Odor Control Bacteria

Abstract

In accordance with the present invention, novel Bacillus strains are provided, more specifically, Bacillus strains that have extracellular hydrolase activity and that exhibit reduced malodor after incubation at least 25°C for at least 5 days in lactose-free whole milk samples compared to uninoculated lactose-free whole milk samples, compositions comprising these strains, and methods of using the novel Bacillus strains are provided.

Description

[Technical Field]

[0001] In accordance with the present invention, novel Bacillus strains are provided, more specifically, Bacillus strains that have extracellular hydrolase activity and that exhibit reduced malodor after incubation at least 25°C for at least 5 days in lactose-free whole milk samples compared to uninoculated lactose-free whole milk samples, compositions comprising these strains, and methods of using the novel Bacillus strains are provided. [Background technology]

[0002] Microorganisms generally live attached to surfaces and encapsulated by extracellular materials, including biopolymers and polymers, in many natural, industrial, and medical environments. When exposed to suitable conditions, these microorganisms can multiply and produce malodors caused by their own metabolic products or by forming biofilms that provide a sticky area for surrounding malodorous molecules.

[0003] For example, when laundry articles are used, they come into contact with sweat and microorganisms from the user's body. Some of these microorganisms, especially bacteria, can adhere to the laundry articles and form biofilms on the articles. Due to the above-mentioned reasons, the presence of microorganisms causes the laundry articles to smell.

[0004] Additionally, food-soiled or soiled household items, such as tiles, plates, cups, and cutlery containing food or drink residue, can exhibit an increase in malodor due to microbial growth over time. For example, milk is an excellent growth medium for microorganisms, especially bacteria. It provides nutrients and moisture and has a nearly neutral pH. Off-flavors and / or malodors in milk are the result of bacterial growth called psychrophiles. Psychrotrophs include many types of bacteria, all of which cause milk spoilage. Their ideal growth temperature is 18-22°C. Low temperatures slow their growth but do not kill them. At temperatures above 7°C, their growth is stable, and off-flavors and / or malodors can develop within 2-3 days.

[0005] Malty flavors in milk taste like Grape-Nuts cereal. They are caused by the presence and growth of Streptococcus lactis in insufficiently cooled milk. These flavors are generally precursors to acidic flavors and rancidity. They rarely occur in pasteurized milk. However, if not stopped by pasteurization, the malty flavors later become acidic.

[0006] Bacteria and biofilms can also occur on surfaces in sanitary facilities such as toilets, showers and sinks, which typically maintain residual moisture that provides excellent conditions for bacterial growth and can therefore also cause bacterial-based malodors.

[0007] WO 2011 / 098579 relates to bacterial deoxyribonuclease formulations and methods for the disruption and prevention of biofilms, thus controlling malodors caused by bacteria. However, the synthesis and purification of the nuclease polypeptides is time-consuming and expensive.

[0008] Therefore, there is a long-standing need to provide formulations that can provide long-term control of bacterial growth and malodour, and that can be produced at low cost and with minimal effort.

[0009] In the present invention, the inventors have surprisingly found that Bacillus strains (i) have extracellular enzyme activity, (ii) provide odor control, and in particular, they reduce or avoid malodors appearing in milk samples, (iii) demonstrate long-term cleaning efficacy on hard surfaces, and (iv) are viable over a wide pH range. Thus, the Bacillus strains of the present invention can be used without additional ingredients or in combination with additional cleaning components to control malodors and / or provide long-term cleaning efficacy caused by microbial growth without the prior need for laborious and expensive protein purification. For application in cleaning and malodor control formulations, Bacillus strains can be used in their spore-forming form. Summary of the Invention [Means for solving the problem]

[0010] In a first aspect, the problem underlying the present invention is solved by a Bacillus strain which has extracellular hydrolase activity and which shows reduced malodour after incubation for at least 5 days at at least 25°C in lactose-free whole milk samples compared to non-inoculated lactose-free whole milk samples.

[0011] In a preferred embodiment, the hydrolase activity is selected from a protease activity, an amylase activity, a lipase activity, a mannanase activity, a cellulase activity, or a combination thereof.

[0012] In a more preferred embodiment, the incubation is at least 30° C. for at least 7 days.

[0013] In a preferred embodiment, the strain has the ability to form spores and is preferably present in the form of spores.

[0014] The present invention also relates to a preferred embodiment, wherein the strain is selected from the group of Bacillus species consisting of Bacillus methylotrophicus, Bacillus subtilis and Bacillus amyloliquefaciens.

[0015] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention. i) as deposited under accession number DSM 34306 or a variant thereof having all of the distinguishing characteristics as described above; ii) as deposited under accession number DSM 34307 or a variant thereof having all of the distinguishing characteristics as described above; iii) as deposited under accession number DSM 34304 or a variant thereof having all of the distinguishing characteristics as above; or iv) as deposited under accession number DSM 34305 or a variant thereof having all of the distinguishing characteristics as described above.

[0016] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention, comprising: i) whether the strain as deposited under accession number DSM 34306 contains a 16S rDNA having the sequence of SEQ ID NO: 1; ii) the strain as deposited under accession number DSM 34307 contains a 16S rDNA having the sequence of SEQ ID NO: 2; iii) the strain as deposited under accession number DSM 34304 contains a 16S rDNA sequence having the sequence of SEQ ID NO: 3; or iv) relates to a Bacillus strain of the invention, wherein the strain as deposited under accession number DSM 34305 comprises a 16S rDNA having the sequence SEQ ID NO: 4.

[0017] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention, comprising: i) whether the variant of the strain as deposited under accession number DSM 34306 contains a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 1; ii) whether the variant of the strain as deposited under accession number DSM 34307 contains a 16S rDNA having at least 98% sequence identity with SEQ ID NO:2; iii) a variant of the strain as deposited under accession number DSM 34304 contains a 16S rDNA sequence having at least 98% sequence identity with SEQ ID NO: 3; or iv) The Bacillus strain of the invention, wherein a variant of the strain as deposited under accession number DSM 34305 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO:4.

[0018] In a second aspect, the present invention relates to a composition comprising at least one Bacillus strain of the present invention and at least one additional component.

[0019] In a preferred embodiment, the composition is a detergent composition, more preferably a laundry detergent composition, a hard surface cleaning detergent composition, a hygiene or kitchen cleaner or an anti-malodor product.

[0020] In a further preferred embodiment, the composition comprises one or more surfactants and / or one or more builders and / or one or more enzymes and / or one or more preservatives, preferably one or more surfactants and / or one or more solvents.

[0021] In a third aspect, the present invention relates to a method for improving the surfactant activity of a detergent formulation by adding at least one Bacillus strain of the present invention to the detergent formulation.

[0022] In a fourth aspect, the present invention relates to a cleaning method comprising contacting at least one Bacillus strain of the invention or a composition of the invention with an object in need of cleaning, preferably a laundry or hard surface household item.

[0023] In a fifth aspect, the present invention relates to a method for reducing malodours comprising contacting an object in need of cleaning, preferably a laundry or hard surface household item, with at least one Bacillus strain of the invention or a composition of the invention.

[0024] In a sixth aspect, the present invention relates to a method for controlling or reducing malodour-causing microorganisms comprising contacting at least one Bacillus strain of the invention or a composition of the invention with an object or a medium in which said microorganisms are present or growing.

[0025] In a seventh aspect, the present invention relates to the use of a Bacillus strain as deposited under accession number DSM 34306, DSM 34307, DSM 34304 or DSM 34305 for malodour inhibition.

[0026] The invention is further illustrated by the following figures and non-limiting sequences and examples. [Brief explanation of the drawings]

[0027] [Figure 1]1 shows the growth and sporulation of eight test strains, namely, Bacillus methylotrophicus (including SEQ ID NO: 1), Bacillus subtilis (including SEQ ID NO: 2), Bacillus amyloliquefaciens (including SEQ ID NO: 3), Bacillus amyloliquefaciens (including SEQ ID NO: 4), Paenibacillus sp., Paenibacillus glycanilyticus, Paenibacillus amylolyticus, and Gulosibacter faecalis, after 72 hours of cultivation in BA055 medium. [Figure 2] 1 shows the evaluation of four different enzyme activities on agar plates for the bacterial strains mentioned above. [Figure 3] Score values ​​for spore count, enzyme score and odor control as well as classification of the overall score for the eight bacterial strains tested are shown. [Figure 4] Shown are DM-03 (Shepherd's Pie) CFT-tiles covered with sporulation of the indicated strains for 7 days at 30°C without mechanical cleaning and rinsed with dH2O to remove the stains. CTRL: surfactant without spores or water without spores. [Figure 5]Figure 5A shows spore viability of selected strains of the present invention (Bacillus methylotrophicus (including SEQ ID NO: 1), Bacillus subtilis (including SEQ ID NO: 2), Bacillus amyloliquefaciens (including SEQ ID NO: 3), and Bacillus amyloliquefaciens (including SEQ ID NO: 4)) in 60% glycerol solutions adjusted to pH 5 (gray + clear), 7 (white + dots), and 9 (black + stripes) stored at room temperature. No significant reduction in viability was observed for any of the strains. Figure 5B shows brightfield observation (100x magnification) of Bacillus amyloliquefaciens (containing SEQ ID NO: 3) stored at pH 9 after 14 days showing only spores without any vegetative cells. DETAILED DESCRIPTION OF THE INVENTION

[0028] In the present invention, the inventors have surprisingly discovered that certain Bacillus strains provide a combination of good sporulation and spore viability, sufficient extracellular enzyme activity, such as protease, lipase, amylase, and mannanase activity, and the ability to control malodors caused by microorganisms, particularly reducing or avoiding malodors occurring in milk samples. These novel Bacillus strains can be used in (hard surface) cleaning, ADW, malodor inhibition, and detergent formulations to provide cleaning benefits and control malodors. In certain embodiments, these Bacillus strains are selected from the group consisting of Bacillus methylotrophicus, Bacillus subtilis, and Bacillus amyloliquefaciens.

[0029] In a first aspect, the problem underlying the present invention is solved by a Bacillus strain which has extracellular hydrolase activity and which shows reduced malodour after incubation for at least 5 days at at least 25°C in lactose-free whole milk samples compared to non-inoculated lactose-free whole milk samples.

[0030] The terms "Bacillus" and "Bacillus strain," when used interchangeably herein, refer to a genus of Gram-positive rod-shaped bacteria that are members of the phylum Firmicutes. Under stressful environmental conditions, Bacillus bacteria produce oval endospores that can remain dormant for extended periods of time. Bacillus bacteria can be characterized and identified based on the nucleotide sequence of their 16S rRNA or fragments thereof (e.g., approximately 1000 nt, 1100 nt, 1200 nt, 1300 nt, 1400 nt, or 1500 nt fragments of the 16S rRNA or rDNA nucleotide sequence). At the time of filing, the genus Bacillus included 266 species, including B. acicliceler, B. acidicola, B. cidiproducens, B. aeoliiis, B. aerius, B. aerophilus, B. agaradaerens, B. aidingensis, B. akibai, B. alcalophilus, B. algicola, B. alkalinilriticilis, and B. alkalisediminis. diminis, B.alkalitelhtris, B.allitudinis, B.alveayuensis, B.amyloliquefaciens, B.anthracis, B.aquimaris, B.arsenicus, B.aryabhatiai, B.asahii, B.atrophaeus, B.aurantiacus, B.azotoformans, B.badius, B.balaricusbarharicus, B. baiaviensi, B. beijingensis, B. beiizoevorans, B. beveridgei, B. bogoriensis, B. boroiiiphilus, B. biiianolivoraiis, B. canaveralius, B. carboniphilus, B. cecembensis, B. cellulosiliishii B.cellulosilyiiciis, B.cereiis, B.chagarmorensis, B.chwigangensis, B.cibi, B.circiilans, B.clarkii, B.clausii, B.coagilaiis, B.coahtiilensis, B.cohiiii, B.decisifroiidis, B.decolor ationis, B.drenlensis, B.farraginis, B.faslidiosus, B.jirmus, B.flexus, B.foramiiiiis, B.fordii, B.fords, B.fuarioli arioli), B. funiculus, B. galactosidilylicus, B. galliciensis, B. geladni, B. gibsonii, B. giisengi, B. giisengihitini, B. graminis, B. harmapalushalmapalus, B.halochares, B.halodurans, B.hemicellulosilyticus, B.herberlslcinensis, B.Iwrikoshi, B.Iwrneckiae, B.horti, B.hminis, B.hwajinpoensis, B.india B. idriensis, B. indicus, B. infantis, B. infermis, B. isabeliae, B. isronensis, B. jeolgali, B. korelensis, B. korlensis, B. kribbensis, B. krulwichiae, B. B.lehensis, B.lenlus, B.licheniformis, B.liloralis, B.locisalis, B.lucifereiisis, B.luleolus, B.macauensis, B.inacyae, B.mannaiiilylicus, B.marisflavi vi), B. marmarcnsis, B. inassiliiisis, B. inegaleiu, B. methanolicz, B. methylotrophicus, B. mojavensis, B. muralis, B. lmirimardni, B. mycoidcs, B. ιιαι / iaieiisis, B.ιιαιι / iaieiisis), B.iicmhaiisedimiiiis, B.nealsoiiii, B.neizhoensis, B.niabensis, B.iiiacini, B.novalis, B.oceaiisediminis, B.odysseyi, B.okhensis, B.okii B. okiihideiisis, B. oleronius, B. oshimensis, B. panaciterrae, B. paiagoiensis, B. persepolensis, B. plakorlidis, B. pocheonensis, B. polygoni, B. pseudoalcalifilus iphilus, B. pseiidofirnnis, B. pseudomycoides, B. psychrosaccharolyticus, B. pumilus, B. qingdaonensis, B. rigid, B. niris, B. safensis, B. salarrhts, B. saliphilus us), B. schlegelii, B. selenatarsenatis, B. selentireducens, B. seohaeanensis, B. shacklelonii, B. siamensis, B. simplex, B. siralis, B. smithii, B. soli, B. solisarsisolisalsi, B.sonoreiisis, B.sporolhermodiirons, B.stralosphericits, B.subteiraneus, B.subtilis, B.taeansis, B.tequilensis, B.thermanlaicticus, B.thermoamylovorans, B.thermocloacae, B.thermolactis Bacillus species may include, but are not limited to, B. lactis, B. thioparans, B. thuringiensis, B. tripoxylicola, B. tusciae, B. vallismortis, B. vedderi, B. vietnamensis, B. vireti, B. wakoensis, B. weienstephanensis, B. xiaoxieiisis, and mixtures or blends thereof. A more complete list of Bacillus species can be found at the following internet address, which is incorporated herein by reference: https: / / lpsn.dsmz.de / genus / bacillus. The present invention also relates to a preferred embodiment in which the strain is selected from the group of Bacillus species consisting of Bacillus methylotrophicus, Bacillus subtilis and Bacillus amyloliquefaciens.

[0031] The term "strain" as used herein refers to a genetic variant, subtype, or culture within a biological species. In a more preferred embodiment, the strain is naturally occurring or isolated from a natural sample, such as soil, water, air, or a biological sample.

[0032] "Malodor," as used herein, refers to an unpleasant odor caused directly or indirectly by naturally occurring bacteria and other microorganisms, such as fungi and archaea. A malodor is considered to be directly caused by a microorganism if it is caused by one or more metabolic intermediates and / or end products of the microorganism. Furthermore, microorganisms, specifically bacteria, can form biofilms. Molecules that surround the biofilm and have an unpleasant odor can adhere to and accumulate in the biofilm. Therefore, a (bacterial) biofilm may be the source of the unpleasant odor of molecules not synthesized by the bacteria (an indirect cause of the malodor). The term "reduced malodor," as used herein, refers to a reduction in or complete absence of an unpleasant odor. In preferred embodiments, the malodor is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the original malodor. The appearance of malodor can be measured by olfaction or by using analytical equipment. The use of analytical equipment is preferred when the chemical molecule causing the malodor is known. In a further embodiment, malodor refers to no or only trace amounts of butyric acid under the conditions described herein, i.e., incubation of the strain of the present invention in a lactose-free whole milk sample at at least 25°C for at least 5 days. In this setting, the butyric acid concentration may be less than 0.2 g / L, preferably less than 0.1 g / L, more preferably less than 0.05 g / L, and most preferably, no butyric acid is detectable using high-performance liquid chromatography (HPLC) measurement. Other methods for investigating milk samples and detecting microbial metabolic products, such as butyric acid, are well known to those skilled in the art and include, but are not limited to, mass spectrometry-based methods, surface-enhanced Raman spectroscopy (SERS), nuclear magnetic resonance spectroscopy (NMR), surface plasmon resonance spectroscopy (SPRS), etc.

[0033] The terms "incubating" and "incubation," as used herein, refer to the process of mixing the Bacillus strain of the present invention with milk and allowing them to interact under the conditions defined below. The Bacillus strain can be in liquid form, i.e., bacteria dissolved in a culture medium, or in solid form as spores or vegetative Bacillus harvested from solid culture plates. Incubation of the bacterial strain and milk can be carried out for at least 5 days, at least 6 days, or at least 7 days. The incubation temperature is at least 18°C, at least 20°C, at least 23°C, at least 25°C, at least 26°C, at least 27°C, at least 28°C, at least 29°C, or at least 30°C. The incubation temperature may be 50°C or less, 45°C or less, 40°C or less, 38°C or less, 35°C or less, or 33°C or less. Preferably, the incubation temperature is constant. Preferred embodiments involve incubating the Bacillus strains of the present invention and milk at 25-40°C for 5 days, 6 days, or 7 days. In preferred embodiments, the Bacillus strains of the present invention and milk are incubated at at least 25°C for at least 5 days, more preferably at least 30°C for at least 7 days. In even more preferred embodiments, the Bacillus strains of the present invention and milk are incubated at 25°C for 5 days, more preferably at 30°C for 7 days. "Non-inoculated," as used herein, refers to a milk sample that is not intentionally contacted with the bacterial strain of interest whose ability to reduce malodor is to be tested. However, bacteria and other microorganisms that cause milk spoilage will be found in this milk sample. Furthermore, "lactose-free whole milk sample," as used herein, refers to milk that contains all of its natural fat content. The raw milk may have a fat content of at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, or at least 5.5%. The milk may be derived from a mammal, preferably from a cow, goat, sheep, or buffalo, more preferably from a cow.Additionally, the milk is substantially lactose-free. More specifically, the term "lactose-free," as used herein, is to be understood to include milk containing less than 0.20% w / w lactose based on the liquid milk, more specifically less than 0.15% w / w lactose based on the liquid milk, even more specifically less than 0.10% w / w lactose based on the liquid milk, and even more specifically 0.01% to 0.05% w / w lactose based on the liquid milk, and even more specifically 0.01% or less lactose based on the liquid milk. Note that measurements to obtain the lactose content of a milk sample may be prepared on a dry solids milk basis and then converted to a liquid milk basis. In an even more preferred embodiment, the milk is not sterilized.

[0034] As used herein, the term "(extracellular) hydrolase activity" refers to the catalytic activity of enzymes that use water to break chemical bonds, typically resulting in the splitting of larger molecules into smaller molecules. The term "extracellular" in this context refers to enzyme activity that can be measured outside the bacterial environment. This extracellular activity is caused by enzymes that are produced within the bacteria and subsequently transported outside the bacterial cell. Some common examples of hydrolase enzymes are lipases, phosphatases, glycosidases, peptidases, and esterases, including nucleosidases. Hydrolases are classified as EC3 in the EC numbering system for enzymes. Hydrolases can be further classified into several subclasses based on the bond they act on: EC 3.1: ester bond (esterases: nucleases, phosphodiesterases, lipases, phosphatases), EC 3.2: sugar (DNA glycosylases, glycoside hydrolases), EC 3.3: ether bond, EC 3.4: peptide bond (proteases / peptidases), EC 3.5: carbon-nitrogen bond other than peptide bond, EC 3.6 anhydride (anhydride hydrolases including helicases and GTPases), EC 3.7 carbon-carbon bond, EC 3.8 halide bond, EC 3.9: phosphorus-nitrogen bond, EC 3.10: sulfur-nitrogen bond, EC 3.11: carbon-phosphorus bond, EC 3.12: sulfur-sulfur bond, and EC 3.13: carbon-sulfur bond. In a preferred embodiment, the hydrolase activity is selected from a protease activity, an amylase activity, a lipase activity, a mannanase activity, a cellulase activity, or a combination thereof.

[0035] The term "protease," as used herein, refers to at least one protease that may be selected from serine proteases (EC 3.4.21). Serine proteases or serine peptidases are characterized by having a serine in the catalytic active site, which forms a covalent adduct with a substrate during catalysis. The serine protease may be selected from the group consisting of chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119), plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5), and subtilisin. Subtilisins are also known as subtilopeptidases, e.g., EC 3.4.21.62, hereinafter also referred to as "subtilisins." In a preferred embodiment, the protease exhibiting extracellular activity is a subtilisin according to EC 3.4.21.62. Protease activity can be determined on agar plates or in a liquid enzyme assay. Assays for measuring protease activity are well known in the art and include, but are not limited to, colorimetric assays, mass spectrometry-based assays, and fluorescence resonance energy transfer (FRET) assays. These assay types may be used to determine lipase, amylase, mannanase, and cellulase activity. In a preferred embodiment, the protease assay uses milk powder as a substrate, and the protease activity is at least 0.002, at least 0.005, at least 0.008, at least 0.01, at least 0.012, or at least 0.015 AU / min*mL.

[0036] The "amylases" (alpha and / or beta) according to the present invention include those of bacterial origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of alpha-amylases (EC 3.2.1.1). Chemically modified or protein-engineered variants are included. The amylases according to the present invention have "amylolytic activity" or "amylase activity," which involves the (endo)hydrolysis of glucosidic bonds in polysaccharides. Alpha-amylase activity can be determined by assays known to those skilled in the art. An example of an assay for measuring alpha-amylase activity is a method utilizing Phadebas tablets (Phadebas Amylase Test, supplied by Magle Life Science) as a substrate. Starch is hydrolyzed by alpha-amylase to produce soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of alpha-amylase activity. The measured absorbance is directly proportional to the specific activity (activity / mg of pure alpha-amylase protein) of the alpha-amylase of interest under a given set of conditions. Alpha-amylase activity can also be determined by a method utilizing ethylidene-4-nitrophenyl-alpha-D-maltoheptaside (EPS). D-maltoheptaside is a block oligosaccharide that can be cleaved by endo-amylases. After cleavage, the alpha-glucosidase included in the kit digests the substrate, liberating free PNP molecules that have a yellow color, which can be measured by visible spectrophotometry at 405 nm. A kit containing the EPS substrate and alpha-glucosidase is manufactured by Roche Costum Biotech (cat. No. 10880078103). The slope of the time-dependent absorbance curve is directly proportional to the specific activity (activity per mg of enzyme) of the alpha-amylase of interest under a given set of conditions.In a preferred embodiment, the amylase assay uses corn starch stained with Lugolsch solution or red starch as a substrate and the amylase activity is at least 1.2, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5 or at least 4.7 U / mL.

[0037] "Lipase," "lipolytic enzyme," and "lipid esterase" all refer to enzymes in EC class 3.1.1 ("carboxyl ester hydrolases"). Lipase refers to an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes with cutinase activity may be referred to herein as cutinases), sterol esterase activity (EC 3.1.1.13), and / or wax-ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin. Methods for determining lipolytic activity are well known in the literature (see, for example, Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, pp. 63-71). For example, lipase activity can be measured by ester bond hydrolysis of the substrate paranitrophenyl palmitate (pNP-palmitate), C:16, releasing the yellow color pNP, which can be detected at 405 nm. "Lipolytic activity" refers to the catalytic effect exerted by a lipase, which can be expressed in lipolytic units (LU). For example, 1 LU can correspond to the amount of lipase that produces 1 μmol of titrated fatty acid per minute during pH stating under the following conditions: temperature 30°C; pH = 9.0; the substrate can be an emulsion of 3.3 wt.% olive oil and 3.3% gum arabic in the presence of 13 mmol / L Ca2+ and 20 mmol / L NaCl in 5 mmol / L Tris buffer. In preferred embodiments, the lipase assay uses tributyrin or methylumbelliferyl heptanoate as a substrate and the amylase activity is at least 48, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700 or at least 790 U / mL.

[0038] The term "mannanase," as used herein, refers to a polypeptide selected from the group of mannan-degrading enzymes. The at least one mannan-degrading enzyme may be selected from the group of β-mannosidases (EC 3.2.1.25), endo-1,4-β-mannosidases (EC 3.2.1.78), and 1,4-β-mannobiosidases (EC 3.2.1.100). Preferably, the at least one mannan-degrading enzyme is selected from the group of endo-1,4-β-mannosidases (EC 3.2.1.78), a group of enzymes that may be referred to herein as endo-β-1,4-D-mannanases, β-mannanases, or mannanases. Polypeptides having mannan-degrading or mannanase activity can be tested according to standard test procedures known in the art, such as by applying the solution to be tested to a 4 mm diameter well punched in an agar plate containing 0.2% AZCL galactomannan (carob), a substrate for the assay of endo-1,4-beta-D-mannanase available as Cat No. I-AZGMA from Megazyme, Inc. (Megazyme's internet address: http: / / www.megazyme.com / Purchase / index.html). Mannan-degrading activity can alternatively be tested in a liquid assay using carob megalactomannan stained with Remazol Brilliant Blue as described in McCleary, BV (1978). Carbohydrate Research, 67(1), 213-221. Another method for testing mannan-degrading activity uses detection of reducing sugars when incubated with a substrate such as guar gum or carob pulp - for a reference see Miller, GL Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Analytical Chemistry 1959;31:426-428.In a preferred embodiment, the mannanase assay uses locust bean gum as a substrate and the amylase activity is at least 1.2, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5 or at least 4.7 U / mL.

[0039] A "cellulase," "cellulase enzyme," or "cellulolytic enzyme" according to the present invention is an enzyme involved in the hydrolysis of cellulose, i.e., having "cellulolytic activity" or "cellulase activity." The at least one cellulase of the present invention may be selected from cellobiohydrolases (1,4-PD-glucan cellobiohydrolases, EC 3.2.1.91), endo-ss-1,4-glucanases (EC 3.2.1.4), and ss-glucosidases (EC 3.2.1.21). Endoglucanases of EC class 3.2.1.4 may be endoglucanases designated as endo-1,4-ss-D-glucan 4-glucanohydrolases, endo-1,4-beta-glucanases, carboxymethylcellulases, and beta-1,4-glucanases. Assays for measuring "cellulase activity" or "cellulolytic activity" are known to those skilled in the art. For example, cellulolytic activity can be determined by the fact that cellulase hydrolyzes carboxymethylcellulose to produce reducing carbohydrates, and the reducing activity is determined colorimetrically by the ferricyanide reaction according to Hoffman, WS, J. Biol. Chem. 120, 51 (1937). Cellulolytic activity can be provided in units per gram of enzyme. For example, one unit can liberate 1.0 μmole of glucose from cellulose at pH 5.0 and 37° C. within 1 hour (2 hours of incubation time). In preferred embodiments, the cellulase activity is at least 1.7, at least 3.0, at least 4.0, at least 10.0, at least 20.0, at least 25.0, at least 30.0, at least 50.0, or at least 58.0 U / mL.

[0040] In a more preferred embodiment, the incubation is at least 30° C. for at least 7 days.

[0041] In a preferred embodiment, the strain has the ability to form spores and preferably exists in the form of spores. The term "bacillus cell" refers to both spores and vegetative bacillus cells. The terms "spore" or "bacillus spore," when used interchangeably herein, refer to spores, which can be characterized as immobile, solid, non-reproductive structures produced by bacillus bacteria. The primary function of spores is generally to ensure bacterial survival during periods when external conditions become unfavorable. They are also resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, starvation, and chemical exposure methods. Spores are typically found in soil and water, where they can persist for long periods of time. The spore membrane is impermeable to many toxic molecules and may also contain enzymes involved in spore growth. The spore nucleus contains normal cellular structures such as DNA and ribosomes, but is metabolically inactive. When the environment becomes unfavorable for the bacterium, it can begin the sporulation process, which takes about 8 hours. In a preferred embodiment, the spores are ellipsoidal spores. The term "vegetative bacillus cell" refers to a functional vegetative bacillus cell that can divide and produce more vegetative cells. Vegetative cells also have an active metabolism.

[0042] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention. i) as deposited under accession number DSM 34306 or a variant thereof having all of the distinguishing characteristics as described above; ii) as deposited under accession number DSM 34307 or a variant thereof having all of the distinguishing characteristics as described above; iii) as deposited under accession number DSM 34304 or a variant thereof having all of the distinguishing characteristics as above; or iv) as deposited under accession number DSM 34305 or a variant thereof having all of the distinguishing characteristics as described above.

[0043] The term "mutant" as used herein refers to a mutated or deleted form of a gene or another section of a genomic sequence. Preferably, the mutation occurs in the sequence of the 16S rDNA. The mutation may be associated with a functional modification of the nucleic acid or the corresponding protein. The functional modification may be a loss of function or may increase the function, for example, the binding activity of the nucleic acid and / or protein to another nucleic acid and / or protein. Thus, a mutation in the 16S rDNA may cause a reduction in binding of the encoded 16S rRNA molecule to the Shine-Dalgarno sequence or to proteins of the 30S subunit of the ribosome, such as S2-20.

[0044] The term "distinguishing characteristics as described above" refers to the characteristics of the Bacillus strains of the present invention of (i) providing extracellular enzyme activity; (ii) reducing malodor, and (iii) forming spores, all as described in more detail above.

[0045] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention, comprising: i) whether the strain as deposited under accession number DSM 34306 contains a 16S rDNA having the sequence of SEQ ID NO: 1; ii) the strain as deposited under accession number DSM 34307 contains a 16S rDNA having the sequence of SEQ ID NO: 2; iii) the strain as deposited under accession number DSM 34304 contains a 16S rDNA sequence having the sequence of SEQ ID NO: 3; or iv) relates to a Bacillus strain of the invention, wherein the strain as deposited under accession number DSM 34305 comprises a 16S rDNA having the sequence SEQ ID NO: 4.

[0046] As used herein, "16S rDNA" refers to the gene encoding the 16S ribosomal RNA (16S rRNA DNA), which is composed of approximately 1500 nucleotides and is the major component of the small prokaryotic ribosomal subunit (30S). Also, as further explained below, the bacteria of the present invention described herein may have a 16S rDNA sequence that has a certain sequence identity to the SEQ ID NOs listed below.

[0047] In a preferred embodiment, the present invention provides a Bacillus strain of the present invention, comprising: i) whether the variant of the strain as deposited under accession number DSM 34306 contains a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 1; ii) whether the variant of the strain as deposited under accession number DSM 34307 contains a 16S rDNA having at least 98% sequence identity with SEQ ID NO:2; iii) a variant of the strain as deposited under accession number DSM 34304 contains a 16S rDNA sequence having at least 98% sequence identity with SEQ ID NO: 3; or iv) The Bacillus strain of the invention, wherein a variant of the strain as deposited under accession number DSM 34305 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO:4.

[0048] In a preferred embodiment, the term "variant" as used herein may refer to at least one fragment or full-length sequence of SEQ ID NO: 1, 2, 3, or 4. Such a fragment or full-length sequence comprises or encodes a 16S rRNA having at least 100, at least 200, at least 300, at least 500, at least 700, at least 900, at least 1100, at least 1300, or at least 1500 consecutive nucleotides of the original sequence. In another preferred embodiment, the term "variant" refers not only to at least one fragment, but also to a nucleotide sequence or fragment thereof that is at least 98, 98.2, 98.4, 98.6, 98.8, 99.0, 99.2, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% identical to the reference nucleotide sequence referred to. Known methods include various methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity. See, for example, Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition. In a preferred embodiment, ClustalW software (Larkin, MA, Blackshields, G., Brown, NP, Chenna, R., McGettigan, PA, McWilliam, H., Valentin, F., Wallace, IM, Wilm, A., Lopez, R., Thompson, JD, Gibson, TJ, Higgins, DG (2007): Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is used with default settings applied. The variant has at least 98% sequence identity with the sequence according to SEQ ID NO: 1, 2, 3 or 4. In a preferred embodiment, the variant has at least 99% sequence identity with the sequence according to SEQ ID NO: 1, 2, 3 or 4. In an even more preferred embodiment, the variant has at least 99.5% sequence identity with a sequence according to SEQ ID NO: 1, 2, 3 or 4.

[0049] In addition to the feature that the variants of the strains as deposited under accession numbers DSM 34306, DSM 34307, DSM 34304 and DSM 34305 contain a 16S rDNA having at least 98% sequence identity with SEQ ID NOs: 1, 2, 3 and 4, respectively, these variants have a genome containing at least one (circular) nucleotide strand that hybridizes to the complementary nucleotide strand of the corresponding wild-type genome under high stringency conditions. Preferably, both strands of the variant hybridize to their corresponding wild-type strands, respectively, under stringent conditions. It is noted that these variants still have the "distinguishing characteristics as described above", i.e., (i) providing extracellular enzyme activity; (ii) reducing malodor; and (iii) forming spores, all as described in more detail above. In a more preferred embodiment, the strain as deposited under accession number DSM 34306 has a genome comprising a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 1 and containing at least one (circular) nucleotide strand that hybridizes under high stringency conditions to a complementary nucleotide strand of the genome of the strain as deposited under accession number DSM 34306. Alternatively, in a more preferred embodiment, the strain as deposited under accession number DSM 34307 has a genome comprising a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 2 and containing at least one (circular) nucleotide strand that hybridizes under high stringency conditions to a complementary nucleotide strand of the genome of the strain as deposited under accession number DSM 34307. Alternatively, in a more preferred embodiment, the strain as deposited under accession number DSM 34304 has a genome comprising a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 3 and containing at least one (circular) nucleotide stretch that hybridizes under high stringency conditions to a complementary nucleotide stretch of the genome of the strain as deposited under accession number DSM 34304.Alternatively, in a more preferred embodiment, the strain as deposited under accession number DSM 34305 has a genome containing at least one (circular) nucleotide stretch that hybridizes under high stringency conditions to a complementary nucleotide stretch in the genome of the strain as deposited under accession number DSM 34305, including a 16S rDNA sequence with at least 98% sequence identity to SEQ ID NO: 4. More preferably, hybridization of variants is tested under very high stringency conditions. For nucleic acids, similar sequences can also be determined by hybridization using the respective stringency conditions. The term "high stringency conditions" refers to prehybridization and hybridization for probes at least 100 nucleotides long, according to standard Southern blotting procedures, in 5x SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C for 12 to 24 hours. The support material is finally washed three times for 15 minutes each using 2x SSC, 0.2% SDS at 65°C. The term "high stringency conditions" refers to prehybridization and hybridization at 42°C in 5x SSPE, 0.3% SDS, 200 micrograms / ml sheared denatured salmon sperm DNA, and 50% formamide for 12-24 hours according to standard Southern blotting procedures for probes at least 100 nucleotides in length. The support material is finally washed three times for 15 minutes each using 2x SSC, 0.2% SDS at 70°C.

[0050] In a second aspect, the present invention relates to a composition comprising at least one Bacillus strain of the present invention and at least one additional component. Preferably, the additional component is selected from the group consisting of one or more surfactants and / or one or more builders and / or one or more preservatives and / or one or more enzymes and / or one or more solvents, preferably one or more surfactants and / or one or more solvents. "Surfactant" (used herein synonymously with "surface-active agent") refers to an organic chemical that, when added to a liquid, alters the properties of that liquid at an interface. Depending on their ionic charge, surfactants are called nonionic, anionic, cationic, or amphoteric.

[0051] The term "builder," as used herein, may refer to a compound or substance that can stabilize another compound or composition by neutralizing the compound or composition. Additionally, builders may sequester calcium and magnesium hardness ions that may otherwise bind to and reduce the effectiveness of a co-surfactant or co-surfactant. A "metal ion-trapping builder," as used herein, differs from a precipitating builder in that a modest amount of precipitate is formed when the builder is initially used in an amount sufficient to combine with all of the calcium ions in an aqueous solution at neutral pH and 7° dH hardness (German hardness). A "strong builder" is a builder that strongly binds calcium ions, having a logarithmic stability constant (Log KCa) of the cation / chelator complex greater than 4, particularly greater than 5, greater than 6, or greater than 7. 2+ It is classified as a highly efficient chelating agent capable of binding to divalent cations such as methyl methacrylate and methyl methacrylate. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25°C. A "powerful metal ion-trapping builder" combines both of the above properties. The solvent can be water and / or an organic solvent. The organic solvent can be a water-miscible solvent. The organic solvent can be one or more selected from the group consisting of glycerol, propanediol, polypropylene glycol, and polyethylene glycol.

[0052] The term "preservative," as used herein, refers to an agent that is a chemical compound that controls or inhibits the growth or reproduction of microorganisms, or even kills them depending on the concentration. Preservatives can be antimicrobial agents that can be added to aqueous products and compositions to maintain the original performance, properties, and integrity of the products and compositions by inhibiting the growth of or killing microorganisms depending on the concentration of the preservative.

[0053] The compositions / formulations may contain one or more preservatives as listed on pages 35-39 of patent WO 2021 / 115912 A1 ("Formulations comprising a hydrophobically modified polyethyleneimine and one or more enzymes"). Of particular interest in cleaning compositions and fabric care and home care products, especially laundry formulations, are any of the following preservatives: 4,4'-Dichloro-2-hydroxydiphenyl ether (also known as 5-chloro-2-(4-chlorophenoxy)phenol, diclosan, DCPP), Tinosan® HP100 (a commercial product from BASF SE, containing 30% active 4,4'-dichloro-2-hydroxydiphenyl ether); 2-phenoxyethanol (also known as phenoxyethanol, methylphenyl glycol, phenoxetol, ethylene glycol phenyl ether, ethylene glycol monophenyl ether, 2-(phenoxy)ethanol, 2-phenoxy-1-ethanol); 2-bromo-2-nitropropane-1,3-diol (also known as 2-bromo-2-nitro-1,3-propanediol, Bronopol) ); glutaraldehyde (also known as 1-5-pentanedial, pentane-1,5-dial, glutaral, glutaric dialdehyde); glyoxal (also known as ethanedial, oxylaldehyde, 1,2-ethanedial); 2-butyl-benzo[d]isothiazol-3-one ("BBIT"); 2-methyl-2H-isothiazol-3-one ("MIT"); 2-octyl-2H-isothiazol-3-one ("OIT"); 5-chloro-2-methyl-2H-isothiazol-3-one 5-chloro-2-methyl-2H-isothiazol-3-one ("CMIT" or "CMIT"); a mixture of 5-chloro-2-methyl-2H-isothiazol-3-one ("CMIT") and 2-methyl-2H-isothiazol-3-one ("MIT") (CMIT / MIT mixture); 1,2-benzisothiazol-3(2H)-one ("BIT"); hexa-2,4-dienoic acid (commonly known as "sorbic acid") and its salts, such as calcium sorbate, sodium sorbate; (E,E)-hexa-2,4-dienoic acid potassium (potassium sorbate); Lactic acid and its salts; L-(+)-lactic acid; in particular sodium lactate; benzoic acid and its salts, such as sodium benzoate, ammonium benzoate, calcium benzoate, magnesium benzoate, MEA-benzoic acid, potassium benzoate; salicylic acid and its salts, such as calcium salicylate, magnesium salicylate, MEA salicylate, sodium salicylate, potassium salicylate, TEA salicylate; benzalkonium chloride, benzalkonium bromide, benzalkonium saccharinate;Didecyldimethylammonium chloride ("DDAC"); N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine ("diamine"); peracetic acid; hydrogen peroxide, and formic acid. At least one preservative may be added to the composition of the present invention at a concentration of 0.001 to 10% based on the total weight of the composition. Preferably, the composition contains 2-phenoxyethanol at a concentration of 0.1 to 2% or 4,4'-dichloro-2-hydroxydiphenyl ether (DCPP) at a concentration of 0.005 to 0.6%. Preferred preservatives for hard surface cleaning compositions are selected from 2-phenoxyethanol, bronopol, and formic acid.

[0054] Additionally, the at least one additional component can be an enzyme added in addition to the enzyme secreted by the Bacillus strain of the invention. In one embodiment, the enzyme not derived from a strain of the invention is classified as an oxidoreductase (EC1), transferase (EC2), hydrolase (EC3), lyase (EC4), isomerase (EC5), or ligase (EC6). EC numbering is according to the Enzyme Nomenclature, Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, including its addenda published 1993-1999. Preferably, the enzyme is a hydrolase (EC3). In a preferred embodiment, the enzyme not derived from a strain of the invention is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, nucleases, DNases, phosphodiesterases, phospholip ... The enzymes are selected from the group consisting of enzymes such as ribonucleases, ribonucleases, transglutaminases, dispersins, ribonucleases ...More preferably, the enzyme is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, xylanases, DNases, dispersins, pectinases, oxidoreductases, and cutinases, as well as combinations of at least two of the above types. Most preferably, the enzyme not derived from a strain of the invention is a protease, preferably a serine protease, more preferably a subtilisin protease.

[0055] In a preferred embodiment, the composition is a detergent composition, more preferably a laundry detergent composition, a hard surface cleaning detergent composition, a hygiene or kitchen cleaner, or an anti-malodor product. Preferably, it is a cleaning composition and / or a fabric care and home care product, preferably for improving the removal of oily and greasy stains, comprising at least one Bacillus strain of the present invention as defined above, preferably a laundry detergent formulation and / or a manual dishwashing detergent formulation, more preferably a liquid laundry detergent formulation and / or a liquid manual dishwashing detergent formulation and / or a hygiene cleaner. In another preferred embodiment of the present invention, the cleaning composition can be used to remove particulate stains and / or oily and greasy stains, and more preferably for maintaining whiteness in laundry care. In another embodiment, the cleaning composition of the present invention is a hard surface cleaning composition that can be used to clean various surfaces, such as hardwood, tile, ceramic, plastic, leather, metal, or glass.

[0056] In another embodiment, the cleaning composition of the present invention is a liquid or solid automatic dishwashing detergent composition, preferably a solid automatic dishwashing detergent composition, that can be used to clean dishes, e.g., dishes such as glass.

[0057] In a third aspect, the present invention relates to a method for improving the surfactant activity of a detergent formulation by adding at least one Bacillus strain of the present invention to the detergent formulation.

[0058] In a fourth aspect, the present invention relates to a cleaning method comprising contacting at least one Bacillus strain of the present invention or a composition of the present invention with an object in need of cleaning, preferably a laundry or hard-surface household item. The term "object (in need of cleaning)" as used herein refers to any object having a surface that attracts dirt. In particular, the object may have a flexible structure, such as laundry. However, in other preferred embodiments, the object is a household item with a solid and hard surface. Such objects include smaller, movable objects such as dishes, glasses, and tiles. Nevertheless, this group of objects also includes larger, non-movable objects, in particular sanitary objects such as toilets, showers, and sinks, or kitchen items such as refrigerators, freezers, worktops, and storage areas. In a preferred embodiment, the Bacillus strains of the present invention are used in cleaning methods and formulations associated with food storage, particularly storage of milk and milk or dairy products such as, but not limited to, cream, butter, yogurt, cheese, condensed milk, kefir, milk powder, sour cream, crème fraîche, immer, custard, chocolate, and ice cream.

[0059] In a fifth aspect, the present invention relates to a method for reducing malodours comprising contacting an object in need of cleaning, preferably a laundry or hard surface household item, with at least one Bacillus strain of the invention or a composition of the invention.

[0060] In a sixth aspect, the present invention relates to a method for controlling or reducing malodour-causing microorganisms comprising contacting at least one Bacillus strain of the invention or a composition of the invention with an object or a medium in which said microorganisms are present or growing.

[0061] The term "malodor-causing microorganisms," as used herein, includes all forms of microorganisms, such as bacteria, archaea, fungi, and protists, that are directly or indirectly responsible for unpleasant odors. In a preferred embodiment, these microorganisms are psychrophiles. "Psychrophiles" or "psychrophilic microorganisms" are extremophilic bacteria that can grow and reproduce at low temperatures ranging from -20°C to 20°C. They have an optimum growth temperature of 15°C. Most such organisms are bacteria or archaea, but some eukaryotes, such as lichens, snow algae, phytoplankton, fungi, and small wingless insects, are also classified as psychrophiles.

[0062] The term "culture medium," as used herein, refers to a solution containing nutrients that nourish growing microorganisms, e.g., bacteria. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. The solution may also contain components that enhance growth and / or survival beyond the minimum rate. The solution has a pH and salt concentration that allows for cell survival and growth.

[0063] In a seventh aspect, the present invention relates to the use of a Bacillus strain as deposited under accession number DSM 34306, DSM 34307, DSM 34304 or DSM 34305 for malodour inhibition.

[0064] The following biological material has been deposited under the terms of the Budapest Treaty at the Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, Germany, and has been given the following accession numbers:

[0065] [Table 1]

[0066] Unless otherwise noted, terms used herein are to be understood in accordance with conventional usage by those of ordinary skill in the art. Unless otherwise stated or apparent from the nature of the definition, the definitions apply to all compounds, methods and uses described herein.

[0067] The compositions of the present invention comprise at least one microorganism as described herein. The microorganism should be present in an effective amount. The terms "effective amount," "effective concentration," or "effective dosage" are defined herein as the amount, concentration, or dosage of one or more odor-controlling microbial strains capable of inhibiting malodors caused by odor-causing organisms or substances derived therefrom on an item, a cleaning device, or an item subjected to a cleaning process, and / or on a cleaning device. In a preferred embodiment, the compositions of the present invention comprise 0.001-5% by weight of a Bacillus strain of the present invention. The weight of spores can be the weight of dry spores or wet biomass, preferably the weight of dry spores.

[0068] The compositions may be solid, semi-solid or gel-like, liquid (including sprays) or aerosol. They may be formulated in all varieties customary for the respective application: bars, powders, granules, aggregates, pastes, gels, solutions, emulsions, suspensions, etc. They may also be formulated as liquid compositions to be absorbed into wipes or pads.

[0069] The composition generally contains a carrier. In liquid, semi-solid, or gel-like compositions, the carrier is or includes a solvent, mostly water, (and / or) an alkanol (generally a C2-C3 alkanol, i.e., ethanol, n-propanol, and / or isopropanol; these generally also act as wetting agents, allowing for better wetting or penetration of the treated substrate by the composition; this latter effect is particularly useful when a surfactant is not included in the composition), a different organic solvent (details of such additional solvents are described below in connection with preferred embodiments of the composition), or a mixture thereof. In solid, semi-solid, or gel-like compositions, the carrier is or includes a solid carrier. In bar soaps, the soap component (e.g., a solid salt of a long-chain fatty acid) is also generally the carrier.

[0070] Depending on the intended use, the composition generally comprises further components. Examples are pH adjusters, sequestering agents, thickeners, antifreeze agents, antifoaming agents, colorants, or fragrances. Further details of such further components are described below in connection with preferred embodiments of the composition.

[0071] In a preferred embodiment, the (liquid) composition comprises (a) 0 to 2% by weight (= 2 to 90 < 100 ppm) of at least one preservative, relative to the total weight of the composition; (b) 0.01 to 10% by weight of at least one nonionic or anionic surfactant, based on the total weight of the composition (c) 0 to 10 wt. % of one or more surfactants, based on the total weight of the composition; (d) 0 to 90% by weight of at least one C2-C3-alkanol, based on the total weight of the composition; (e) 0 to 10 wt. % of at least one organic solvent, based on the total weight of the composition; (f) 0 to 10 wt. % of at least one sequestering agent, based on the total weight of the composition; (g) 0 to 10% by weight of further additives, relative to the total weight of the composition; and / or (h) up to 100% but at least 30% by weight of water based on the total weight of the composition.

[0072] Preferably, at least one of components (c) to (g) is present, and more preferably, at least component (c) is present.

[0073] The surfactant (or surface-active compound) (referred to as component (c) in the above and below embodiments) can be anionic, cationic, nonionic, or amphoteric (zwitterionic). Anionic, cationic, nonionic, and amphoteric surfactants are widely known in the art.

[0074] The anionic surfactants may be, for example, sulfate, sulfonate, or carboxylate types, or mixtures thereof. -Alkyl sulfates (generally of the formula RO-SO3 - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di-, or triethanolammonium); for example, sodium lauryl sulfate; - alkyl ether sulfates (generally of the formula R-(CH2CH2-O) x -O-SO3 - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, x is 1 to 10, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium laureth sulfate (SLES); - alkylbenzenesulfonates (generally of the formula R-(C6H4)-SO3 - M +(wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium dodecylbenzenesulfonate; -olefin sulfonates (generally of the formula R-SO3 - M + wherein R is a long chain monoolefin group, e.g., C 12 ~C 24 -alkenyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, C 14 / C 16 -sodium α-olefin sulfonate; -alkanesulfonates (generally of the formula R-SO3 - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 is alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, C 13 ~C 17 alkali metal or ammonium paraffin sulfonates; - sulfated monoglycerides (generally of the formula R-COO-CH2-CH(OH)-CH2-O-SO3 - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium cocoglyceryl sulfate; alkyl sulfosuccinates, such as disodium N-octadecyl sulfosuccinamate, diammonium lauryl sulfosuccinate, tetrasodium N-(1,2-dicarboxyethyl)-N-octadecyl sulfosuccinate; sodium diamyl sulfosuccinate, dihexyl sulfosuccinate, or dioctyl sulfosuccinate; acyl taurates, for example N-alkyltaurines, prepared by reacting dodecylamine with sodium isethionate or N-acyltaurines obtained by reacting N-methyltaurine with fatty acids; - acyl isethionate (generally of the formula R-COO-CH2CH2-SO3 - M + wherein R is a long chain alkyl group, e.g., C 10 ~C 30 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, ammonium cocoyl isethionate, sodium cocoyl isethionate or sodium lauroyl isethionate; - alkyl glyceryl ether sulfonates (generally represented by the formula RO-CH2-CH(OH)-CH2-SO3 - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, cocoglyceryl ether sulfonate; - sulfonated fatty acids and sulfonic acid fatty acid methyl esters (generally of the formula R-CH(SO3M) + )-COOH and R-CH(SO3M + )-COOCH3 (R is a long chain alkyl group, for example, C8-C 24 -alkyl, and M +is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, α-sulfonated coconut fatty acid or lauryl methyl ester; - acyl glutamate (generally of the formula R-CO-N(COOH)-CH2CH2-COO - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium lauroyl glutamate or sodium cocoyl glutamate; -acyl sarcosine salts (generally of the formula R-CO-N(CH3)-CH2-COO - M + (wherein R is a long chain alkyl group, for example, C8 to C 24 -alkyl, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium lauroyl sarcosinate, sodium cocoyl sarcosinate or ammonium lauroyl sarcosinate; alkyl sulfoacetates, - fatty acid salts having 8 to 24 carbon atoms in the alkyl / alkenyl moiety, generally derived from the saponification of oils or fats, for example palm oil or tallow (thus containing, inter alia, oleates, linoleates, palmitates, myristates, stearates, etc.), where the counter cation is generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); - alkyl and alkenyl ether carboxylates (generally of the formula R-(OCH2CH2) x -OCH2-COO -M + wherein R is a long chain alkyl or alkenyl group, e.g., C8 to C 24 -alkyl or -alkenyl, x is 1 to 10, M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium laureth carboxylate; acylated peptides, - acyl lactylates (generally of the formula R-CO-[OCH(CH3)-CO] x -COO - M + wherein R is a long chain alkyl or alkenyl group, e.g., C8 to C 24 -alkyl or -alkenyl, x is 3, and M + is the cation equivalent, generally Na + , K. + , NH4 + , mono-, di- or triethanolammonium); for example, sodium cocoyl lactylate.

[0075] Another class of suitable anionic surfactants are the polyalkoxylate polycarboxylated surfactants, as described, for example, in U.S. Pat. No. 5,376,298, EP-A-0129328, WO 03 / 018733, and U.S. Pat. No. 5,120,326. Polyalkoxylate polycarboxylated surfactants have the formula RO-(CHO) x -[CH(L)CH(L)] y -[CH2CH(CH3)O) z Q (wherein R is a hydrophobic hydrocarbon group, preferably an alkyl, containing 6 to 16, preferably 8 to 14, carbon atoms; x is a number from 0 to 60, preferably from 4 to 50, more preferably from 6 to 50; L is a C1 to C3 alkyl group or a group of the formula -CH(COO - )-CH2(COO -) and at least one L group in each molecule is a group having the formula -CH(COO - )-CH2(COO - ), y is 1 to 12, preferably 2 to 10, more preferably 3 to 8; z is a number from 0 to 20, preferably 0 to 15, more preferably 0 to 10; and Q is selected from the group consisting of H and sulfonate groups, the compound being electrically neutralized by the presence of a cationic group, preferably selected from the group consisting of sodium, potassium, and substituted ammonium, e.g., monoethanolammonium, cations. Such polyalkoxylate polycarboxylate surfactants are commercially available under the Plurafac® brand from BASF, e.g., Plurafac® CS-10.

[0076] The cationic surfactant may be, for example, C8 to C 16 - ammonium salts such as dialkyldimethylammonium halides, dialkoxydimethylammonium halides, or imidazolinium salts with long-chain alkyl groups.

[0077] Nonionic surfactants are typically condensation products of one or more alkylene oxides, primarily ethylene oxide, and various reactive hydrogen-containing compounds, for example, having a hydrophobic chain with 8 to 24 carbon atoms, such as condensation products of polyethylene oxide with fatty alcohols, long-chain branched alkyl alcohols, fatty acids, fatty amines, polyhydric alcohols, or polypropylene oxide.

[0078] Suitable alkoxylated alcohols are listed below, provided that they are different from the alkoxylated alkanols of component (b), i.e., C6-C6 alkoxylated with an average of 3 to 5 units of ethylene oxide. 14- they are suitable as component (c) only if they are not alkanols, for example if they contain on average more than 5 or less than 3 ethylene oxide units or are alkoxylated with an average of 3 to 5 ethylene oxide and propylene oxide units in total; if they have fewer than 6 or more than 14 carbon atoms in the alcohol moiety, if the alcohol moiety is not derived from an alkanol, and / or if they are alternatively or additionally alkoxylated with another alkylene oxide (e.g., only with propylene oxide, PO) or alkylenediols (e.g., only with propylene-1,2- or 1,3-diol).

[0079] Suitable alkoxylated, advantageously ethoxylated, alcohols are especially alkoxylated, advantageously ethoxylated, primary alcohols, preferably having 8 to 18 carbon atoms and an average of 1 to 20, preferably 1 to 12, moles of ethylene oxide (EO) per mole of alcohol, where the alcohol radical may be linear or branched, particularly 2-methyl-branched, or may contain a mixture of linear and methyl-branched groups, as typically present in oxoalcohol groups. Also suitable are alkyl alcohols synthesized by the Guerbet method, such as 2-ethylhexanol, 2-n-propylheptanol, 2-isopropylheptanol, 2-n-butyloctanol, and 2-n-pentylnonanol, preferably 2-ethylhexanol, 2-n-propylheptanol, and 2-isopropylheptanol. 2-n-propylheptanol is more preferred. Nonionic surfactants synthesized from this latter alcohol are sold by BASF under the brand names Lutensol® XP and Lutensol® XL.

[0080] Other preferred ethoxylated alkyl alcohols have a high degree of branching, especially those available under the BASF brand names Lutensol® TO, Lutensol® ON and Lutensol® TDA.

[0081] Also suitable are straight chain radicals formed from naturally occurring alcohols having 12 to 18 carbon atoms, such as coconut alcohol, palm alcohol, tallow fatty alcohol or oleyl alcohol, and alcohol ethoxylates having an average of 2 to 12 EOs per mole of alcohol. Preferred ethoxylated alcohols include, for example, C ethoxylates having 7 EOs or 10 EOs. 12 ~C 14 Alcohols, C9-C with 7 EO or 10 EO 11 -alcohols, C with 7 EO, 8 EO or 10 EO 13 ~C 15 -alcohols, C with 7 EO or 10 EO 12 ~C 18 These include alcohols and mixtures thereof. The stated degree of ethoxylation is a statistical average value, which may be an integer or a fraction, for a particular product. Alcohol ethoxylates with a narrow homolog distribution (narrow range ethoxylates, NRE) are also suitable. In addition to these alkoxylated alcohols, it is also possible to use fatty alcohols with more than 12 EOs. Examples include tallow fatty alcohols with 14, 25, or 30 EOs. It is also possible to use alkoxylated alcohols containing both EO and PO groups in the molecule. In this case, it is also possible to use block copolymers with EO-PO or PO-EO block units, but also EO-PO-EO or PO-EO-PO copolymers. It will be understood that it is also possible to use mixed alkoxylated nonionic surfactants in which the EO and PO units are not present in blocks but in a random distribution. Such products can be obtained by the simultaneous action of ethylene oxide and propylene oxide on fatty alcohols.

[0082] Suitable alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters preferably have 1 to 4 carbon atoms in the alkyl chain, and are especially fatty acid methyl esters.

[0083] Non-ethoxylated nonionic surfactants are, for example, sugar surfactants, glycerol monoethers, polyhydroxyamides (glucamides) or amine oxides.

[0084] Sugar surfactants are, for example, alkyl and / or alkenyl polyglycosides, sugar or alkyl sugar fatty acid esters, and fatty sugar amides.

[0085] Alkyl and / or alkenyl polyglycosides are nonionic surfactants having carbohydrates as the hydrophilic portion and fatty alcohols or fatty acids as the hydrophobic component. Examples are those of the formula ROG p where R is a long-chain alkyl or alkenyl group, mostly having 4 to 22 carbon atoms; G is an aldose or ketose moiety, mostly a glucose moiety; and p is 1 to 10.

[0086] G is preferably derived from an aldose or ketose having 5 or 6 carbon atoms. In one embodiment, the component G is selected from the group of hexoses, preferably selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose, more preferably glucose. In another embodiment, the component G is selected from the group of pentoses, preferably selected from the group consisting of ribulose, xylulose, ribose, arabinose, xylose, and lyxose, more preferably selected from xylose and arabinose.

[0087] The index p in the above formula gives the degree of polymerization (DP) and is a number between 1 and 10. In one embodiment, p is between 1.1 and 3.0.

[0088] R can be linear or branched. For example, the group R is derived from a linear primary alcohol, such as a fatty alcohol, or a branched primary alcohol, in particular a so-called oxoalcohol. Examples of R derived from a linear primary alcohol are n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-heptadecyl, or n-octadecyl. Examples of R derived from a branched primary alcohol are isoamyl, isohexyl, isoheptyl, 2-ethylhexyl, and 2-propylheptyl.

[0089] It is also possible to use mixtures of different alkyl and / or alkenyl polyglycosides, thus all combinations of different aldoses or ketoses with all possible alkyl and / or alkenyl groups can be used.

[0090] Commercially available alkyl and / or alkenyl polyglycosides are, for example, the products sold under the PLANATAREN® and PLANTACARE® brands from Henkel, such as PLANTAREN 1200, PLANTAREN 1300, PLANTAREN 2000, PLANTACARE 2000, PLANTACARE 818, PLANTACARE 1200; the products sold under the TRITON® CG brand from Seppic, such as TRITON CG 110 (or ORAMIX CG 110) and TRITON CG 312 (or ORAMIX NS 10); the products sold as LUTENSOL® GD 70 from BASF SE; the products sold under the Glucopon® brand from BASF SE, such as Glucopon 100 DK, Glucopon 215 UP, Glucopon 225 DK, Glucopon 425. N / HH, Glucopon GD 70, Glucopon 50 G, Glucopon 600 CSUP or Glucopon 650 EC; as well as the product Plantatex® LLE from BASF SE.

[0091] Sugar or alkyl sugar fatty acid ester is a sugar or alkyl sugar C4-C 22 Fatty acid esters, among which mention may be made in particular of: (C1-C4) alkyl glucoside esters, such as methyl glucoside monostearate, for example the product sold under the name GRILLOCOSE® IS by Grillowerke; methyl glucoside sesquistearate, for example the product sold under the name GLUCATE SS by Amerchol; 6-ethyl glucoside decanoate, for example the product sold under the name BIOSURF 10 by Novo; mixtures of 6-ethyl glucoside mono- and dicoconut fatty acids (82 / 7), for example the product sold under the name BIOSURF® COCO by Novo; mixtures of 6-ethyl glucoside mono- and dilauric acids (84 / 8), for example the product sold under the name BIOSURF® 12 by Novo; butyl glucoside C, such as mono-coconut butyl glucoside. 12 ~C 18 Fatty acid monoesters, such as the product sold under the name REWOPOL® V3101 or REWOSAN® V3101, and polyoxyethylenated monococobutyl glucosides with 3 moles of ethylene oxide, such as the product sold under the name REWOPOL® V3122 by Rewo; glucose esters, such as 6-O-hexadecanoyl-[alpha]-D-glucose, 6-O-octanoyl-D-glucose, 6-O-oleyl-D-glucose, 6-O-linoleyl-D-glucose, which can be prepared from the corresponding acid chloride and D-glucose; sucrose monoesters, such as sucrose monolaurate, such as the product sold under the name GRILLOTEN® LES 65, and sucrose monococoate sold under the name GRILLOTEN® LES 65K by Grillo-Werke.

[0092] Fatty sugar amides are compounds containing at least one amide functional group and containing at least one sugar or sugar derivative moiety and at least one fatty chain; such compounds can result, for example, from the action of a fatty acid or fatty acid derivative on the amine functional group of an amino sugar, or from the action of a fatty amine on a sugar containing a carboxylic acid functional group (free or lactone form) or a functional group derived from a carboxylic acid or a carbonyl functional group, optionally in the presence of a suitable auxiliary reagent. Examples are N-substituted aldonamide polyhydroxylated fatty acid amides or mixtures thereof.

[0093] N-substituted aldonamides include, for example, N-substituted lactobionamides, N-substituted maltobionamides, N-substituted cellobionamides, N-substituted melibionamides and N-substituted gentiobionamides, for example: N-alkyl lactobionamides, N-alkyl maltobionamides, N-alkyl cellobionamides, N-alkyl melibionamides or N-alkyl gentibionamides which are mono- or di-substituted with saturated or unsaturated, linear or branched aliphatic hydrocarbon groups which may contain aromatic hydrocarbon groups (e.g., benzyl, aniline, substituted benzyl, phenylethyl, phenoxyethyl, vinylbenzyl) or alicyclic groups (e.g., cyclopentyl, cyclohexyl), as well as heteroatoms preferably having up to 36 carbon atoms, more preferably up to 24 carbon atoms, and even more specifically 8 to 18 (e.g., methyl, ethyl, amyl, hexyl, heptyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl; allyl, undecenyl, oleyl, linoleyl, propenyl, heptenyl) heteroatoms; N-lactobionyl amino acid esters (amino acids may mean in particular: alanine, valine, glycine, lysine, leucine, arginine, aspartic acid, glutamic acid, threonine, serine, cysteine, histidine, tyrosine, methionine, or may be selected, for example, from [beta]alanine, sarcosine, gamma-aminobutyric acid, ornithine, citrulline or their equivalents), said N-lactobionyl amino acid esters having the formula -(CH)n -C(=O)-OR (wherein R is an aliphatic hydrocarbon group which may contain up to 36 carbon atoms and n is an integer greater than 1), and the corresponding N-maltobionyl amino acid esters, N-melibionyl amino acid esters, N-cellobionyl amino acid esters and N-gentiobionyl amino acid esters; Group-(CH2) n N-(alkyloxy)alkyl lactobionamides mono- or di-substituted with —OR′, where R′ is an aliphatic, aromatic, or alicyclic hydrocarbon group; The group -R'-(OR') n mono- or di-substituted with R'R'' (where R' is an alkylene group such as ethylene, propylene or mixtures thereof, n is an integer greater than 1, and R'' is a lactobionamide, maltobionamide, cellobionamide, melibionamide, or gentiobionamide group); N-(polyalkyloxy)alkyl lactobionamides, N-(polyalkyloxy)alkyl maltobionamides, N-(polyalkyloxy)alkyl cellobionamides, N-(polyalkyloxy)alkylmelibionamide or N-(Polyalkyloxy)alkylgentibionamide is.

[0094] Examples of polyhydroxylated fatty amides are those of the formula TC(=O)-N(V)-W (Wherein, T is C5 to C 31 Hydrocarbon groups, preferably C7 to C 15V means a linear alkyl or alkenyl chain; V means hydrogen, a C1-C4 hydrocarbon group, 2-hydroxyethyl, 2-hydroxypropyl or mixtures thereof, preferably C1-C4 alkyl such as methyl, ethyl, propyl, isopropyl, N-butyl and more particularly methyl; W means a polyhydroxy hydrocarbon-containing group having a linear hydrocarbon chain with at least three hydroxyl groups directly attached to the chain, or an alkoxylated derivative (preferably ethoxylated or propoxylated) of said group.

[0095] W is preferably a reducing sugar derivative obtained by reductive amination, more preferably a glycityl group. Reducing sugars may include glucose, maltose, lactose, galactose, mannose, and xylose. Preferably, W is a group of the following formula: -(CH2)-(CHOH) n -CHOH; -CH-(CHOH)-(CHOH) n-1 and -CH-(CHOH)(CHOR')-(CHOH)-CHOH, where n is an integer ranging from 3 to 5 and R' is hydrogen, a cyclic or aliphatic monosaccharide or one of its alkoxylated derivatives. Glycityl groups where n is 4 are preferred, in particular the group -(CH)-(CHOH)-CHOH.

[0096] The group TC(=O)-N- can be, for example, cocamide, stearamide, oleamide, lauramide, myristyramide, capricamide, palmitamide, tallowamide.

[0097] Amine oxide type nonionic surfactants are generally represented by the formula R a R b R c N + -O - (In the formula, R a is a long chain alkyl group, e.g., C 10 ~C 18 -Alkyl, preferably C 12 ~C 16 -alkyl, and R b and R cis a short-chain alkyl or hydroxyalkyl group, e.g., methyl, ethyl, or 2-hydroxyethyl. A specific example is lauryl dimethyl amine oxide. Additionally, long-chain alkyl groups can be derived from natural sources (oils or fats), resulting in a mixture of such amine oxides, e.g., N-cocoalkyl-N,N-dimethyl amine oxide and N-tallow alkyl N,N-dihydroxyethyl amine oxide.

[0098] Amphoteric surfactants are, for example, derivatives of secondary or tertiary amines, such as C6-C 18 - alkyl betaines (e.g., cocoamidopropyl betaine; disodium cocoamphodiacetate (DSCADA)) or C6-C 18 alkyl sulfobetaines, or amine oxides such as alkyl dimethyl amine oxides.

[0099] C2-C3-alkanols [component (d)] are ethanol, n-propanol and isopropanol. Mixtures thereof are also suitable.

[0100] An organic solvent, different from component (d) [component (e)], generally aids in providing a stable composition, especially when the composition is a concentrate containing a large amount of organic matter.

[0101] Suitable solvents are therefore polar protic or polar aprotic. Examples of suitable solvents (e) are alkanols other than C2-C3-alkanols, such as n-butanol or tert-butanol; C2-C8-alkanediols; C1-C8-alkyl monoethers of C2-C8-alkanediols; diglycol, C1-C8-alkyl monoethers of diglycol, polyetherpolyols; C1-C8-alkyl monoethers of polyetherpolyols; aminoalcohols such as ethanolamine, diethanolamine and triethanolamine; monophenyl ethers of C2-C3-alkanediols, for example 2-phenoxyethanol or phenoxypropanol.

[0102] Among the above solvents, C2-C8 alkanediols, C1-C8 alkyl monoethers of C2-C8 alkanediols, and C1-C8 alkyl monoethers of diglycol are preferred. C2-C4 alkanediols, particularly ethylene glycol and propylene glycol, and C1-C4 alkyl monoethers of C2-C3 alkanediols, such as C1-C4 alkyl monoethers of ethylene glycol or propylene glycol, are more preferred. Specific examples include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol mono-n-propyl ether, ethylene glycol mono-n-butyl ether (also known as butyl glycol), propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol mono-n-propyl ether, and propylene glycol mono-n-butyl ether; and C1-C4 alkyl monoethers of diglycol, such as butyl diglycol, and n-hexanol ethoxylated with 1 to 3 EO groups, and mixtures thereof.

[0103] Sequestrants [component (f)], also known as builders, structural materials, framework materials, complexing agents, chelators, chelating agents, or softeners, bind alkaline earth metal and other water-soluble metal salts without precipitation. They aid in soil breakdown, disperse soil components, aid in soil release, and may have cleaning properties themselves. Many of the sequestrants listed below are multifunctional, meaning that they have additional functions, such as dispersing activity.

[0104] Suitable sequestering agents may be organic or inorganic, examples being aluminosilicates, carbonates, phosphates and polyphosphates, polycarboxylic acids, polycarboxylates, hydroxycarboxylic acids, phosphonic acids, such as hydroxyalkylphosphonic acids, phosphonates, aminopolycarboxylic acids and their salts, and polymeric compounds containing carboxylic acid groups and their salts.

[0105] Suitable inorganic sequestering agents are, for example, crystalline or amorphous aluminosilicates with ion exchange properties, such as zeolites. Crystalline silicates suitable as sequestering agents are, for example, disilicates or layered silicates, such as δ-Na2SiO5 or β-Na2SiO5 (SKS6 or SKS7). Suitable inorganic sequestering agents based on carbonates are carbonates and bicarbonates. These can be used in the form of their alkali metal salts, alkaline earth metal salts, or ammonium salts. Typical phosphates used as inorganic sequestering agents are alkali metal orthophosphates and / or polyphosphates, such as pentasodium triphosphate.

[0106] Suitable organic sequestering agents include, for example, C4-C 30 -di-, -tri- and -tetracarboxylic acids, such as succinic acid, propanetricarboxylic acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, and C2-C6 20 Alkyl- and alkenyl succinic acids containing alkyl or alkenyl groups are also suitable organic sequestering agents, such as hydroxycarboxylic acids and polyhydroxycarboxylic acids (sugar acids). These include C4-C 20These include hydroxycarboxylic acids such as malic acid, tartaric acid, glutonic acid, mucic acid, lactic acid, glutaric acid, citric acid, tartronic acid, glucoheptonic acid, lactobionic acid, and sucrose mono-, di-, and tricarboxylic acids. Among these, citric acid and its salts are preferred. Another class is carboxylated fructans. Fructans are polymers of fructose molecules. They are typically constructed from fructose residues with a sucrose unit (i.e., glucose-fructose disaccharide) at the otherwise reducing end. The bond between fructose residues typically occurs at one of the two primary hydroxyls (OH-1 or OH-6). In inulin, fructosyl residues are linked by β-2,1-linkages. In levan and phlein, fructosyl residues are linked by β-2,6-linkages. Graminin-type fructosyl residues contain both β-2,1- and β-2,6-linkages. Preferably, the carboxylated fructan is derived from inulin. Particular examples are carboxymethyl linulin and carboxyethyl linulin. Suitable carboxylated fructans are described in EP 3561032 A1 and WO 2010 / 106077.

[0107] Suitable organic sequestering agents are also phosphonic acids, such as hydroxyalkylphosphonic acids or aminophosphonic acids, and their salts. These include, for example, phosphonobutanetricarboxylic acid (2-phosphinobutane-1,2,4-tricarboxylic acid; PBTC), aminotris-methylenephosphonic acid (N[CHPO(OH)]), aminotris(methylenephosphonate), sodium salt (ATMP; N[CHPO(ONa)]), ethylenediamine(methylenephosphonic acid) (EDTMPA), hexamethylenediamine(tetramethylenephosphonic acid), hexamethylenediamine(tetramethylenephosphonate), potassium salt (C 10 H (28-x) N2K x O 12P4 (x=6)), bis(hexamethylene)triamine(pentamethylenephosphonic acid) ((HO2)POCH2N[(CH2)2N[CH2PO(OH)2]2]2), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP; (HO)2POCH2N[CH2CH2N[CH2PO(OH)2]2]2), diethylenetriaminepenta(methylenephosphonate), sodium salt (CH9H (28-x) N3Na x O 15 P5 (x=7); 1-hydroxy-C1-C such as tetramethylene-triamine-pentaphosphonic acid, hydroxyethylamine diphosphonic acid, 2-hydroxyethyliminobis(methylenephosphonic acid) (HOCH2CH2N[CH2PO(OH)2]2), morpholinomethane diphosphonic acid, and 1-hydroxyethane-1,1-diphosphonic acid (HEDP; CH2C(OH)[PO(OH)2]2). 10-alkyl-1,1-diphosphonic acids are also suitable. Another suitable organic sequestering agent is polyaspartic acid. Polyaspartic acid includes salts of polyaspartic acid. The salt-forming cations may be monovalent or polyvalent, and examples include sodium, potassium, magnesium, calcium, ammonium, and the ammonium salts of mono-, di-, and triethanolamine. Such polymers may be copolymers, particularly (a) L- or D-aspartic acid (preferably L-aspartic acid), (b) a carboxylic acid, and (c) a diamine or amino alcohol. Such copolymers generally contain 70 to 95 mol % of (a), 5 to 30 mol % of (b), and 2 to 20 mol % of (c). The molar ratio of the carboxyl-containing compound (b) to the diamine or amino alcohol (c) is preferably 5:1 to 1:1.5 or 3:1 to 1:1.2, more preferably 3:1 to 1:1 or 2:1 to 1:1. Suitable organic sequestering agents are also aminopolycarboxylic acids, such as nitrilotriacetic acid (NTA), nitrilomonoacetic acid / propionic acid, nitrilotripropionic acid, β-alaninediacetic acid (β-ADA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,3-propylenediaminetetraacetic acid, 1,2-propylenediaminetetraacetic acid, N-(alkyl)ethylenediaminetriacetic acid, N-(hydroxyalkyl)ethylenediaminetriacetic acid, ethylenediaminetriacetic acid, cyclohexylene-1,2-diaminetetraacetic acid, iminodisuccinic acid, ethylenediaminedisuccinic acid, serinediacetic acid, isoserinediacetic acid, L-asparaginediacetic acid, L-glutaminediacetic acid, methylglycinediacetic acid (MGDA), and salts of the aforementioned aminopolycarboxylic acids. Suitable organic sequestering agents are also polymeric compounds containing carboxylic acid groups, such as acrylic acid homopolymers. The term "acrylic acid homopolymer" also includes polymers in which some or all of the carboxylic acid groups are present in neutralized form. Suitable polymeric compounds containing carboxylic acid groups are also oligomaleic acids. Suitable polymeric compounds containing carboxylic acid groups are also terpolymers of unsaturated C4-C8 dicarboxylic acids.Suitable unsaturated C4-C8 dicarboxylic acids in this context are, for example, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, methylenemalonic acid and citraconic acid. Suitable polymeric compounds containing carboxylic acid groups are also homopolymers of monoethylenically unsaturated C3-C8 monocarboxylic acids, such as acrylic acid, methacrylic acid, crotonic acid, 2-ethylacrylic acid, 2-phenylacrylic acid, cinnamic acid, vinylacetic acid and sorbic acid, copolymers of dicarboxylic acids, such as maleic acid and acrylic acid, terpolymers of maleic acid, acrylic acid and vinyl esters of C1-C3 carboxylic acids, copolymers of maleic acid with C2-C8 olefins.

[0108] Further additives [component (g)] are, for example, pH adjusters (pH regulators), thickeners, antifreeze agents, antifoaming agents, colorants and fragrances [i.e., different from components (a) and (b)].

[0109] Depending on the desired pH of the composition, the pH adjuster (pH modifier) ​​is an acid or a base. The pH can also be adjusted by a buffer system.

[0110] The acid may be inorganic or organic. Suitable inorganic acids include sulfuric acid, hydrochloric acid, and phosphoric acid, with sulfuric acid being generally preferred. Suitable organic acids include saturated, unsubstituted aliphatic C1-C6 mono-, di-, and tricarboxylic acids, such as formic acid, acetic acid, propanoic acid, oxalic acid, succinic acid, glutaric acid, and adipic acid; saturated aliphatic C1-C6 mono-, di-, and tricarboxylic acids having one or more OH groups, such as glycolic acid, lactic acid, tartaric acid, and citric acid; unsaturated aliphatic C1-C6 mono-, di-, and tricarboxylic acids, such as sorbic acid; aromatic carboxylic acids, such as benzoic acid, salicylic acid, and mandelic acid; and sulfonic acids, such as methanesulfonic acid or toluenesulfonic acid. Organic acids primarily function to adjust the pH of the composition, but some of them, such as di- and tricarboxylic acids, can also act as sequestering agents.

[0111] Suitable bases are, in particular, inorganic bases such as carbonates, for example sodium or potassium carbonate, as described in connection with the sequestering agents; further ammonium carbonate, alkali metal and alkaline earth metal bicarbonates, for example sodium or potassium bicarbonate, alkali metal and alkaline earth metal hydroxides, for example NaOH or KOH, or ammonium hydroxide. Organic bases may also be used; examples are alkanolamines such as monoethanolamine, triethanolamine or aminomethylpropanol, or guanidine derivatives such as 1,1,3,3-tetramethylguanidine or triazabicyclodecene.

[0112] Suitable buffers are typical systems such as hydrogen phosphate / dihydrogen phosphate buffer, carbonate / bicarbonate buffer, acetic acid / acetate or Tris buffer. Furthermore, most of the acids mentioned above, which are weak acids and whose anionic salts are not strong, also have buffering capacity.

[0113] The thickener serves to impart a desired viscosity to the compositions of the present invention.

[0114] Any known thickener (rheology modifier) ​​is in principle suitable, provided that it does not adversely affect the efficacy of the composition. Suitable thickeners may be either naturally occurring or synthetic.

[0115] Thickeners of natural origin are mostly derived from polysaccharides. Examples are xanthan, gellan gum, carob flour, guar flour or gum, carrageenan, agar, tragacanth, gum arabic, alginates, modified starches such as hydroxyethyl starch, starch phosphate esters or starch acetate, dextrin, pectin, and cellulose derivatives such as carboxymethylcellulose, hydroxyethylcellulose, hydrophobically modified hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, etc. Natural thickeners are also inorganic thickeners such as polysilicic acids and clay minerals, e.g., layered silicates, as well as the silicates mentioned as builders.

[0116] Examples of synthetic thickeners are polyacrylic and polymethacrylic acid compounds, for example (partially) crosslinked homopolymers of acrylic acid, for example with allyl ethers of sucrose or pentaerythritol, or with propylene (carbomer), for example Carbopol® brands from BF Goodrich (e.g. Carbopol® 676, 940, 941, 934, etc.) or 3V Polygel® brands from Sigma (e.g. Polygel® DA), copolymers of ethylenically unsaturated mono- or dicarboxylic acids, for example terpolymers of acrylic acid, methacrylic acid or maleic acid with methyl acrylate or ethyl acrylate and (meth)acrylates derived from long-chain ethoxylated alcohols, for example Acusol® brands from Rohm & Haas (e.g. Acusol® 820 or 1206A), copolymers of two or more monomers selected from acrylic acid, methacrylic acid and their C1-C4-alkyl esters, for example copolymers of methacrylic acid, butyl acrylate and methyl methacrylate or butyl acrylate and methyl methacrylate, for example Aculyn® and Acusol® brands from Rohm & Haas (e.g. Aculyn® 22, 28 or 33 and Acusol® 810, 823 and 830), or crosslinked high molecular weight acrylic acid copolymers, for example acrylic acid C 10 ~C 30 and one or more comonomers selected from acrylic acid, methacrylic acid and their C1-C4-alkyl esters, said copolymers being crosslinked with allyl ethers of sucrose or pentaerythritol (e.g., Carbopol® ETD 2623, Carbopol® 1382 or Carbopol® AQUA 30 from Rohm & Haas). Another preferred group of substances is the Rheovis® brand from BASF, e.g., Rheovis® AT120.

[0117] Examples of suitable antifreeze agents are ethylene glycol, propylene glycol, urea and glycerin.

[0118] Examples of suitable antifoaming agents are silicones, long chain alcohols and salts of fatty acids.

[0119] Suitable colorants (e.g., red, blue, or green) are low- and water-soluble dyes. Examples are inorganic colorants (e.g., iron oxide, titanium oxide, ferricyanide) and organic colorants (e.g., alizarin, azo, and phthalocyanine colorants).

[0120] Fragrances may be of natural or synthetic origin; their nature is generally not critical.

[0121] The Bacillus strains of the present invention can be produced via fermentation. As used herein, the term "fermentation" generally refers to any activity or process involving the breakdown (digestion) of organic material by the Bacillus strains of the present invention, resulting in their growth. The term fermentation, as used herein, should be understood to include any type of fermentation, including, but not limited to, substantially zero-growth fermentation, i.e., a fermentation process in which the cell density of the microorganisms applied to the fermentation process is substantially zero and the microorganisms do not undergo significant population growth; under-fermentation, i.e., a fermentation process in which the fermentation is carried out for an insufficient period; and over-fermentation, e.g., a fermentation process in which the fermentation is carried out for too long a period. The term "fermentation" encompasses both anaerobic and aerobic processes, as well as processes involving a combination or sequence of one or more anaerobic and / or aerobic stages. Furthermore, fermentation can be a continuous or batch process.

[0122] There are two determinants that induce sporulation: nutrient starvation and cell density. Thus, to produce spores of the Bacillus strains of the present invention, vegetative bacterial cells must reach an OD of greater than 0.3, greater than 0.4, greater than 0.5, or greater than 0.6. 600Alternatively, the cell density can be increased to 10 or 20. 6 >10 cells / mL 7 >10 cells / mL 8 More than cells / mL, 5*10 8 cells / mL or greater than 10 9 May be greater than cells / mL.

[0123] As used herein and in the appended claims, the singular forms "a" and "an" also include the respective plural forms, unless the context clearly dictates otherwise. In the context of the present invention, the terms "about" and "approximately" indicate a margin of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically denotes a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value.

[0124] Furthermore, the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," etc. in the specification and claims are used to distinguish between similar components and do not necessarily describe a sequential or chronological order. It is to be understood that terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein can be performed in orders other than those described or illustrated herein. When the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," "i," "ii," etc. refer to steps of a method, use, or assay, unless otherwise specified in this application, as described herein above or below, there is no time gap or inconsistency between the steps, i.e., the steps may be performed simultaneously, or there may be a time gap of seconds, minutes, hours, days, weeks, months, or even years between such steps.

[0125] Throughout this application, various publications are referenced. The entire disclosures of these articles and the references cited within them in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0126] The term "comprising" should be understood to be open-ended. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising." Hereinafter, when a group is defined as comprising at least a certain number of members, this means that the group also includes groups consisting only of these members.

[0127] The present invention is further illustrated by the following examples, sequences, and figures, from which additional features, embodiments, aspects, and advantages of the present invention may be obtained. Furthermore, the materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting. [Example]

[0128] Each strain in the collection, which included approximately 50 strains from a variety of different bacterial species, was tested for its ability to form spores, provide extracellular enzyme activity, and reduce malodor according to the following protocol: A score for each criterion was assigned, and the strain with the highest overall score was further tested for cleaning efficacy on soiled tiles and long-term spore viability.

[0129] Example 1 - Spore count Prior to use, all strains were streaked onto TSAY agar plates (30 g / L tryptic soy broth, 3 g / L yeast extract, 20 g / L agar) and incubated at 30°C for 2 days. All sporulation experiments were performed in BA055 medium containing 10 g / L glucose*H2O, 5 g / L yeast extract, 10 g / L Sofarin Soymeal, 10 g / L corn starch, 3.7 g / L K2HPO4, and 2.5 g / L KH2PO4, adjusted to pH 7, and sterilized at 121°C for 30 min. To determine the ability of bacterial cultures to produce viable spores, spore counts were performed by inoculating 1 mL of BA055 medium in flower plates (m2p-labs) with a single colony. Plates were incubated at 30°C and 1000 rpm for 72 h. A dilution series was then performed by mixing 100 μL of the sample with 900 μL of dilution solution containing 0.9% NaCl and 0.1 g / L Tween 80 and vortexing for 30 seconds. The dilution was repeated until sufficient dilution was achieved. 10 μL of the diluted sample was spotted onto an agar plate, incubated at 30°C for 24 hours, and the cells were counted. To differentiate vegetative cells from spores, 100 μL of the diluted cell suspension was transferred to a 96-well plate and incubated at 60°C for 30 minutes in a thermocycler. 10 μL of the heat-treated sample was spotted onto an NB agar plate, incubated at 30°C for 24 hours, and then the cells were counted. For semiquantitative evaluation, 1*10 8 spores / mL is evaluated as 1, and 2*10 8 spores / mL is evaluated as 2, 3*10 8 spores / mL was rated as 3 and continued as the spore count increased. After 72 hours, spore counts >10 9 Strains that reached spores / mL were scored as 10, whereas strains that reached <10 8 Strains with spores / mL were scored as 0.

[0130] 1 shows the spore counts of four bacterial strains, namely, Bacillus methylotrophicus (containing SEQ ID NO: 1), Bacillus subtilis (containing SEQ ID NO: 2), Bacillus amyloliquefaciens (containing SEQ ID NO: 3), and Bacillus amyloliquefaciens (containing SEQ ID NO: 4), which had the highest overall score of all bacterial strains tested. As comparative examples, data are shown for four additional strains, namely, Paenibacillus sp., Paenibacillus glycanilyticus, Paenibacillus amylolyticus, and Glosibacter faecalis. The spore count scores achieved by each strain are shown in the "Spore Count" column of Figure 3.

[0131] Example 2 - Agar Plate Enzyme Assay For agar plate enzyme assays, strains were streaked onto NB-agar-based plates (8 g / L NB (Difco), 20 g / L agar) containing additional additives to allow semiquantitative visual assessment of enzyme activity. To assess amylase activity, plates additionally contained 2.5 g / L red starch. In a second approach, agar plates contained 5 g / L soluble corn starch and were stained with Lugolsch solution after 48 h of incubation. For protease activity, NB-agar plates additionally contained 10 g / L skim milk powder. To assess lipase activity, NB-agar was mixed with 10 ml / L tributyrin before pouring the agar plates. For mannanase activity, 1 g / L locust bean gum was added to NB-agar. After 48 h of incubation on the plates, the respective enzyme activities were visually assessed. High enzyme activity resulted in a large, clear halo around the bacterial colony due to degradation of the respective substrate. Enzyme activity was scored semiquantitatively from 0 (no activity) to 3 (high activity). The values ​​of the two different assays for amylase activity were added and the sum was divided by 2 to obtain the average amylase activity.

[0132] Figure 2 shows the scores for the strains described in Example 1 for each enzyme assay tested. An overall enzyme score was generated by adding the scores for the average amylase, protease, lipase, and mannanase activities. This score is shown in the "Enzyme Score" column in Figure 3.

[0133] Example 3 - Odor Control The strains were cultivated in 1 mL of BA055 medium on flower plates (m2p-lab) at 30°C and 1000 rpm. The biomass was separated by centrifugation (4,500 g, 10 min) and the supernatant was discarded. The flower plates were resealed with AirPore lids and allowed to dry at room temperature. After 7 days, approximately 1*10 9The pellets containing viable spores were resuspended in 1 mL of lactose-free whole milk (unsterilized, lactose-free, 3.5% fat) and transferred to sterile 50 mL Falcon tubes. The tubes were incubated at 30°C for 1 week without stirring. Odor formation was assessed olfactorily by sniffing (10 = no odor / milky odor, 8 = weakly acidic odor, 6 = very acidic and mildly malodorous, 4 = moderately malodorous, 2 = strong malodorous).

[0134] The respective scores for the different strains (Bacillus methylotrophicus (including SEQ ID NO: 1), Bacillus subtilis (including SEQ ID NO: 2), Bacillus amyloliquefaciens (including SEQ ID NO: 3), Bacillus amyloliquefaciens (including SEQ ID NO: 4), Paenibacillus sp., Paenibacillus glycanilyticus, Paenibacillus amylolyticus and Gulosibacter faecalis) are shown in Figure 3, column "Odor Control".

[0135] Based on the overall score, the strains Bacillus methylotrophicus (including SEQ ID NO: 1), Bacillus subtilis (including SEQ ID NO: 2), Bacillus amyloliquefaciens (including SEQ ID NO: 3) and Bacillus amyloliquefaciens (including SEQ ID NO: 4) were selected for further testing.

[0136] Example 4 - Long-term cleaning effect (CFT tiles) 20 mL of BA055 medium in a 100 mL baffled shake flask was inoculated with the target strain and incubated at 30°C and 220 rpm for 72 hours. Spores were collected by centrifugation, washed with dH2O, and resuspended in either dH2O or 1x Novozymes Deep Clean Multi EU 5x EP based sterile filtered detergent. At least 3*10 8 4 mL of solution containing cfu / mL was spread on different types of CFT tiles to evaluate their long-term cleaning efficacy on different substrates (DM-40: hard surface cleaner soil according to IKW protocol; DM-177: corn / starch mix on melamine, colored, extra heavy; DM-03: shepherd's pie on melamine). All CFT tiles were placed on Corning agar plates and incubated at 30°C for 7 days. Plates were then rinsed with dH2O, dried, and evaluated visually or by photodensitometry using a Mach5 instrument.

[0137] The long-term cleaning effect for Bacillus subtilis (DSM 34307), Bacillus amyloliquefaciens (DSM 34304), and Bacillus amyloliquefaciens (DSM 34305) can be seen in Figure 4, which shows the cleaning effect of the indicated bacterial strains on tiles soiled with Shepherd's Pie (Paenibacillus glycanilyticus, an additional strain from the library tested, shows no effect on long-term cleaning). Significant cleaning effect was observed for all four bacterial strains on DM-03 CFT tiles soiled with complex medium (Shepherd's Pie), which contains a mixture of protein, starch, and fat.

[0138] Example 5 - Long-term spore viability Long-term spore viability was assessed by inoculating 50 mL of BA055 medium with the bacterial strain and incubating at 30°C and 200 rpm. After 5 days of incubation, the biomass was collected by centrifugation, washed three times with 50 mL of dH2O, and then resuspended in 60% glycerol. The pH of the glycerol solution was adjusted to pH 5 / 7 / 9 using 0.1 M NaOH or 0.1 M HCl, respectively. The spore-containing glycerol solution was stored in 15 mL plastic tubes at room temperature. Spore counts were measured at 7-day intervals as described above.

[0139] For all four selected test strains, spore counts remained stable over a 4-week period, demonstrating the strains' high stability over the selected pH range (Figure 5A). Furthermore, no spore germination was observed microscopically as long as the spores remained in the glycerol solution. Figure 5B shows bright-field observations (100x magnification) of Bacillus amyloliquefaciens (containing SEQ ID NO: 3) stored at pH 9 after 14 days, demonstrating the sole presence of spores without any vegetative cells.

[0140] Example 6: Comparison of the strains of the invention with reference strains via sensory malodour assessment and HPLC measurements 1 mL of lactose-free milk (3.5% fat) was inoculated with 1*10^9 spores / mL in a 15 mL Falcon tube and incubated at 30°C for 7 days. Odor formation was then assessed by a sensory panel (0 = strong odor formation, 10 = no odor formation; see also Example 3). After sensory evaluation, the sample was diluted with 1 mL of dH2O, mixed by pipetting, and centrifuged (6,000 g, 10 min). The supernatant was filtered using a 0.22 μm PES filter, transferred to a glass vial, and measured via HPLC (Aminex HPX-87-H (BIO-RAD), 300 x 7.8 mm, 9 μm particle size, with 0.5 M H2SO4 (Roth) as eluent, 0.5 ml / min flow rate, 30°C).

[0141] The correlation between the malodor caused by microorganisms and the presence of certain metabolite compounds is well known in the art. Along these lines, Greenman et al. (Greenman, John et al. Journal of the American Dental Association (1939), 136, 6 (2005): 749-57) devised an organoleptic model that correlates the sensory evaluation of a given sample with the concentration of different compounds. For example, a commonly found malodorous compound is butyric acid, which in its protonated form shows a strong malodor perception at relatively low concentrations. Already low concentrations of >0.8 g / L cause a strong odor perception. The sensory malodor assessment of the strains of the present invention and the control and reference strains correlates well with the proposed butyric acid concentration and olfactory perception as described by Greenman et al. (See Figure 6). B. subtilis ATCC 6633, previously described as a suitable strain for probiotic cleaning applications (Vehapi, M., Oezcimen, D. Bulletin of Biotechnology 1 (2020): 1-7), exhibited a significantly negative odor profile in terms of both butyric acid formation (0.44 g / L) in milk samples and sensory perception by the panel. An additional publicly available Bacillus amyloliquefaciens strain (DSM 1060) also demonstrated odor-control behavior. Conversely, milk samples inoculated with the strains of the present invention (containing SEQ ID NOS: 1-4) demonstrated an overall positive odor profile, analytically confirmed by the composition of organic acids in the samples. Thus, milk samples inoculated with these strains showed no or only low concentrations of butyric acid (up to 0.2 g / L), resulting in no or only a slight detectable odor and an overall positive organoleptic perception by the test panel.

[0142] Example 7 - ADW Formulation Below are exemplary ADW (automatic dishwasher) formulations containing the inventive strains of the present application:

[0143] Automatic dishwashing detergent

[0144] [Table 2]

[0145] Automatic dishwashing unit - portioned product (pouch) Particle composition wt% STPP 0 Silicates 1-5 Sodium carbonate 25-50 MGDA 5~25 Polymers with zinc ions 1 5~10 polymer dispersants 2 0~5 Nonionic surfactants 3 3~10 Enzymes 0-6 Bleaching agents, bleaching catalysts, activators 5-15 Fragrance 0.05~0.2 Sodium sulfate 0-20 Bacterial strain of the present invention 0.001 to 5 liquid composition DPG 40~50 Nonionic surfactants 3 40~50 Neodol C11E9 0~5.0 Glycerin 0~5.0 Dye 0.1~1.0 1 Any mixture of copolymers as defined in the polymer section or copolymers with zinc ions 2 ACUSOL® 445N from Dow 3 SLF-18 POLY TERGENT from BASF Corporation

[0146] Example 8 - Cleaning Formulation Below are exemplary cleaning formulations containing the inventive strains of this application.

[0147] Strong acidic hygiene cleaner based on lactic acid / methanesulfonic acid (%)

[0148] [Table 3]

[0149] The following I-VIII are ready-to-use (RTU) cleaner formulations (i.e., formulations that are not further diluted with water before use) containing the Bacillus spores of the present invention. Such formulations provide good initial cleaning results. The spores are sufficiently stable in the formulations that germination of the spores is not inhibited during use. Such formulations can be used to treat common hard surfaces in homes or offices, or in commercial, industrial, or agricultural environments, but particularly surfaces in bathrooms, kitchens, dairy farms, and other places where food is stored or processed.

[0150] [Table 4]

[0151] Formulations Ia-VIII-a correspond to the same formulations as I-VIII, but with Bacillus subtilis (DSM 34307) instead of Bacillus methylotrophicus (DSM 34306).

[0152] Formulations Ib-VIII-b correspond to the same formulations as I-VIII, but with Bacillus amyloliquefaciens (DSM 34304) instead of Bacillus methylotrophicus (DSM 34306).

[0153] Formulations Ic-VIII-c correspond to the same formulations as I-VIII, but with Bacillus amyloliquefaciens (DSM 34305) instead of Bacillus methylotrophicus (DSM 34306).

[0154] The following are dilutable concentrated cleaning formulations (i.e., formulations that are diluted 10-100 times with water before use) containing the XYZ spores of the present invention. The spores are sufficiently stable in the formulations. After dilution with water, such formulations provide good initial cleaning results and do not inhibit spore germination during use. Such formulations can be used to treat common hard surfaces in homes or offices, or in commercial, industrial, or agricultural environments, but particularly surfaces in bathrooms, kitchens, dairy farms, and other places where food is stored or processed.

[0155] [Table 5]

[0156] [Table 6]

[0157] Formulations IX-a to XVI-a correspond to the same formulations as IX to XVI, but with Bacillus subtilis (DSM 34307) instead of Bacillus methylotrophicus (DSM 34306).

[0158] Formulations IX-b to XVI-b correspond to the same formulations as IX to XVI, but with Bacillus amyloliquefaciens (DSM 34304) instead of Bacillus methylotrophicus (DSM 34306).

[0159] Formulations IX-c to XVI-c correspond to the same formulations as IX to XVI, but with Bacillus amyloliquefaciens (DSM 34305) instead of Bacillus methylotrophicus (DSM 34306).

[0160] Example 9 - Detergent Formulation In a preferred embodiment, the bacterial strains of the present invention are used in laundry detergents.

[0161] The liquid laundry detergent according to the present invention comprises: 0.05 to 20% of at least one polymer (anionic, nonionic, or amphoteric) 1-50% surfactant 0.1 to 40% of builders, cobuilders and / or chelating agents 0.1~50% of other adjuvants 0.001 to 5% of the bacterial strain of the present invention Water that adds up to 100% It consists of:

[0162] A preferred liquid laundry detergent according to the present invention comprises (wt%): 0.2 to 6% of at least one polymer (anionic, nonionic, or amphoteric) Anionic surfactants selected from 5-40% C10-C15-LAS and C10-C18 alkyl ether sulfates containing 1-5 ethoxy units Nonionic surfactants selected from C10-C18 alkyl ethoxylates containing 1.5-10% of 3-10 ethoxy units 2-20% of soluble organic builders / cobuilders selected from C10-C18 fatty acids, di- and tricarboxylic acids, hydroxydi- and hydroxytricarboxylic acids and polycarboxylic acids An enzyme system containing 0.05 to 5% of at least one enzyme suitable for detergent applications and preferably also an enzyme stabilizing system. 0.5 to 20% of a mono- or diol selected from ethanol, isopropanol, ethylene glycol, or propylene glycol 0.1~20% of other adjuvants 0.001 to 5% of the bacterial strain of the present invention Water that adds up to 100% It consists of:

[0163] The solid laundry detergent (e.g., as a powder, granule or tablet) according to the present invention comprises: 0.05 to 20% of at least one polymer (anionic, nonionic, or amphoteric) 1-50% surfactant 0.1 to 80% of builders, cobuilders and / or chelating agents 0-50% bulking agent 0-40% bleaching active substance 0.1-30% of other adjuvants and / or water 0.001 to 5% of the bacterial strain of the present invention The total of the components is 100%.

[0164] Preferred solid laundry detergents according to the present invention include: 0.2 to 6% of at least one polymer (anionic, nonionic, or amphoteric) Anionic surfactants selected from 5-30% C10-C15-LAS, C10-C18 alkyl sulfates and C10-C18 alkyl ether sulfates containing 1-5 ethoxy units Nonionic surfactants selected from C10-C18 alkyl ethoxylates containing 1.5-7.5% of 3-10 ethoxy units 5-50% of inorganic builders selected from sodium carbonate, sodium bicarbonate, zeolite, soluble silicates, and sodium sulfate 0.5 to 15% of cobuilders selected from C10 to C18 fatty acids, di- and tricarboxylic acids, hydroxydi- and hydroxytricarboxylic acids and polycarboxylic acids An enzyme system containing 0.1 to 5% of at least one enzyme suitable for detergent applications and preferably also an enzyme stabilizing system. 0.5 to 20% of a mono- or diol selected from ethanol, isopropanol, ethylene glycol, or propylene glycol 0.1~20% of other adjuvants 0.001 to 5% of the bacterial strain of the present invention Water that adds up to 100% It consists of:

[0165] In a preferred embodiment, the bacteria according to the present invention are used in manual dishwashing detergents.

[0166] The liquid manual dishwashing detergent according to the present invention comprises: 0.05 to 10% of at least one polymer (anionic, nonionic, or amphoteric) 1-50% surfactant 0.1~50% of other adjuvants 0.001 to 5% of the bacterial strain of the present invention Water that adds up to 100% It consists of:

[0167] Preferred liquid manual dishwashing detergents according to the present invention include: 0.2 to 5% of at least one polymer (anionic, nonionic, or amphoteric) Anionic surfactants selected from 5-40% C10-C15-LAS, C10-C18 alkyl ether sulfates containing 1-5 ethoxy units, and C10-C18 alkyl sulfates. 2-10% Cocamidopropyl Betaine 0-10% lauramine oxide 0-2% nonionic surfactant, preferably C10 Guerbet alcohol alkoxylate 0-5% enzyme, preferably amylase, and preferably also an enzyme stabilizing system 0.5 to 20% of a mono- or diol selected from ethanol, isopropanol, ethylene glycol, or propylene glycol 0.1~20% of other adjuvants 0.001 to 5% of the bacterial strain of the present invention Water that adds up to 100% It consists of:

[0168] The following table shows certain types of common cleaning compositions corresponding to typical compositions associated with typical cleaning conditions typically employed in various regions and countries of the world. At least one bacterial strain of the present invention may be added to such formulations in a suitable amount, for example, 0.1-5% by total weight.

[0169] General formula for laundry detergent compositions:

[0170] [Table 7]

Claims

1. A Bacillus strain that has extracellular hydrolase activity and that exhibits reduced malodor after incubation for at least 5 days at at least 25°C in lactose-free whole milk samples compared to uninoculated lactose-free whole milk samples.

2. 2. The Bacillus strain of claim 1, wherein the hydrolase activity is selected from a protease activity, an amylase activity, a lipase activity, a mannanase activity, a cellulase activity, or a combination thereof.

3. 3. The Bacillus strain of claim 1 or 2, wherein the incubation is at least 30°C for at least 7 days.

4. A Bacillus strain according to any one of claims 1 to 3, wherein the strain has the ability to form spores and preferably exists in the form of spores.

5. 5. The Bacillus strain of any one of claims 1 to 4, wherein the strain is selected from the group of Bacillus species consisting of Bacillus methylotrophicus, Bacillus subtilis and Bacillus amyloliquefaciens.

6. i) as deposited under accession number DSM 34306 or a variant thereof having all the distinguishing characteristics of claims 1 to 4; ii) as deposited under accession number DSM 34307 or a variant thereof having all of the distinguishing characteristics of claims 1 to 4; iii) as deposited under accession number DSM 34304 or a variant thereof having all of the distinguishing characteristics of claims 1 to 4; or iv) as deposited under accession number DSM 34305 or a variant thereof having all the distinguishing characteristics of claims 1 to 4. The Bacillus strain according to any one of claims 1 to 5,

7. i) said strain as deposited under accession number DSM 34306 contains a 16S rDNA having the sequence of SEQ ID NO: 1; ii) said strain as deposited under accession number DSM 34307 comprises a 16S rDNA having the sequence of SEQ ID NO:2; iii) said strain as deposited under accession number DSM 34304 comprises a 16S rDNA having the sequence of SEQ ID NO: 3; or iv) The Bacillus strain of claim 6, wherein said strain as deposited under accession number DSM 34305 comprises a 16S rDNA having the sequence of SEQ ID NO:

4.

8. i) said variant of said strain as deposited under accession number DSM 34306 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO: 1; ii) said variant of said strain as deposited under accession number DSM 34307 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO:2; iii) said variant of said strain as deposited under accession number DSM 34304 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO:3; or iv) The Bacillus strain of claim 6, wherein said mutant of said strain as deposited under accession number DSM 34305 comprises a 16S rDNA having at least 98% sequence identity with SEQ ID NO:

4.

9. A composition comprising at least one Bacillus strain according to any one of claims 1 to 8 and at least one additional component.

10. 10. The composition of claim 9, wherein the composition is a detergent composition, preferably a laundry detergent composition, a hard surface cleaning detergent composition, a hygiene or kitchen cleaner or an anti-malodor product.

11. 11. A composition according to claim 9 or 10, wherein the composition comprises one or more surfactants and / or one or more builders and / or one or more preservatives and / or one or more enzymes and / or one or more solvents, preferably one or more surfactants and / or one or more solvents.

12. A method for improving the surfactant activity of a detergent formulation by adding at least one Bacillus strain according to any one of claims 1 to 8 to said detergent formulation.

13. 12. A cleaning method comprising contacting at least one Bacillus strain according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 11 with an object in need of cleaning, preferably a laundry or hard surface household item.

14. 12. A method for reducing malodours comprising contacting an object in need of cleaning, preferably a laundry or hard surface household item, with at least one Bacillus strain according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 11.

15. 12. A method for controlling or reducing malodor-causing microorganisms, the method comprising contacting at least one Bacillus strain according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 11 with an object or a medium in which said microorganisms are present or growing.

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