syloid, clay, sodium bicarbonate, salt, e.g., sodium chloride, granulated sucrose, wheat bran, xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose, polydextrose, dextrin, alpha- cyclodectrin, inulin, acacia gum Senegal, pectin, guar gum, and / or water, preferably wherein the or one of the carrier material(s) is calcium carbonate.

[0025] It is advantageous to consider various carrier materials, as this causes a higher flexibility in production and creates a wider range of potential applications. Test results indicated that the addition of clay has a special effect and furthermore reduces the ammonia production. Carrier materials are defined as materials used for applying the active probiotic strain(s).

[0026] Another preferred embodiment according to the invention is a composition as defined above, wherein the or one of the carrier material(s) is clay and / or syloid and / or calcium carbonate. Another preferred embodiment according to the invention is a composition as defined above, wherein the carrier materials) is or comprises syloid and calcium carbonate.

[0027] Another preferred embodiment according to the invention is a composition as defined above, wherein the composition comprises one or more fragrance material(s) and wherein one or the, several or all fragrance material(s) is / are selected from the group showing in human sensory testing to reduce the perception of cat urine and / or feces malodor a value of 3 or greater on an 1 1 pt. scale [(0) = no odor - (10) = strongest imaginable odor], and / or selected from the group consisting of ethyl isovalerate, (5E)-2,6,10-Trimethylundeca-5,9- dienal (profarnesal), thyme oil, magnolia, 2-Heptylcyclopentanone (projasmone P), 4- Methyl-2-phenyl-3,6-dihydro-2H-pyran & 4-Methyl-6-phenyl-3,6-dihydro-2H-pyran & 4- Methylene-2-phenyltetrahydro-2H-pyran (mixture of isomers, commercially known as rosyrane super), ethyl 2-methylbutyrate, isoeugenyl methyl ether, acetoin (natural), isoborneol, benzyl isovalerate, , (+)-cis-rose oxide (rose oxide high cis), thymol (thymol cryst.), gamma-octalactone, 1-(ethoxymethyl)-2-methoxybenzene (corps eglantine), ethyl caprylate, grapefruit oil terpenes, herbarosen, 2,4-Dimethylcyclohex-3-ene-1- carbaldehyde (vertocitral C), Ethyl 2-methyl-1 ,3-dioxolane-2-acetate (applinal), DL- camphor, Ethyl-2-methyl-1 ,3-dioxolan-2-acetat (Fructone), Ethyl (2-methyl-1 ,3-dioxolan-2- yl)acetate (jasmaprunat), 2-Phenoxyethyl-2-methylpropanoate (Phenirat®), blood orange oil, 3-Methyl-2-pentyl-2-cyclopenten-1-on (dihydro jasmone), ethyl caprinate, 4-(4- Methoxyphenyl)butan-2-one (Frambinon®), geraniol (natural), geraniol super, grapefruit oil, 2-Phenyl propionaldehyde dimethyl acetal (Hydratropic aldehyde dimethyl acetal), isoamyl isovalerate, abrialis lavandin oil (natural), ligustral, beta,beta-3-Trimethyl- benzenepropanol (MAJANTOL®), orange oil, tangerine cravo oil brazil.

[0028] The addition of the above defined fragrances is particularly advantageous, as these support the effect of reducing or avoiding malodor by additionally providing a pleasant odor. The application of probiotics in cat litter according to the invention and in combination with fragrances offers a more holistic malodor control technology.

[0029] Another preferred embodiment according to the invention is a composition as defined above , wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is at least 6 x 105CFU / g.

[0030] Furthermore, a preferred embodiment according to the invention, is a composition as defined above, wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is in the range of from 5 x 105to 1 x 107CFU / g, preferably 1 x 106to 9 x 106CFU / g, more preferably 1 x 106to 5 x 106CFU / g, based on the total amount of the cat litter composition. It is particularly advantageous to choose the above-mentioned dosage ranges. The present invention is efficient at very low dosages, yielding in a cost-effective application.

[0031] Particularly preferred is a composition as defined above, wherein component (c) is a solid, and wherein the total amount of the probiotic strain(s) is in the range of from 1 x 106to 9 x 106CFU / g, based on the total weight of component (c).

[0032] The present invention also relates to a method for producing a cat litter composition, preferably a composition as defined above, comprising or consisting of the following steps:

[0033] (i) Providing

[0034] (a) one or more litter material(s),

[0035] (b) one or more probiotic strain(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium,

[0036] (c) one or more carrier material(s) for the probiotic strain(s), and

[0037] (d) optionally one or more fragrance(s),

[0038] (ii) applying the probiotic strain(s) to the carrier materials), optionally in two, three, or four dilution steps,

[0039] (iii) mixing the mixture obtained in step (ii) with the litter material(s),

[0040] (iv) optionally adding one or more fragrance(s).

[0041] Another preferred embodiment according to the invention is a method as defined above, wherein the mixing in step (iii) results in a homogenous mixture. In connection with the present invention, the adding of one or more fragrances (mentioned as “step (iv)”) can be performed any time during the method, i.e. not necessarily subsequently to step (iii).

[0042] Another preferred embodiment according to the invention is a method as defined above, wherein applying the probiotic strain(s) to the carrier material(s) in step (ii) is carried out in a bioreactor, wherein the strain(s) are grown in a liquid culture, (optionally) and wherein after complete sporulation the liquid culture is stabilized by addition of citric acid, (optionally) and wherein the stabilized liquid is concentrated via centrifugation to obtain a slurry, (optionally) and wherein the obtained slurry is stored for liquid formulations or spray dried.

[0043] Another preferred embodiment according to the invention is a method as defined above, wherein the mixing of the mixture obtained in step (ii) with the litter material(s) in step (iii) is performed in a dry-blender, preferably in a V-blender, together with the carrier material(s) for an appropriate time to achieve the desired wt.-% and / or CFU / g, wherein the blending time depends on the volume of the blend and the blender size.

[0044] In connection with the present invention, the applying the probiotic strain(s) to the carrier materials), (mentioned as “step (ii)”) can be successively and / or simultaneously performed with one or more different carrier materials. Another preferred embodiment according to the invention is a method as defined above, wherein in step (ii) applying the probiotic strain(s) to the carrier material(s) is performed in two, three, or four dilution steps, preferably wherein in the first dilution step calcium carbonate is used as carrier material or wherein calcium carbonate and maltodextrin are used in the first step as carrier materials.

[0045] In connection with the present invention, the applying the probiotic strain(s) to the carrier materials), (mentioned as “step (ii)”) in two, three, or four dilution steps is particularly advantageous, since it results in a more homogenous distribution of the bacterial spores within the primed carrier material, and if applying liquid carrier material(s), it provides a better handling of the volumes.

[0046] Another preferred embodiment according to the invention is a method as defined above, wherein one or the, several or all litter materials) is / are selected from the group consisting of sand, clay, diatomite, sepiolite, bentonite, crystalline silica, silica gel, pine wood pellets, recycled newspaper, clumping sawdust, Brazilian cassava, corn, wheat, walnuts, barley, soy pulp, dried orange peel, grass seed, paper, tofu, chalk, and quartz. Another preferred embodiment according to the invention is a method as defined above, wherein the or one of the probiotic strain(s) is Bacillus paralicheniformis BL-01 and / or Priestia megaterium 1-70.

[0047] Another preferred embodiment according to the invention is a method as defined above, wherein one or the, several or all carrier material(s) is / are selected from the group consisting of maltodextrin, calcium carbonate, syloid, clay, sodium bicarbonate, sodium chloride, granulated sucrose, wheat bran, xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose, polydextrose, dextrin, alpha-cyclodectrin, inulin, acacia gum Senegal, pectin, guar gum, and / or water, preferably, wherein the or one of the carrier material(s) is calcium carbonate.

[0048] Another preferred embodiment according to the invention is a method as defined above, wherein the or one of the carrier material(s) is clay and / or syloid and / or calcium carbonate.

[0049] Another preferred embodiment according to the invention is a method as defined above, wherein the carrier materials) is or comprises syloid and calcium carbonate.

[0050] Another preferred embodiment according to the invention is a method as defined above, wherein the composition comprises one or more fragrances and wherein one or the, several or all fragrance(s) provided is / are selected from the group showing in human sensory testing to reduce the perception of cat urine and / or feces malodor a value of 3 or greater on an 11 pt. scale [(0) = no odor - (10) strongest imaginable odor]], and / or selected from the group consisting of ethyl isovalerate, (5E)-2,6,10-Trimethylundeca-5,9-dienal (profarnesal), thyme oil, magnolia, 2-Heptylcyclopentanone (projasmone P), 4-Methyl-2- phenyl-3,6-dihydro-2H-pyran & 4-Methyl-6-phenyl-3,6-dihydro-2H-pyran & 4-Methylene-2- phenyltetrahydro-2H-pyran (mixture of isomers, commercially known as rosyrane super), ethyl 2-methylbutyrate, isoeugenyl methyl ether, acetoin (natural), isoborneol, benzyl isovalerate, , (+)-cis-rose oxide (rose oxide high cis), thymol (thymol cryst.), gammaoctalactone, 1-(ethoxymethyl)-2-methoxybenzene (corps eglantine), ethyl caprylate, grapefruit oil terpenes, herbarosen, 2,4-Dimethylcyclohex-3-ene-1-carbaldehyde (vertocitral C), Ethyl 2-methyl-1 ,3-dioxolane-2-acetate (applinal), DL-camphor, Ethyl-2- methyl-1 ,3-dioxolan-2-acetat (Fructone), Ethyl (2-methyl-1 ,3-dioxolan-2-yl)acetate (jasmaprunat), 2-Phenoxyethyl-2-methylpropanoate (Phenirat®), blood orange oil, 3- Methyl-2-pentyl-2-cyclopenten-1-on (dihydro jasmone, ethyl caprinate, 4-(4- Methoxyphenyl)butan-2-one (Frambinon®), geraniol (natural), geraniol super, grapefruit oil, 2-Phenyl propionaldehyde dimethyl acetal (Hydratropic aldehyde dimethyl acetal), isoamyl isovalerate, abrialis lavandin oil (natural), ligustral, beta,beta-3-Trimethyl- benzenepropanol (MAJANTOL®), orange oil, tangerine cravo oil brazil.

[0051] Another preferred embodiment according to the invention is a method as defined above, wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is at least 6 x 105CFU / g.

[0052] Furthermore, a preferred embodiment according to the invention, is a method as defined above, wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is in the range of from 5 x 105to 1 x 107CFU / g, preferably 1 x 106to 9 x 106CFU / g, more preferably 1 x 106to 5 x 106CFU / g, based on the total amount of the cat litter composition.

[0053] For such a method according to the invention, preferably, the above described in context regarding a cat litter composition according to the invention, in particular the preferred embodiments thereof, apply accordingly.

[0054] The present invention also relates to cat litter composition, preferably a composition as defined above, obtained or obtainable by a method as defined above.

[0055] The present invention also relates to the use of one or more probiotic strain(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium, in a cat litter composition according to the present invention, for reducing or avoiding malodors, preferably for reducing or avoiding cat urine malodor.

[0056] The use of probiotic strain(s) from the species Bacillus paralicheniformis and / or Priestia megaterium is particularly advantageous, since within the tested species they showed high stability in the cat litter composition and high efficacy for ammonia prevention even at low dosages.

[0057] Another preferred embodiment according to the invention is the use as defined above, wherein the or one of the probiotic strain(s) is Bacillus paralicheniformis BL-01 and / or Priestia megaterium 1-70, preferably wherein the or one of the carrier materials) is calcium carbonate. The application of bacterial strain BL-01 and / or 1-70 is particularly advantageous, since it showed the highest stability in the cat litter composition and the highest efficacy for ammonia prevention, even at low dosages.

[0058] The invention will now be described in more detail hereinafter with references to selected examples. Preferred aspects of the present invention are disclosed in the accompanying claims.

[0059] Description of the Figures:

[0060] Figure 1 shows the arrangement of an ammonia production assay established to measure ammonia production over time.

[0061] Figure 2 shows a diagram of the detectable ammonia amount [ppm] measured over time (days) with the ammonia production assay of batches with a (comparative) active, with an active ingredient according to the invention (probiotic strain) and without any active. Figure 2 shows averaged data of all performed test results.

[0062] Figure 3 shows a diagram of the dose response results of probiotic strain Bacillus paralicheniformis BL-01 with concentrations of 5 x 105and 5 x 106CFU / g.

[0063] Figure 4 shows a diagram of the dose response results of probiotic strain Bacillus paralicheniformis BL-01 with concentrations of 1 x 107and 5 x 106CFU / g.

[0064] Figure 5 shows the sensory testing results, where the ammonia release of samples without any active, with active, and with probiotics according to the invention were compared.

[0065] Figure 6 shows average intensities of perfume vs. malodor of the sensory test results, where the ammonia release of samples without any active, with active, and with probiotics according to the invention were compared.

[0066] Figure 7 shows a ranking of the sensory test results, where the ammonia release of samples without any active, with active, and with probiotics according to the invention were compared.

[0067] Figure 8 shows a column chart of the ammonia release of the tested samples, where maltodextrin was used as carrier material according to the invention in comparison with other cat litters, e.g., without maltodextrin. Figure 9 shows a column chart of the ammonia release of the tested samples, where syloid was used as carrier material according to the invention in comparison with other cat litters, e.g., without syloid.

[0068] Figure 10 shows a diagram of the detectable ammonia amount [ppm] measured over time (days) within the ammonia production assay of batches containing no chemical active, urease positive bacteria, real urine, and with or without an active ingredient according to the invention (here: probiotic strain BL-01).

[0069] Examples

[0070] 1) Target bacteria identification

[0071] In order to identify urease positive bacteria on cat litter and to determine the bacterial population, unused and used cat litter were directly compared using cell culture and RNA sequencing. 16S RNA sequencing was performed on bacteria cultured from the dirty litter and thereby >20 species have been identified as urease positive bacteria in greater amounts (103CFU). Contamination with urease positive bacteria was also found in unused cat litter. The following six bacteria were subsequently used in the ammonia production assay:

[0072] • Staphylococcus equorum

[0073] ® Staphylococcus saprophyticus

[0074] ® Staphylococcus epidermidis

[0075] ® Staphylococcus nepalensis

[0076] ® Staphylococcus hominis

[0077] ® Micrococcus luteus

[0078] 2) Ammonia production assay

[0079] A robust and reproducible model system for measurement of ammonia production over time was developed (Figure 1). A suitable amount, e.g. 200g, cat litter was mixed with various amounts of test bacteria (or, respectively, a comparative substance) contained in different carrier systems. From the obtained mixture, a portion (e.g. 90g) cat litter were placed in a desiccator and incubated with 3 mL pooled overnight cultures of ammonia producing bacteria at a concentration of 103CFU / mL. The pooled bacteria were mixed with 12 mL synthetic urine and applied using sterile microbiological techniques to each desiccator. The synthetic urine was composed of the following further components: Caso Bouillon (Merck VM666359445), K3PO4 (Sigma P5629), NaHCO3(Sigma 792519), K2SO4 (Sigma 60528), KCI (Sigma P9541), NaCI (Sigma 31434), Urea (Sigma U5378), MgSO4(Sigma 63136) and distilled water at a pH 6.2. The prepared mixture of synthetic urine and the test probiotics on the cat litter was then placed in a 5 L Duran bottle. The setup was stored under controlled atmosphere conditions at 25 °C. The ammonia production over time was monitored daily with an analytical detector (MultiRAE).

[0080] Example 1 (comparative example)

[0081] The above described assay was performed with a comparative substance (“active”, here: Acticide® MBS microbiocide, a blend of 1 ,2-benzisothiazolin-3-one (2.5%) and 2-methyl- 4-isothiazolin-3-one (2.5%) in water), dosed in cat litter at a concentration of 0.2-0.5%. Figure 2 shows the prevention of ammonia production over 16 days in comparison with litter without active ingredient, where ammonia production occurs after eight days and increases rapidly. Figure 2 shows averaged data of all performed test results.

[0082] Example 2

[0083] The above described assay was performed probiotic strain BL-01 as active ingredient. Figure 2 shows the effective prevention of ammonia production over 16 days in comparison with dirty litter without active ingredient, where ammonia production occurs after eight days and increases rapidly, and with dirty litter, where a chemical “active” (see comparative example above) was applied. Figure 2 shows averaged data of all performed test results.

[0084] 3) Probiotic strain selection

[0085] In order to determine an effective probiotic strain various strains were tested within the ammonia production assay. Some of the tested probiotics, e.g. Bacillus subtilis, showed effectiveness in the first days but after this period the ammonia production increased rapidly. (Table 1). Bacillus paralicheniformis exhibited a particularly advantageous and outstanding performance, since it showed effectiveness up to 11 days. In further experiments, effectivity even up to over 16 days was observed. Table 1 : Exemplary results of ammonia production assay (1)

[0086] 3.2 x

[0087] * Mixture of all 1.8 x 108103

[0088] CFU / g CFU / g mentioned Bacillus Lacto- bacteria ssp. bacillus ssp

[0089] ** Commercial product with undisclosed bacteria

[0090] 8.0 x

[0091] * Mixture of all 1.0 x 10® 102mentioned CFU / g CFU / g

[0092] Bacillus Lacto- bacteria ssp. bacillus ssp

[0093] ** Commercial product with undisclosed bacteria

[0094] In another experimental setup, probiotic strain Bacillus paralicheniformis BL-01 was investigated in more detail, more precisely, different concentrations of BL-01 were tested. The results shown in Table 2 reveal a particularly suitable performance of this strain. In further experiments, effectivity even up to over 14 days was observed.

[0095] Table 2: Exemplary results of ammonia production assay (2)

[0096] Furthermore, probiotic strains BL-01 , BL-02, BL-03 of the species Bacillus paralicheniformis and of probiotic strain 1-70 of the species Priestia megaterium have been tested, showing also an effective malodor prevention (Table 3).

[0097] Table 3: Exemplarily results of ammonia production assay with single effective dose of probiotic strains BL-01 , BL-02, BL-03 of the species Bacillus paralicheniformis and of probiotic strain 1-70 of the species Priestia megaterium control litter probiotic

[0098] 4) Dose response tests

[0099] Various dosages have been tested in order to obtain the most effective concentration of probiotic strain(s). For example, BL-01 was tested in varying concentrations in the ammonia production assay. In Table 3, 4 and 5 one can observe that the best performance is obtained at a dosage of 1 - 5 x 106CFU / g. These results are also depicted in Figures 3 and 4.

[0100] Table 4: Exemplarily results of ammonia production assay with various dosages of probiotic strain BL-01 of the species Bacillus paralicheniformis (I)

[0101] Table 5: Exemplarily results of ammonia production assay with various dosages of probiotic strain BL-01 of the species Bacillus paralicheniformis (II) 5) Sensory testing

[0102] Sensory tests were performed to confirm the analytical results and to demonstrate a consumer perceivable benefit of malodor control on dirty litter. Figure 5 proves ammonia reduction by commercial litter with active (Acticide®) and active according to the invention

[0103] (probiotic strain) while only fragrance without active does not promote ammonia reduction actually causing the strongest intensity of malodor perception (Figure 6). Furthermore, with application of a probiotic strain according to the invention to a litter, the malodor intensity decreases even more (Table 6). In Figure 6 the average intensities of perfume vs. malodor obtained from the sensory test results are depicted and shows the best result for the cat litter, where a probiotic strain according to the invention was applied. These results were underlined in Figure 7 with a ranking. Table 6: Sensory testing results

[0104] *healing herb fragrance (Symrise).

[0105] 6) Carrier material variation

[0106] Various possible carrier materials for the application of probiotic strain(s) were tested. The best result was achieved by the use of calcium carbonate, whereby using clay was also observed to be particularly advantageous. Surprisingly, the calcium carbonate control without probiotic strain(s) showed reduced ammonia production in the first 6 days, but afterwards the malodor increased rapidly. This effect was also observed for the clay control without probiotic strain(s) and furthermore, the ammonia production only increased slowly over time (Table 7).

[0107] Table 7: Carrier material variation Time PostWith c = 107c = 106c = 107c = 106

[0108] Without pure pure

[0109] Inoculation Active on on on on

[0110] Active Clay CaCO3

[0111] (Days) (MIT) Clay Clay CaCO3CaCO3

[0112] 4 2 2 1 1 2 2 1 0

[0113] 6 4 1 1 0 1 1 0 1

[0114] 10 8 5 5 11 5 5 4 4

[0115] 12 15 4 10 15 3 4 4 2

[0116] 14 22 3 15 28 2 2 2 1

[0117] 17 45 3 20 50 2 3 2 2

[0118] 19 70 4 21 64 4 4 4 3

[0119] Further experiments have been performed, wherein the influence of various carrier materials was investigated, for example maltodextrin (Table 8 and Figure 8).

[0120] Table 8: Impact of maltodextrin on cat litter control litter Analyte in litter

[0121] Litter without

[0122] Litter active withoutactive

[0123] Litter without supplement,

[0124] Litter without Litter with supplement, active urease pos active active urease pos supplement, bacteria,

[0125] Analyte supplement, supplement, bacteria, urease pos synthetic in litter urease pos urease pos synthetic urine bacteria, urine plus B. bacteria, bacteria, plus B. para- synthetic para- synthetic synthetic licheniformis urine plus licheniformis urine urine BL-01 [4 x 10 maltodextrin BL-01 [4 x6CFU / g] in 106CFU / g] in maltodextrin maltodextrin

[0126] Urease producin + + + + + g bacteria

[0127] Time post inocula¬

[0128] Ammonia detection in [ppm] tion [days]

[0129] 3 5 6 5 5 5

[0130] 5 4 8 6 0 2

[0131] 7 7 6 13 2 1

[0132] 10 17 7 32 6 11 Another set of experiments was performed, wherein the influence of syloid as carrier material was investigated. As it is illustrated in Table 9 and Figure 9, syloid has a positive effect on the cat litter by reducing the ammonia release.

[0133] Table 9: Impact of syloid on cat litter control liter Analyte in liter

[0134] Litter without active Litter without active supplement, urease

[0135] Analyte in supplement, with urease pos bacteria, synthetic urine plus B. liter pos bacteria, synthetic paralicheniformis BL-01 [5 x 106CFU / g] in urine Syloid ® AL-1 FP

[0136] Urease producing + + bacteria

[0137] Time post inoculation Ammonia detection in [ppm] [days]

[0138] 0 3.5 3

[0139] 1 4 3

[0140] 4 4 3

[0141] 5 3.5 3

[0142] 6 5 4

[0143] 7 6 4

[0144] 8 10 5.5

[0145] 11 29 14

[0146] 12 41 ,5 17

[0147] 7) Production of primed carrier material

[0148] 7a) Cultivation of probiotic strains:

[0149] In a stirred bioreactor (10,000 or 20,000 L volume), probiotic strain BL-01 was grown in liquid culture using yeast extract and glucose as the primary nitrogen and carbon source respectively. In addition to carbon and nitrogen, the liquid medium included all necessary components to support Bacillus growth and sporulation including phosphates, calcium, magnesium, and manganese. The culture was grown at 37 °C with constant agitation from multiple impellors and aeration via air through a sparge ring. The pH was controlled and kept between 6.0 and 8.8. After complete sporulation, the liquid culture was stabilized by the addition of citric acid to adjust the pH of the culture to approximately 4.5. The stabilized culture was then concentrated via continuous centrifugation (typically with a rate of 2,000 to 3,000 liters / hours) in a disk stacked centrifuge to obtain a concentrated slurry that is approximately 10 to 20 times more concentrated than the stabilized culture (related to CFU). Exemplarily, if the final stabilized culture was 5 x 109CFU / mL, the slurry had a concentration of 50 to 100 billion CFU / mL.

[0150] 7b) solid formulation:

[0151] The generated concentrated slurry of probiotic bacteria was spray dried to obtain approximately 100 kg of powder at a concentration of 5 x 1011to 1 x 1012CFU / g. After spray drying, inert carrier (e.g., calcium carbonate) was added to the concentrated BL-01 spray dry to achieve a final concentration of 2 x 101° CFU / g . Components were mixed in a V-blender at room temperature for 30 minutes to obtain the primed carrier material. Typically, the final primed carrier material is 1 to 10% BL-01 spores and 90 to 99% calcium carbonate or other suitable inert carrier. id formulation:

[0152] After addition of water (balance up to 100%), potassium sorbate (0.2% wt / wt), sodium benzoate (0.2% wt / wt), and EDTA (01 .% wt / wt), to the slurry of bacteria (obtained in 7a)). The components were mixed in an overhead mixer at room temperature for 15 minutes to obtain the primed carrier material at a concentration of 2 x 101° CFU / mL. Typically, the final primed carrier is 1 to 3% BL-01 spores, 0.5% preservatives, and 96.5 to 98.5% water.

[0153] 8) Cat litter preparation with solid carrier material

[0154] The primed carrier material (obtained in step 7b); 1x101° CFU / g) is mixed (40 rpm) for 30 min at room temperature (TR CRYSTAL Revolver rotating agitator) in a carrier material matrix (calcium carbonate or e.g., clay, maltodextrin) in order to obtain a primary concentration (Cf1) at 1 x 109CFU / g. 1.25 g of Cf1 is mixed with the carrier material matrix (the same as used in the step before) for a final weight of 10 g and then added to 250 g bentonite as litter material for an average concentration of 5 x 106CFU / g (Cf1 L). In order to obtain a lower concentration in the litter, an additional 1 / 10 dilution is carried out from Cf1 (Cf2 = 1x108CFU / g). 1 .25 g of Cf2 is mixed with 8.75 g of carrier material matrix (the same as used I the step before) and then added to the litter for a final Cf2L concentration of 5 x 105CFU / g. The litter supplemented with matrix and probiotic powder is mixed using a roller mixer at 60 rpm at ambient temperature for 1 hour with manual stirring every 15 min (Thermo Scientific™). with id as solid carrier material

[0155] 1g of the primed carrier material at 1 x 1 O10CFU / g is suspended in 9 g Syloid AL-1 FP to a final concentration of 1 x 109CFU / g mixing head over head at (Rotator Genie, Scientific industries) for 20 minutes at room temperature. The working stock at 1 x 109CFU / g is blended into 200 g bentonite as litter material to obtain the final cat litter (5 x 106CFU / g). All components were mixed for 10 minutes at 49 rpm, 21 °C using a Turbula® T2F 3D Shaker Mixer [WAB Group] in a 2 L stainless steel mixing container. with lii carrier material

[0156] From the primed carrier material (8 x 109CFU / ml), a dissolution was carried out in saline water in order to obtain a concentration of 8 x 108CFU / ml (Cf1). Another dilution was performed in order to obtain another concentration of 8 x 107CFU / ml (Cf2). Then, 31 pl of Cf2 were added in saline water (qsp 5 ml) and sprayed in 250g of litter bentonite to obtain a final concentration of 5x105CFU / g. The spraying was carried out in 2 times of 2.5g with manual agitation then agitation using a roller stirrer at 60 rpm at room temperature for 1 hour. The litter remained 24 hours at 20°C in order to stabilize the water activity before adding urine. with addition

[0157] In a similar manner as described above, the primed carrier material at 1 x 109CFU / g is blended into 250 g bentonite as cat litter material to obtain a final viability in of 5 x 106

[0158] CFU / g cat litter. To this litter fragrance was added to obtain a use level of 0.11 wt.-% of fragrance in litter. The fragranced litter is mixed in a V-Blender at room temperature for 15 min to ensure proper distribution.

[0159] 9) Natural cat urine tests

[0160] In the further course of the investigations, the experiments were also carried out with real cat urine. The results observed in the artificial model could be successfully reproduced, and the observed effect could hereby be demonstrated under real conditions (see Table 10 and Figure 10). Table 10: Ammonia production assay with real urine

[0161] PCT

[0162] (Original in Electronic Form)

[0163] (This sheet is not part of and does not count as a sheet of the international application) (Original in Electronic Form)

[0164] (This sheet is not part of and does not count as a sheet of the international application)

[0165] FOR RECEIVING OFFICE USE ONLY

[0166] FOR INTERNATIONAL BUREAU USE ONLY

Claims

Claims1 . Cat litter composition comprising or consisting of the following components:(a) one or more litter material(s),(b) one or more probiotic strain(s),(c) one or more carrier material(s) for the probiotic strain(s), and(d) optionally one or more fragrance(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium, and preferably, wherein the litter material does not consist of zeolite, preferably wherein the cat litter composition comprises at least one additional litter material in case the cat litter composition comprises zeolite as litter material, more preferably wherein the cat litter composition does not comprise zeolite.

2. Composition according to claim 1 , wherein one or the, several or all litter material(s) is / are selected from the group consisting of sand, clay, diatomite, sepiolite, bentonite, crystalline silica, silica gel, pine wood pellets, recycled newspaper, clumping sawdust, Brazilian cassava, corn, wheat, walnuts, barley, soy pulp, dried orange peel, grass seed, paper, tofu, chalk, and quartz.

3. Composition according to claim 1 or 2, wherein the probiotic strain(s) is / are Bacillus paralicheniformis BL-01 and / or Priestia megaterium 1-70.

4. Composition according to any one of claims 1 to 3, wherein one or the, several or all carrier materials) is / are selected from the group consisting of maltodextrin, calcium carbonate, syloid, clay, sodium bicarbonate, salt, e.g. sodium chloride, granulated sucrose, wheat bran, xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose, polydextrose, dextrin, alpha-cyclodectrin, inulin, acacia gum Senegal, pectin, guar gum, and / or water,preferably wherein the or one of the carrier material(s) is calcium carbonate.

5. Composition according to any one of the preceding claims, wherein the composition comprises one or more fragrance material(s) and wherein one or the, several or all fragrance material(s) is / are selected from the group showing in human sensory testing to reduce the perception of cat urine and / or feces malodor a value of 3 or greater on an 11 pt. scale [(0) = no odor - (10) strongest imaginable odor], and / or selected from the group consisting of ethyl isovalerate, (5E)-2,6,10-Trimethylundeca-5,9-dienal (profarnesal), thyme oil, magnolia, 2-Heptylcyclopentanone (projasmone P), 4-Methyl-2-phenyl-3,6- dihydro-2H-pyran & 4-Methyl-6-phenyl-3,6-dihydro-2H-pyran & 4-Methylene-2- phenyltetrahydro-2H-pyran (mixture of isomers, commercially known as rosyrane super), ethyl 2-methylbutyrate, isoeugenyl methyl ether, acetoin (natural), isoborneol, benzyl isovalerate, (+)-cis-rose oxide (rose oxide high cis), thymol (thymol cryst.), gammaoctalactone, 1-(ethoxymethyl)-2-methoxybenzene (corps eglantine), ethyl caprylate, grapefruit oil terpenes, 2,4-Dimethylcyclohex-3-ene-1-carbaldehyde (vertocitral C), Ethyl 2- methyl-1 ,3-dioxolane-2-acetate (applinal), DL-camphor, Ethyl-2-methyl-1 ,3-dioxolan-2- acetat (Fructone), Ethyl (2-methyl-1 ,3-dioxolan-2-yl)acetate (jasmaprunat), 2- Phenoxyethyl-2-methylpropanoate (Phenirat®), blood orange oil, 3-Methyl-2-pentyl-2- cyclopenten-1-on (dihydro jasmone), ethyl caprinate, 4-(4-Methoxyphenyl)butan-2-one (Frabinon®), geraniol (natural), geraniol super, grapefruit oil, 2-Phenyl propionaldehyde dimethyl acetal (Hydratropic aldehyde dimethyl acetal), isoamyl isovalerate, abrialis lavandin oil (natural), ligustral, beta,beta-3-Trimethyl-benzenepropanol (MAJANTOL®), orange oil, tangerine cravo oil brazil.

6. Composition according to any one of the preceding claims, wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is at least 6 x 105CFU / g.

7. Method for producing a cat litter composition, preferably a composition according to any one of claims 1 to 6, comprising or consisting of the following steps:(i) Providing(a) one or more litter material(s),(b) one or more probiotic strain(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium,(c) one or more carrier materials) for the probiotic strain(s), and(d) optionally one or more fragrance(s),(ii) applying the probiotic strain(s) to the carrier materials), optionally in two, three, or four dilution steps,(iii) mixing the mixture obtained in step (ii) with the litter material(s),(iv) optionally adding one or more fragrance(s).

8. Method according to claim 7, wherein one or the, several or all litter materials) is / are selected from the group consisting of sand, clay, diatomite, sepiolite, bentonite, crystalline silica, silica gel, pine wood pellets, recycled newspaper, clumping sawdust, Brazilian cassava, corn, wheat, walnuts, barley, soy pulp, dried orange peel, grass seed, paper, tofu, chalk, and quartz.

9. Method according to claim 7 or 8, wherein the probiotic strain(s) is / are Bacillus paralicheniformis BL-01 and / or Priestia 1-70.

10. Method according to any one of claims 7 to 9, wherein one or the, several or all carrier material(s) is / are selected from the group consisting of maltodextrin, calcium carbonate, syloid, clay, sodium bicarbonate, sodium chloride, granulated sucrose, wheat bran, xylitol, sorbitol, mannitol, maltitol, lactitol, isomalt, erythritol, d-tagatose, isomaltulose, sucralose, polydextrose, dextrin, alpha-cyclodectrin, inulin, acacia gum Senegal, pectin, guar gum, and / or water, preferably, wherein the or one of the carrier material(s) is calcium carbonate.11 . Method according to any one of claims 7 to 10, wherein the composition comprises one or more fragrances and wherein one or the, several or all fragrance(s) provided is / are selected from the group showing in human sensory testing to reduce the perception of cat urine and / or feces malodor a value of 3 or greater on an 11 pt. scale [(0) = no odor - (10) strongest imaginable odor]], and / or selected from the group consisting of ethyl isovalerate, (5E)-2,6,10-Trimethylundeca-5,9-dienal (profarnesal), thyme oil, magnolia, 2- Heptylcyclopentanone (projasmone P), 4-Methyl-2-phenyl-3,6-dihydro-2H-pyran & 4- Methyl-6-phenyl-3,6-dihydro-2H-pyran & 4-Methylene-2-phenyltetrahydro-2H-pyran(mixture of isomers, commercially known as rosyrane super), ethyl 2-methylbutyrate, isoeugenyl methyl ether, acetoin (natural), isoborneol, benzyl isovalerate, (+)-cis-rose oxide (rose oxide high cis), thymol (thymol cryst.), gamma-octalactone, l-(ethoxymethyl)- 2-methoxybenzene (corps eglantine), ethyl caprylate, grapefruit oil terpenes, 2,4- Dimethylcyclohex-3-ene-1-carbaldehyde (vertocitral C), Ethyl 2-methyl-1 ,3-dioxolane-2- acetate (applinal), DL-camphor, Ethyl-2-methyl-1 ,3-dioxolan-2-acetat (Fructone), Ethyl (2- methyl-1 ,3-dioxolan-2-yl)acetate (jasmaprunat), 2-Phenoxyethyl-2-methylpropanoate (Phenirat®), blood orange oil, 3-Methyl-2-pentyl-2-cyclopenten-1-on (dihydro jasmone), ethyl caprinate, 4-(4-Methoxyphenyl)butan-2-one (Frabinon®), geraniol (natural), geraniol super, grapefruit oil, 2-Phenyl propionaldehyde dimethyl acetal (Hydratropic aldehyde dimethyl acetal), isoamyl isovalerate, abrialis lavandin oil (natural), ligustral, beta,beta-3- Trimethyl-benzenepropanol (MAJANTOL®), orange oil, tangerine cravo oil brazil.

12. Method according to any one of claims 7 to 11 , wherein component (c) is a solid and / or liquid, and wherein the total amount of the probiotic strain(s) is at least 6 x 105CFU / g.

13. Cat litter composition, preferably cat litter composition according to any one of claims 1 to 6, obtained or obtainable by a method according to any one of claims 7 to 12.

14. Use of one or more probiotic strain(s), wherein one or the, several or all of the probiotic strain(s) is I are selected from the species Bacillus paralicheniformis and Priestia megaterium, in a cat litter composition as defined in any one of claims 1 to 6 or claim 13, for reducing or avoiding malodors, preferably for reducing or avoiding cat urine malodor.

15. Use according to claim 14, wherein the probiotic strain(s) is / are Bacillus paralicheniformis BL-01 and / or Priestia megaterium 1-70, preferably wherein the or one of the carrier material(s) is calcium carbonate.

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