Microbial product and products thereof

Abstract

The present invention relates to strains of Bacillus velezensis and products derived thereof and compositions comprising at least one of them. The invention also pertains to their application, in particular with regard to coating compositions, pharmaceutical compositions as well as cosmetic compositions.

Description

[0001] 202400023 Foreign Filing 1

[0002] Microbial product and products thereof

[0003] The invention relates to strains of Bacillus velezensis and products derived thereof and compositions comprising at least one of them. In particular, the invention relates to the provision of coating compositions as well as compositions comprising raw materials for said coating compositions. The invention further relates to use of Bacillus velezensis and products derived thereof as a preservative. Another aspect of the invention are pharmaceutical compositions comprising Bacillus velezensis and products derived thereof as well as their use against diseases associated with pathogenic fungi, in particular Penicillium chrysogenum and Fusarium solani, and for treating plant diseases. The invention also pertains to a cosmetic composition comprising strains of Bacillus velezensis and products derived thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Water-based coating compositions, in particular paint- and varnish compositions, have been developed as a solution to decrease the use of organic solvents and the associated emission of volatile organic compounds (VOCs). Similarly, the market has seen a trend for water-based additives, e.g. defoamers. However, the drawback of aqueous compositions is that these are beneficial for the survival and growth of undesired microorganisms such as bacteria, fungi or yeasts. These undesired microorganisms may be introduced to such compositions via the raw materials, the process water, within the production plant, or in the filled containers of the product. According to own findings Pseudomonas and bacteria of related genera can easily contaminate the production process water. Furthermore, contamination with persistent forms of environmental germs, especially mold spores, occurs via the air.

[0006] The degradation of paints and varnish raw materials in the presence of undesired microorganisms manifests itself through a change in color, a penetrating odor, gas formation, reduced stability, pH variations and viscosity reduction. The loss of quality of the coating raw materials, such as defoamer emulsions, leads to a decrease in functionality and ultimately to product damage and time delays in the formulated color. Although the presence of microorganisms can be controlled through improved raw material selection, improved process water treatment and hygiene measures in operation, they cannot be completely avoided. Therefore, stabilization of the aqueous products is necessary.

[0007] This problem has been generally solved using chemical biocides. However, the use of toxic biocides to stabilize aqueous products can impair human health. The use of classic biocides for the pot preservation of water-based coatings is therefore under increasing regulatory pressure. First, organomercury compounds and formaldehyde-releasing biocides were banned due to their carcinogenic effect. The isothiazoline derivatives were introduced as formaldehyde-free alternatives for the preservation of aqueous products, including 1 ,2-benzisothiazolin-3-one (BIT), 2-methylisothiazole-3(2H)-one (MIT), 2-octyl-2H- isothiazole-3-one (OIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), and 4,5-dichloro-2-n-octyl-3(2H)- isothiazolone (DCOIT) (P. Samyn et al. (2021), Materials Proceedings 7(1):18, "Current Alternatives for InCan Preservation of Aqueous Paints: A Review"). There are indications that MIT in particular can lead to allergic skin reactions and that it has therefore been classified as a skin sensitizer and the concentration limit has been set to below 15 ppm according to the Risk Assessment Committee (RAC) of the European Chemicals Agency (ECHA). In view of a harmonized classification, it is assumed that the use concentrations 202400023 Foreign Filing 2 of the other isothiazolines will also be reduced to <15 ppm. The problem is that these concentrations are ineffective against a wide range of microorganisms, especially molds. At the same time, the current options for alternative preservatives are limited by the Article 95 list. Currently, 52 biocidal active ingredients are listed, of which only 15 are approved for use in the paint and coatings industry and often do not lead to long-term stabilization of the aqueous products.

[0008] It is therefore an object of the present invention to provide a bio-based solution to the preservation of and storage stability of paint- and varnish compositions, as well as water-based raw materials used therein.

[0009] BRIEF DESCRIPTION OF FIGURES

[0010] Figure 1 shows a comparison of Bacillus velezensis DSM 34979and DSM 34982 and benchmark preservative antifungal (inhibitory) capacity on phytopathogenic contaminants of defoamer solutions (Penicillium chrysogenum field isolate A and B, Fusarium solani field isolate A and B) in well diffusion antagonism assays, values in mm clearance of pathogen.

[0011] Figure 2 is a visualization of the clearance of fungal pathogens in well antagonism diffusion assays by DSM 34979 (A-D) and DSM 34982 (E-H). The fungal pathogen is Penicillium chrysogenum field isolate A in Figs. 2 A and E, Penicillium chrysogenum field isolate B in Figs. 2 B and F, Fusarium solani field isolate A in Figs. 2C and G, and Fusarium solani field isolate B in Figs. 2 D and H.

[0012] Figure 3 A) shows pO2 curves of Penicillium chrysogenum (P.c.) of liquid well antagonism assay. Antifungal activity of Bacillus velezensis DSM 34979 and DSM 34982 fermentation broths are shown against Penicillium chrysogenum. Here 12.5% (v / v) sterile filtered fermentation broth of each strain was used. The mean data of technical duplicates were shown and represents the results of three biological experiments. Figure 3 B) is a visual overview of wells from the Woulter-Duetz plate after 7 days of incubation. Penicillium chrysogenum (P.c.) growth is only shown in the first well (growth control).

[0013] Figure 4 A) shows pO2 curves of Fusarium solani of liquid well antagonism assay. Anti-fungal activity of Bacillus velezensis DSM 34979 and DSM 34982 fermentation broths are shown against Fusarium solani. Here 12.5% (v / v) sterile filtered fermentation broth of each strain was used. The mean data of technical duplicates were shown and represents the results of three biological experiments. Figure 4 B) is a visual overview of wells from the Woulter-Duetz plate after 7 days of incubation. Fusarium solani growth is only shown in the first well (growth control).

[0014] Figure 5 shows the growth of Penicillium chrysogenum in different preserved defoamer emulsion. SN3 (DSM 34979 sterile filtered fermentation broth) and SN7 (DSM 34982 sterile filtered fermentation broth) represent cell free supernatant, regular describes a common biocide mixture.

[0015] Figure 6 shows the result of a PVC30 coating comprising A) neither defoamer nor Bacillus velezensis cell free supernatant, B) 0.5 wt.-% comparative defoamer, C) 0.5 wt.-% inventive defoamer with SN3, D) 0.5 wt.-% inventive defoamer with SN7. 202400023 Foreign Filing 3

[0016] Figure 7 shows the result of microbial growth in A) demineralized water (mostly bacterial), B) Heliogen Blue L 7085 pigments (mostly fungal), as well as pigment concentrate comprising C) only a siloxane-based dispersing agent, as well as a pigment concentrates in which the siloxane-based dispersing agent was partially replaced by D) 10 wt.-%, E) 20 wt.-%, F) 30 wt.-% with an inventive fermentation broth.

[0017] DETAILED DESCRIPTION OF THE INVENTION

[0018] It was surprisingly found that strains of the species Bacillus velezensis can be used to inhibit the growth of undesired microorganisms, in particular fungi, as found in contaminated paint and varnish compositions, wherein Bacillus velezensis strains DSM 34979 and DSM 34982 turned out to be very suitable according to the invention.

[0019] Thus, a first subject of the present invention is a microorganism or microbial preparation selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.

[0020] The microorganisms and microbial preparations of the invention preferably possess inhibitory activity against at least one fungus, preferably against both fungi, selected from Fusarium solani and Penicillium chrysogenum.

[0021] The microorganisms and microbial preparations of the invention further preferably possess inhibitory activity against pathogenic bacteria, in particular selected from E. coli and pathogenic Salmonella bacteria.

[0022] The deposited Bacillus velezensis strains of the invention which turned out to be very suitable as preservative were identified by screening of naturally occurring isolates and were deposited at the DSMZ (Leibniz-lnstitute DSMZ-German Collection of Microorganisms and Cell Cultures, InhoffenstraBe 7B, 38124 Braunschweig, Germany) under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure on March 20, 2024under the Accession Numbers DSM 34979 and DSM 34982 in the name of the applicant Evonik Operations GmbH.

[0023] The Bacillus velezensis strain as deposited under DSM 34979 at the DSMZ exhibits the following characterizing sequences: a 16S rDNA sequence according to SEQ ID NO: 1 , an rpoB sequence according to SEQ ID NO: 2, a gyrB sequence according to SEQ ID NO: 3, an yqfD sequence according to SEQ ID 202400023 Foreign Filing 4

[0024] NO: 4, a groL sequence according to SEQ ID NO: 5, a sequence encoding a hypothetical protein according to SEQ ID NO: 6.

[0025] Accordingly, the mutant of the strain DSM 34979 preferably exhibits a 16S rDNA sequence, an rpoB sequence, a gyrB sequence, an yqfD sequence, a groL sequence and / or a sequence encoding a hypothetical protein according to SEQ ID NO: 6, which are at least 99 or 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 % identical to the respective sequences of the strain DSM 34979 as mentioned above.

[0026] The Bacillus velezensis strain as deposited under DSM 34982 at the DSMZ exhibits the following characterizing sequences: a 16S rDNA sequence according to SEQ ID NO: 7, an rpoB sequence according to SEQ ID NO: 8, a gyrB sequence according to SEQ ID NO: 9, an yqfD sequence according to SEQ ID NO: 10, a groEL sequence according to SEQ ID NO: 1 1 .

[0027] Accordingly, the mutant of the strain DSM 34982 preferably exhibits a 16S rDNA sequence, an rpoB sequence, a gyrB sequence, an yqfD sequence and / or a groEL sequence, which are at least 99 or 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 % identical to the respective sequences of the strain DSM 34982 as mentioned above.

[0028] The mutants of the invention can principally be obtained by any kind of method, i.e., by GMO- or non-GMO methods, but preferably the mutants are not genetically modified, i.e., non-GMO. This means that the mutants of the deposited strains are preferably either also naturally occurring microorganisms or spontaneous mutants of such naturally occurring microorganisms, in particular of the deposited strains, or microorganisms which are obtained by another method which is classified as non-GMO. The term “spontaneous mutant” refers to mutants that arise from naturally occurring microorganisms and / or parent strains without genetically modifying the microorganisms by applying classical gene technological and / or biotechnological methods like site-directed mutagenesis. Such spontaneous mutants may be obtained by classical methods of natural selection, such as growing the microorganisms in the presence of UV light and / or by applying high temperature or protoplast formation and / or in the presence of a certain antibiotic to which the parent strain is susceptible. Suitable spontaneous mutants may in particular be generated by applying so-called Adaptive Laboratory Evolution (“ALE”) (Hirasawa and Maeda (2023) Microorganisms 11 (1), 92).

[0029] Spontaneous mutants might further, but less preferably, be obtained by using mutagens, i.e., chemical substances which induce the formation of mutants. As formation of spontaneous mutants by using mutagens is less preferred, in a preferred embodiment of the invention the spontaneous mutants and / or non-GMO mutants are obtained without the use of such mutagens. If the mutants are obtained by applying gene technological and / or biotechnological methods like site-directed mutagenesis, then preferably methods are applied which are classified as non-GMO. A non-GMO method according to the invention is preferably characterized in that the method does not involve introduction of heterologous genetic information into the microorganism. In a particularly preferred embodiment of the invention the 202400023 Foreign Filing 5 microorganisms of the invention are naturally non-occurring mutants, in particular non-GMO and / or spontaneous mutants as defined before.

[0030] The microorganisms of the invention have preferably the same or very similar characteristics like the parent strain from which they are derived, in particular the ability to inhibit fungal spore outgrowth, above all outgrowth of Penicillium chrysogenum and Fusarium solani, wherein the mutants have preferably at least 90 %, more preferably at least the same inhibitory activity on fungal spore outgrowth, in particular outgrowth of spores of Penicillium chrysogenum and Fusarium solani, like the parent strain from which they are derived, in particular if tested in a liquid well antagonism assay as disclosed in working example 2.

[0031] The cells of the Bacillus strains of the invention may be present, in particular in the preparations, articles, materials, compositions and products of the invention, as spores (which are dormant), as vegetative cells (which are growing), as transition state cells (which are transitioning from growth to sporulation phase) or as a combination of at least two, in particular all of these types of cells.

[0032] Further, the microorganisms of the invention may also be used in non-living, inactivated form, or as a combination of living and inactivated cells, as also the non-living cells are expected to still have a malodor control effect.

[0033] Accordingly, preparations of the microorganisms of the invention, which are a preferred subject of the invention, may be preparations containing intact cells, inactivated cells, cell debris or mixtures thereof. Further, the preparations may also be cell-free preparations, wherein the cell-free preparations may contain cell debris or may be free of cell debris. Particularly preferred examples for preparations of the microorganisms of the invention are the fermentation broth, as obtained after finishing the fermentation of the cells, the supernatant of the fermentation broth, which is obtained by separating all or the major part of the cells from the fermentation broth, as well as cell lysates and cell extracts, i.e. cytosol preparations, which can be obtained by breaking the microbial cells. Such preparations may also be used in concentrated or dried form, wherein the dried form has preferably a total dry matter content of at least 90 wt.-%, more preferably of at least 95 wt.-%.

[0034] The term “fermentation broth” according to the invention refers to the product of a cultivation of bacteria in a suitable fermentation medium. Methods for producing such fermentation broths are well known to those skilled in the art. The fermentation broths of the present invention can for example be obtained by culturing the strains by using the media, conditions and methods as described in US 6,060,051 , EP 0 287 699 A2, US 2014 / 0010792 A1 or in the FAO Report 179 (2016): “Probiotics in Animal Nutrition”. Conventional large- scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. The fermentation broths preferably contain cells in an amount of 1x103to 1x1011CFU, more preferably in an amount of 1x105to 1x1010CFU, above all in an amount of 1x107to 1x109CFU per ml of fermentation broth. 202400023 Foreign Filing 6

[0035] The term “fermentation broth” according to the invention refers to the direct product of the cultivation, i.e. a suspension preferably containing cells in the amount as mentioned in the previous paragraph, as well as to concentrated and dried forms of such a fermentation broth, wherein the dried form (“dried fermentation broth”) preferably has a total dry matter content of at least 95 wt.-%.

[0036] Drying of the preparations of the invention can be carried out by applying methods as generally known to those skilled in the art, in particular by evaporation of water, freeze-drying, lyophilization, spray drying, spray granulation, fluidized bed drying, vacuum drying and combinations thereof. After drying, the dried preparations as obtained can be further worked up, in particular by grinding and / or granulation.

[0037] Cell-free preparations of the microorganisms of the invention can be obtained by centrifugation, filtration and / or decantation of the fermentation broth and / or by centrifugation, filtration and / or decantation of the suspension as obtained after breaking the microbial cells. Depending on the technique used, these cell- free preparations may not be completely devoid of cells, but may still comprise a smaller amount of cells, in particular up to 10 wt.-%, preferably up to 5 wt.-%, more preferably up to 1 wt.-% of cells based on the total dry weight of the preparation. As the cells produce and secret compounds like metabolites, enzymes and / or peptides into the surrounding medium, the supernatants, extracts and lysates of the cells comprise a mixture of such compounds, in particular metabolites, enzymes and / or peptides, as secreted and / or produced by the cells. But in a particular embodiment of the invention, enzymes and / or peptides as secreted by the cells are removed in a subsequent step, for example by chromatography, so that a worked-up supernatant is obtained which comprises mainly or only the metabolites and is preferably essentially free of other components.

[0038] Cell lysates can be prepared either directly by breaking the cells of an optionally dried fermentation broth or by breaking the cells after first separating the cells from the fermentation broth mechanically, in particular by filtration, centrifugation and / or decantation.

[0039] Breaking of the cells can be carried out by applying techniques as known to those of skill in the art, for example by mechanical means or by applying high pressure. Depending on the degree of feree applied, a composition comprising only ruptured cells or a composition comprising a mixture of cell debris and intact cells is obtained. Homogenization of the cells may be realized for example by utilizing means selected from French cell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill, pebble mill, bead mill, high pressure grinding roll, vertical shaft impactor, industrial blender, high shear mixer, paddle mixer, and / or polytron homogenizer. Suitable alternatives are enzymatic and / or chemical treatment of the cells.

[0040] After breaking of the cells, the cell debris and remaining cells, if any, can optionally be separated from the cytosol preparation thus obtained to obtain a cell extract, i.e. a cytosol preparation, which is free of cells and cell debris.

[0041] The fermentation broths of the invention, from which the cells have been removed, i.e. the cell-free supernatant and metabolites containing fractions of the fermentation broth, are also denoted as “postbiotics”, „metabiotics” or “biogenics”. 202400023 Foreign Filing 7

[0042] A further specific preparation of the invention is a preparation containing inactivated cells, i.e. cells which are not able to grow anymore. Preparations containing inactivated cells are also known as “paraprobiotics”. Such preparations can be obtained for example by heat and / or pH inactivation of the cells, preferably by heat and / or pH inactivation of a fermentation broth containing the cells.

[0043] Inactivation of the cells can be carried out, e.g., by heat treatment and / or by adjusting an acid or an alkaline pH. Heat inactivation of the cells is preferably carried out by increasing the temperature to between 60°C and 80°C and incubation for at least 30 minutes. pH inactivation at acidic pH is preferably carried out by lowering the pH to at least 3 by addition of an acid like 5M H2SO4 and incubation for at least 30 minutes, preferably at least one hour. pH inactivation at alkaline pH is preferably carried out by increasing the pH to at least 10 and incubation for at least 30 minutes, preferably at least one hour.

[0044] Alternatively, inactivation of the cells may also be carried out by applying gamma- or UV-irradiation. In one specific embodiment of the invention a fermentation broth is used, in which at least 90 %, more preferably at least 95 or 99 %, in particular all bacterial cells are inactivated. Correspondingly, in this embodiment of the invention a fermentation broth is used which preferably contains no viable cells, at all. Such a product, which contains mainly or exclusively inactivated cells, is preferably used in cosmetical and / or topical applications and is called “ferment” in the context of the present invention.

[0045] The microbial preparations of the invention may contain metabolites, enzymes and / or peptides as produced and / or secreted by the microorganisms of the invention. In a preferred embodiment the microbial preparations contain secondary metabolites, as produced by the microorganisms, based on the dry weight of the preparation preferably in an amount of at least 1 wt.-%, more preferably in an amount of at least 3 wt.-%, in particular in an amount of at least 5 wt.-%.

[0046] The preparations of the invention preferably comprise an effective mixture of at least three, more preferably of at least 5, 8, or 12, in particular of all metabolites of at least one microorganism of the invention. The metabolites possess preferably a molecular weight of between 400 and 4000 Dalton, more preferably of between 500 and 3500 Dalton, in particular of between 500 and 1500 Dalton. The above holds true in particular for secondary metabolites.

[0047] A preparation containing an effective mixture of metabolites as contained in the microorganisms of the invention and / or as contained in the microbial preparations as mentioned before, can be obtained for example according to the methods set forth in US Patent No. 6,060,051. The preparation can in particular be obtained by precipitating the metabolites as contained in the preparations mentioned before by using organic solvents like ethyl acetate and subsequent redissolving of the precipitated metabolites in an appropriate solvent. The metabolites may subsequently be purified by size exclusion filtration that groups metabolites into different fractions based on molecular weight cut-off.

[0048] The microorganisms and microbial preparations of the invention can also be provided in combination with a suitable carrier, wherein the carrier is preferably an inert formulation ingredient added to improve 202400023 Foreign Filing 8 recovery, efficacy, or physical properties and / or to aid in packaging or administration of the microorganisms or microbial preparations. Such carriers may be used individually or in combination and can be added either as part of the fermentation medium, in the course of the fermentation or after the fermentation of the microorganisms has been ended.

[0049] The carrier is preferably selected from anti-caking agents, antioxidants, bulking agents, binders, structurants, coatings and / or protectants. Examples of useful carriers include polysaccharides (in particular starches, maltodextrins, celluloses, methylcelluloses, gums like guar gum, xanthan gum and gum arabic, wheat middlings, corn cob meal, chitosan and / or inulins), protein sources (in particular skim milk powder, sweet-whey powder, gelatine and / or soy flour), protein hydrolysates (in particular gelatine, yeast extract and / or peptones like soy peptone), peptides, sugars (in particular lactose, trehalose, sucrose, dextrose and / or maltose), lipids (in particular lecithin, vegetable oils and / or mineral oils), salts (in particular sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and / or sodium citrate), silicates (in particular clays, zeolites, Fuller’s earth, clintpolite, montmorillonite, perlite, vermiculite, diatomaceous earth, talc, bentonites, kaolin clay, silica in particular precipitated silica, hydrophobic silica and / or hydrophilic silica, and / or silicate salts like aluminium, magnesium and / or calcium silicate), silica gel, silica dioxide, activated carbon, lignite, magnesium and calicum oxide.

[0050] It was surprisingly found that a composition comprising one of the components a) to d) as described above can preserve the coating composition or an additive composition therefore. It was further surprisingly found that this inventive composition can be used to counter-balance negative effects associated with the addition of a defoamer to a coating composition, thereby increasing the defoamers compatibility with the coating composition.

[0051] A further subject of the present invention is also coating composition, preferably an aqueous coating composition, comprising at least one Bacillus velezensis strain or a preparation thereof. The coating composition is preferably a paint, a varnish, or a pigment paste.

[0052] Preferably the coating composition contains a Bacillus velezensis strain or a preparation thereof selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism. 202400023 Foreign Filing 9

[0053] Here, the preparation mentioned in point d) is particularly preferred.

[0054] A further subject of the invention is therefore also the use of a Bacillus velezensis strain or of a preparation thereof as a preservative, in particular for a coating composition, preferably pigment paste, paint, varnish, wherein the Bacillus velezensis strain or preparation thereof is preferably selected from the strains and preparations as mentioned before.

[0055] In one embodiment of the invention, the coating composition comprises a Bacillus velezensis strain or preparation thereof, which possesses inhibitory activity against fungi, in particular against the fungi Fusarium solani and Penicillium chrysogenum, or fungal outgrowth.

[0056] In a further embodiment, the Bacillus velezensis strain or preparation thereof is contained in the coating composition in a concentration of from 0.01 to 30 wt.-%, preferably in a concentration of from 0.05 to 15 wt.-%, in particular in a concentration of from 0.1 to 10 wt.-%, above all in a concentration of from 0.2 to 5 wt.-%.

[0057] In a preferred embodiment the coating composition or an additive composition used for said coating composition is an aqueous composition. The aqueous composition preferably comprises water based on the total weight of composition by at least 1 wt.-%, more preferably at least 5 wt.-%, even more preferably at least 15 wt.-%, most preferably at least 40 wt.-%.

[0058] Additives for coating composition are generally known the skilled person . Preferred additives are selected from the group consisting additives to regulate the surface tension (surfactant), anti-crater agent, dispersant, effect agent, defoamer, texturing agent, scratch resistance agent, anti-blocking agent, lubricant, adhesion promoter, hydrophobic agent, hydrophilic agent, inhibitor, catalyst, corrosion inhibitor, light stabilizer, matting agent, wetting agent, structuring agent, biocide, leveling agent, and / or additives to improve water resistance, and / or chemical resistance.

[0059] The coating compositions can comprise an organic solvent. Preferably the organic cosolvent is present in relation to the total weight of the composition in less than 15 wt.-%, even more preferably less than 10 wt.- %, yet more preferably less than 5 wt.-%, most preferably free from an organic solvent. When an organic solvent is present, it is preferably used as a cosolvent with water. Thus, the organic solvent is preferably a water-miscible solvent. The water-miscible solvent is preferably selected from the group consisting of mono- or polyhydric alcohols, amides, ketones, keto alcohols, cyclic ethers, glycols, polyhydric alcohol lower alkyl ethers, polyalkylene glycols, glycerin, N-methyl-2-pyrrolidone, 2-pyrrolidone, 1 ,3-dimethyl-2- imidazolidinone, glycerin, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, 1 ,3-propanediol, 1 ,4-butanediol, 1 ,5- pentanediol, 1 ,6-hexanediol, 1 ,2,6-hexanetriol, trimethylolpropane, pentaerythritol, Polyhydric alcohol alkyl ethers such as ethylene glycol monobutyl ether and tetraethylene glycol monomethyl ether, polyhydric alcohol aryl ethers such as ethylene glycol monophenyl ether and ethylene glycol monobenzyl ether, and polyhydric alcohol aralkyl ethers, 2-pyrrolidone, Lactams such as N-methyl-2-pyrrolidone and e- 202400023 Foreign Filing 10 caprolactam, 1 ,3-dimethylimidazolidinone acetone, N-methyl-2-pyrrolidone, m-butyrolactone, polyoxyalkylene adduct of glycerin, propylene glycol monomethyl ether acetate, dimethyl sulfoxide, diacetone alcohol, dimethylformamide, propylene glycol monomethyl ether.

[0060] In a preferred embodiment the coating compositions according to the invention comprises at least one component selected from of binders, pigments, fillers, dispersant, defoamers, surfactants, complexing agent, and / or pH regulator, preferably at least a binder, more preferably at least a binder and a pigment.

[0061] Binders used in the inventive coating compositions are preferably water-soluble or -dilutable. Waterdilutable binders are preferably aqueous polymer dispersions. Preferred polymer dispersions are selected from the group consisting of a polyolefin dispersion, acrylic latex, vinyl acrylic latex, epoxy resin dispersion, polyurethane dispersion, alkyd resin dispersion, vinyl acetate dispersion, and ethylene vinyl acetate dispersion, polyester dispersion.

[0062] Pigments can be inorganic or organic. Preferred inorganic pigments are selected from the group consisting of carbon blacks, titanium dioxides, zinc oxides, Prussian blue, iron oxides, cadmium sulfides, chromium pigments, for example chromates, molybdates and mixed chromates and sulfates of lead, zinc, barium, calcium and mixtures thereof. Preferred organic pigments are selected from the group of azo, diazo, condensed azo, naphthol, metal complex, thioindigo, indanthrone, isoindanthrone, anthanthrone, anthraquinone, isodibenzanthrone, triphendioxazine, quinacridone, perylene, diketopyrrolopyrrole and phthalocyanine pigments.

[0063] Preferred fillers are selected from the group consisting of talc, kaolin, silicas, barytes and lime.

[0064] Preferred surfactants can be divided into polysiloxanes and silicone-free surfactants.

[0065] Preferred polysiloxanes comprise polyethers, in particular polyoxyalkylenes. A preferred polysiloxane is disclosed in EP 2 094 761 A1 and adheres to general formula (III):

[0066] C-B-(AB)a-C1

[0067] (formula (III)) in which

[0068] A is a polyoxyalkylene block of the general formula -(CnH2nO)b-,

[0069] B is a polysiloxane block of the general formula -(SiR82-O)c-,

[0070] C and C1are identical or different alkoxypolyoxyalkylene radicals of the general formula Z- O-[CH2-CH(R9)O]d-,

[0071] R8are identical or different C1-4 alkyl radicals or phenyl radicals, with the proviso that at least 90% of the radicals R8are methyl radicals,

[0072] R9are identical or different hydrogen, C1-12 alkyl radicals or phenyl radicals, 202400023 Foreign Filing 11

[0073] Z is an alkyl, alkylene, aryl or alkylaryl radical, a is 1 to 20, b is an average value from 10 to 130, c is 3 to 100, d is independently at each occurrence in C / C1values from 2 to 20, with the proviso that the average value is situated in the range 2 to < 15, n is 2 to 12, with an average numerical value of 2.7 to 4.0.

[0074] Among silicone-free surfactants biosurfactants are particularly preferred. Rhamnolipids, sophorolipids, and mixtures thereof are preferred biosurfactants, as it was found that they can be utilized to reliably stabilize pigments in a dispersion, thereby suppressing unwanted particle reagglomeration.

[0075] Preferred rhamnolipids are disclosed in EP 4 015 584 A1 and EP 4 015 589 A1 , which are compounds general formula (I) or salts thereof:

[0076] (formula (I)) where m = 2, 1 or 0, especially 1 or 0, n = 1 or 0, especially 1 ,

[0077] R6and R7= independently an identical or different organic radical having 2 to 24, preferably 5 to 13, carbon atoms, especially optionally branched, optionally substituted, especially hydroxysubstituted, optionally unsaturated, especially optionally mono-, di- or triunsaturated, alkyl radical, preferably those selected from the group consisting of pentenyl, heptenyl, nonenyl, undecenyl and tridecenyl and (CH2)x-CH3 with x = 1 to 23, preferably 4 to 12.

[0078] Preference is given to a mixed composition of rhamnolipids containing > 90% diRL.

[0079] Preference is given to a mixed composition of rhamnolipids containing

[0080] 51 % by weight to 95% by weight of diRL-C10C10 and 202400023 Foreign Filing 12

[0081] 0.5% by weight to 9% by weight of monoRL-C10C10, where the percentages by weight are based on the sum total of all rhamnolipids present, with the proviso that the weight ratio of di-rhamnolipids to monorhamnolipids is greater than 91 :9, preferably greater than 97:3, more preferably greater than 98:2.

[0082] For the use according to the invention, preference is given to using a rhamnolipid mixture containing 0.5% by weight to 15% by weight of diRL-C10C12:1 , where the percentages by weight are based on the sum total of all rhamnolipids present.

[0083] The sophorolipids, also disclosed in EP 4 015 584 A1 and EP 4 015 589 A1 , are preferably compounds of the formula (Ila) or (lib):

[0084] (formula (lib)) where

[0085] R1and R2are independently either H or an acetyl group,

[0086] R3is H, a methyl, ethyl or hexyl group,

[0087] R4is independently a saturated or unsaturated divalent branched or unbranched organic group,

[0088] R5is H or a methyl group, 202400023 Foreign Filing 13 with the proviso that the total number of the carbon atoms in the groups R4and R5do not exceed the number 29.

[0089] Rhamnolipids are available under the Natsurfact name from Stepan (Northfield, IL, USA) and RHEANCE® One from Evonik Operations GmbH.

[0090] Sophorolipids are available under the HoneySurf name from Holiferm Limited (Manchester, UK) and REWOFERM® SL ONE from Evonik Operations GmbH.

[0091] Preference is given to using rhamnolipids and / or sophorolipids within a range from 0.01 % by weight to 10.0% by weight, more preferably 0.1% by weight to 5.0% by weight, based on the overall coating composition.

[0092] Preferred complexing agents is selected from the group of phosphonic acids, phosphonates, polyphosphates, hydroxycarboxylic acids, dicarboxylic acids, amino acids, porphyrins and mixtures thereof, preferably phosphonic acids, phosphonates and mixtures thereof. Particularly preferred complexing agents are selected from the group of hydroxyethane-(1 ,1-diphosphonic acid) (HEDP), pentasodium aminotrimethylenephosphonic acid (Na5ATMP)), tetrasodium hydroxyethane diphosphonic acid (Na4HEDP) and mixtures thereof.

[0093] The pH regulator may be a base, preferably a base as ammonium hydroxide, sodium hydroxide, or potassium hydroxide. Other neutralizing agents can include lithium hydroxide, for example. In another alternative, the neutralizing agent may, for example, be a carbonate. In another alternative, the pH regulator may, for example, be any amine such as monoethanolamine, or 2-amino-2-methyl-1 -propanol (AMP). Amines useful in embodiments disclosed herein may include diethanolamine, triethanolamine, and TRIS AMINO™ (each available from Angus), NEUTROL™ TE (available from BASF), as well as triisopropanolamine, diisopropanolamine, and N,N-dimethylethanolamine (each available from The Dow Chemical Company, Midland, Ml). Other useful amines may include ammonia, monomethylamine, dimethylamine, trimethylamine, monoethylamine, diethylamine, triethylamine, mono-n-propylamine, butylamine, dibutylamine, tributylamine, dimethyl benzyl amine, dimethyl n-propylamine, N-methanol amine, N-aminoethylethanolamine, N-methyldiethanolamine, monoisopropanolamine, N,N-dimethyl propanolamine, 2-amino-2-methyl-1 -propanol, 1 ,2-diaminopropane, tris(hydroxymethyl)-aminomethane, ethylenediamine N,N,N’N’-tetrakis(2-hydroxylpropyl) ethylenediamine, N,N,N’,N’ tetramethylpropanediamine, 3-methoxypropyl amine, imino bis-propyl amine and the like. In some embodiments, mixtures of amines or mixtures of amines and other surfactants may be used. In one embodiment, the pH regulator may be a polymeric amine, e.g. diethylene triamine. Those having ordinary skill in the art will appreciate that the selection of an appropriate neutralizing agent depends on the specific composition formulated, and that such a choice is within the knowledge of those of ordinary skill in the art.

[0094] Although the inventive coating composition has anti-fungal properties by itself, the conservatory effect can be supplemented using biocides. Biocides may be selected from one or more members of the group consisting of chlorinated hydrocarbons, organometallics, halogen-releasing compounds, metallic salts, 202400023 Foreign Filing 14 quaternary ammonium compounds, phenolics and organic sulfur compounds. Exemplary of organic sulfur compounds are compounds based on an isothiazolinone (also known as isothiazolothione) structure. Preferred isothiazolinones or isothiazolothiones are represented by the general formula (IV) or salts, thereof, in particular sodium salts:

[0095] (formula (IV)) wherein

[0096] X is oxygen or sulfur;

[0097] R10is hydrogen, a substituted or unsubstituted hydrocarbyl group, a substituted or unsubstituted hydrocarbylthio group, a substituted or unsubstituted hydrocarbyloxy group or a carbamoyl group;

[0098] R11and R12are independently hydrogen, a halogen atom, a cyano group, a substituted or unsubstituted hydrocarbyl group or a direct bond to the other of R11or R12.

[0099] When R10, R11and R12are, or contain, substituted hydrocarbyl groups, the substituents are preferably independently halogen, alkoxy or alkylthio where the alkyl groups contain 1 to 4 carbon atoms. If R10is a carbamoyl group, preferably it is of the general type -CON(H)(R13) where R13is a hydrogen atom or a hydrocarbyl group, which may be substituted with halogen, alkoxy or alkylthio substituents. It is generally preferred that R10is a hydrogen atom or a lower alkyl group of 1 to 4 carbon atoms. Most preferably, R10is hydrogen or a methyl group.

[0100] Preferred biocidal isothiazolinone compounds are selected from the group consisting of 5-chloro-2-methyl- 4-isothiazolin-3-one, 2-methyl-2H-isothiazolin-3-one, 4,5-dichloro-2-methylisothiazolin-3-, 2-n- octylisothiazolin-3-one, 1 ,2-benzisothiazolin-3-one, 4,5-trimethylene-4-isothiazolin-3-one, and 2-methyl- 4,5-trimethylene-4-isothiazolin-3-one.

[0101] Preferably the biocide is present in an amount relative to the total mass of the coating composition of 0.5 to 200 ppm, more typically 0.5 to 100 ppm or 1 to 100 ppm, or 0.5 to 25 ppm, total composition. It is further preferred for the biocide to be present in an amount of less than 15 ppm. The term “ppm” refers to parts per million on a weight by total weight of the composition basis.

[0102] A further subject of the invention is, due to the identified antifungal activities, also a B. velezensis strain or a preparation thereof or a composition containing a B. velezensis strain or preparation thereof, in particular selected from the microorganisms and microbial preparations as mentioned before, for use in a method of treating, preventing or mitigating the course of a disease associated with fungal infection, in particular associated with fungal infection by Penicillium chrysogenum, wherein the disease is preferably selected 202400023 Foreign Filing 15 from allergies and lung infections, or associated with fungal infection by Fusarium solani, wherein the disease is preferably selected from mycoses, fusariosis, corneal infections, fungal keratitis, disseminated disease, osteomyelitis, skin infection, fungemia and endophthalmitis.

[0103] A further subject of the invention is therefore also the use of a B. velezensis strain or of a preparation thereof or of a composition containing a B. velezensis strain or a preparation thereof, in particular selected from the microorganisms and microbial preparations as mentioned before, for preparing a pharmaceutical composition for treating, preventing or mitigating the course of a disease associated with fungal infection, in particular associated with fungal infection by Penicillium chrysogenum, wherein the disease is preferably selected from allergies and lung infections and / or for treating, or associated with fungal infection by Fusarium solani, wherein the disease is preferably selected from mycoses, fusariosis, corneal infections, fungal keratitis, disseminated disease, osteomyelitis, skin infection, fungemia and endophthalmitis.

[0104] A further subject of the invention is therefore also a pharmaceutical composition, in particular for treating allergies, lung infections, mycoses, fusariosis, corneal infections, fungal keratitis, disseminated disease, osteomyelitis, skin infection, fungemia or endophthalmitis, containing a pharmaceutically acceptable carrier and at least one microorganism or microbial preparation selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.

[0105] A further subject of the invention is therefore also a cosmetic composition comprising at least one microorganism or microbial preparation selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism. 202400023 Foreign Filing 16

[0106] The cosmetic composition is preferably an emulsion, in particular a W / O or an O / W emulsion. This means that the composition contains preferably at least one surface-active substance as emulsifier or as dispersion agent. Emulsifiers act at the interphase to produce water or oil-stable adsorption layers that protect the dispersed droplets against coalescence and thereby stabilize the emulsion. Thus, emulsifiers, like surfactants, are composed of hydrophobic and hydrophilic molecular moieties. Hydrophilic emulsifiers preferably form O / W emulsions and hydrophobic emulsifiers preferably form W / O emulsions. An emulsion is understood to mean a dispersion of a liquid in the form of droplets in another liquid using an energy input to afford interphases stabilized with surfactants. The choice of this emulsifying surfactant or emulsifier depends on the materials being dispersed and the respective external phase as well as the fineness of the emulsion.

[0107] The cosmetic or pharmaceutical composition according to the invention can be in any form suitable for application, e.g., a soap, a lotion, a serum, a spray, a jelly, a cream, a gel, a paste, a pomade, a balm, an ointment, a foam, a mousse, an emulsion, a stick, a patch, a powder, a cleaning fluid or cleaning milk, a deodorant, an anti-perspirant, a salve, a hair conditioner or a shampoo. The composition can also be applied in a mask or in a band-aid, particularly in a gel reservoir mask or band-aid or matrix mask or bandaid.

[0108] The cosmetic or pharmaceutical composition according to the invention preferably has a pH in the range of between 3 to 8, in particular in the range of 4.0 to 8.0, more preferably in the range of 4.5 to 7.4, particularly preferably in the range of 5.0 to 7.2. The pH of the composition is preferably determined at 22°C after stirring for five minutes using a pH electrode calibrated in accordance with ISO 4319 (1977).

[0109] A further subject of the invention is in particular also the use of the microorganisms or microbial preparations of the invention as preservative for any kind of compositions, in particular for cosmetic or pharmaceutical compositions, preferably for cosmetic or pharmaceutical compositions to be applied topically.

[0110] EXAMPLES

[0111] The following examples illustrate the present invention but are not intended to limit the scope of the invention.

[0112] Example 1 Anti-fungal activity of vegetative DSM 34979 and DSM 34982 cells

[0113] The active cells of DSM34979 and DSM34982 show inhibitory effects on the outgrowth and growth of Penicillium chrysogenum and Fusarium solani spores. DSM34979 and DSM34982 are naturally occurring organisms of the species Bacillus velezensis which were isolated from an environmental soil sample or an open product sample, respectively. In a screening 11 pre-selected natural isolates of the species Bacillus velezensis were used to inhibit the growth of the two phytopathogens on solid potato-dextrose agar-plates (PDA, 4 g / L potato extract, 20 g / L dextrose, 15 g / L agar-agar, add 1000 ml; Sigma Aldrich Sigma P6685- 250G) in a well diffusion antagonism test but DSM34979 and DSM34982 were figured out as best performing strains. 202400023 Foreign Filing 17

[0114] The natural isolates of Penicillium chrysogenum and Fusarium solani were isolated from different defoamer product samples and were grown on PDA for 14 days at room temperature (RT). For the collection of spores from a matured mycelium, a 0.85% (w / v) NaCI solution with 0.5% polysorbate 80 (TWEEN ® 80, P4780 Sigma Aldrich) were prepared and the sterile solution were used to flooding (5-6 ml) overgrown agar-plates. After the plates had been flooded, the surfaces of the mycelia were scraped, and the liquid spore solutions were collected. This was done carefully to avoid contamination and to ensure that a high concentration of spores was maintained within the collected solution. After the spore collection the spore solutions were heat-inactivated for 10 min at 80°C. The concentration of spores in solution was determined and adjusted to 1 .0E+07 CFU / ml. 135p I were spread with a sterile spatula on the surface of a PDA plate. 9 mm diameter wells were cut into the dried plates and these holes were filled with 10OpI test substance. The test substance was either a non-inoculated media control without culture, a benchmark reference substance or the prepared biomass solution of candidate strains.

[0115] Bacillus isolates (DSM34979 and DSM34982) were pre-cultivated in 100 ml shaking flasks with 10 ml of a medium supplemented with trace elements (trace element solution of DSMZ medium 1032) described in Scholz et al. (201 1), which will be referred to as LB-Kelly medium from now on (R. Scholz et al. (2011), J Bacteriol 193(1):215-224, "Plantazolicin, a novel microcin B17 / streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42").

[0116] 1 Opl from a cryo culture was used as inoculum. The cultures were cultivated for 16 h at 37°C and 200 rpm agitation. Both cultures were adjusted to an optical density (OD) 20 at 600 nm (appr. 5*109CFU / ml) in LB- Kelly. Plates with overlayed fungi spores and test-wells (holes) that were filled with Bacillus biomass, LB- Kelly medium control or the common preservative blend of 0.093% MV / B20 (MV: 5-chloro-2-methyl-4- isothiazolin-3-one and 2-methyl-2H-isothiazol-3-one (3:1); B20: 1 ,2-benzisothiazol-3(2H)-one and sodium-hydroxide) diluted in water were incubated for at least 4 days. After 4 days the zones of clearance in mm were determined measuring from the edge of the cut test-well to the border of the cleared lawn. Each well was measured twice (horizontally, vertically), then averaged (Figure 1 and 2). The results are shown in Figures 1 and 2.

[0117] Example 2 Anti-fungal activity of the fermentation broth of DSM 34979 and DSM 34982

[0118] In a next step, the stabilizing effect and therefore antifungal activity of the ferments of different concentrations from the strains DSM34982 and DSM34979 were shown in a liquid well antagonism assays against Penicillium chrysogenum and Fusarium solani isolates. The ferment is a composition of the cell- free and sterile supernatant (polyethersulfone membrane filter type 15407, 0.2 pm pore size, Satorius) of a grown LB-Kelly culture with either DSM34982 or DSM34979. Strains were cultivated in 100 ml shaking flasks with 10 ml LB-Kelly medium (for composition see Example 1) after inoculation of 10 pl cryo-culture. The inoculated culture was incubated at 37°C at 200 rpm for 96 h. The supernatant was harvested by centrifugation and was filtered sterile afterwards. 25%, 12.5%, 10% and up to 1 % (v / v) supernatant were used as ferment with the property to stabilize a fungi spore spiked approach.

[0119] For the liquid well antagonism assay a Woulter-Duetz oxidish plate (PreSens Precision Sensing GmbH; 200001453) was used for monitoring the O2 saturation in the system [pO2 in %]. Therefore, spore suspensions, prepared in Example 1 , were inoculated to a final concentration of 1.0E+05 CFU / ml. Each cultivation-well contains a final blend of 2.5 ml potato-dextrose medium (PD, 4 g / L potato extract, 20 g / L 202400023 Foreign Filing 18 dextrose, pH 8) alone, or with the different spore solutions or with the different spore solutions in combination with the different ferments. As control, only medium without fungi spores and the benchmark sample (0.093% MV / B20 (MV: 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-2H-isothiazol-3-one (3:1); B20: 1 ,2-benzisothiazol-3(2H)-one and sodium-hydroxide) in combination with fungi spores were used. The assays were performed under RT for 7 days and if fungi spores could outgrow an O2 saturation could be measured by the O2 sensor (control). The difference of pO2 from the beginning over time shows fungi growth. If no pO2 saturation is monitored then the ferment is stabilizing the sample. These ferments can be used as stabilizer.

[0120] The results shown in Figure 3 demonstrate that both bacterial fermentation broths (sterile filtered), here used as 12.5% (v / v), could effectively inhibit the outgrowth of Penicillium chrysogenum spores and were as effective as the fresh prepared benchmark (biocide mentioned above). The experiments were performed in three biological replicates and a data set of only one is used for demonstration. Moreover, various ferment concentrations up to 1 % of the final ferment could be tested to effectively delay the outgrowth of fungal spores in this test system.

[0121] Likewise, Figure 4 demonstrate that both bacterial fermentation broths, here used as 12.5% (v / v), could effectively inhibit the outgrowth of Fusarium solani spores and were as more effective than the fresh prepared benchmark (biocide mentioned above). The experiments were performed in three biological replicates and a data set of only one is used for demonstration. As well, various ferment concentrations up to 1 % of the final ferment could be tested to effectively delay the outgrowth of fungal spores in this test system.

[0122] Example 3 Anti-fungal activity of the fermentation broth in a defoamer formulation

[0123] The composition of the fermentation broth (for preparation see Example 2), mentioned above, can be used in a defoamer emulsion to replace common biocides from the formulation without losing the preserving properties.

[0124] A challenge test for the preservation of liquid test substances was performed according to the test protocol from Thor D730 which was adjusted as described in the following part:

[0125] A predetermined spore suspension of Penicillium chrysogenum was diluted in phosphate-buffered saline solution (Merck, PBS) to a spore concentration of 106spores / ml. This suspension was then added to the emulsion in a concentration of 1 wt.-% and homogenized properly by mechanical shaking with Vortex shaker.

[0126] 100 pL of the sample were diluted into 900pL PBS and further processed in decadic dilution until one reaches a suitable dilution. For these trials the twice decadic diluted sample had provided the most significant results. The sampling was performed in triplicate. The diluted sample was then plated onto malt broth agar plates (20g / L malt broth, Sigma Aldrich; 6,25g / L Agar-Agar, Merck) and incubated for 48h at 21 °C. 202400023 Foreign Filing 19

[0127] The cell concentration was determined by colony counting of the grown colonies. The cells per milliliter . have i been ca ilcu ilat .ed i accord ..ing t .o f ro Hllowing equat ..ion - cells = - 7 - counted colonies*dilution - f -actor * — susp - -ension volume ml N samples^plated volume sample volume .

[0128] Only samples in the range of 10 to 200 colonies have been taken into calculation due to reliability. The samples were taken directly after the inoculation and homogenization of the spore suspension and 5 days, 7 days and 14 days after the inoculation. To have good oxygen saturation and homogeneous distribution the samples were shaken every 24h with a Vortex shaker.

[0129] Looking at the overtime development of the cell count, all samples start at a comparable level of approx. 3x104cells / ml. Within the 14 days of testing the samples without preservative and with regular chemical preservation showed increasing amounts of cells / ml with 8.20x104and 8.60x104. In comparison the used fermentation broth showed a concentration dependent inhibition of growth of P. chrysogenum. Used with a volumetric percentage of 10% one could see a slight increase in cell growth after 14 days up to 4.50x104(SN3) and 3.20x104(SN7) cells / mL which was far smaller than in the non-preserved or commonly preserved sample. Looking at the samples with 25 vol.% added supernatant the amount of outgrowing spores remained at the initial level of 2.90x104(SN3) and 2.80x104(SN7) which could be seen as total inhibition of the growth of spores within these samples.

[0130] As shown in the results portrayed in Figure 5, CIT / MIT (5-chloro-2-methyl-4-isothiazolin-3-one / 2-methyl- 4-isothiazolin-3-one; labeled “regular”) shows no preservation of the emulsion against Penicilium chrysogenum, whereas the inventive cell free supernatants (SN3 and SN7 at 10 and 25 vol.-%, respectively) show good inhibition of growth over 14 days.

[0131] Example 4 Positive effect of the fermentation broth on defoamer performance

[0132] Another important point was that the performance of the defoamer emulsion which was stabilized by the fermentation broth and the main properties of the coating are likely the product with the current preservation. The main characteristics which had been analyzed were the defoaming behavior, compatibility and color change.

[0133] For testing of the defoaming behavior the different defoamer samples had been tested at a concentration of 0.5 wt.-% of the polyether-modified polysiloxane-based defoamer TEGO® Foamex 1488 in an architectural coating with a low pigment volume concentration (PVC) which has a high foaming tendency (cf. table 1)

[0134] Table 1 Formulation of a low PVC architectural coating (PVC30) 202400023 Foreign Filing 20

[0135] During the manufacturing of the coating the defoamer was firstly skipped and then added afterwards under stirring into the coating with a dissolver with a blade stirrer (0 40mm) at 2000 rpm for 3 min. The samples were than left over night and further treated after 24h. 50g of each sample were stirred at 4200 rpm with a blade stirrer (0 40mm) for 3 min. 30g of the stirred sample were poured into a 100 mL graduated cylinder and the resulting volume was measured to evaluate the foam height. The remaining sample was poured down on a clear polyvinylchloride foil to observe the defoaming over time and in the cured coating visually. Both results were then put together and show the defoaming behavior.

[0136] To evaluate the compatibility of the defoamer emulsion in the coating mostly the cratering, but also other surface defects, like e.g. orange peel, had been observed with a draw down on glass panels. The samples were created identically to the defoaming samples, but instead of stirring, the samples had been drawn down on glass panels with a 100pm doctor blade. After curing the surface was evaluated regarding surface defects. The resulting coatings are shown in Figure 6.

[0137] In this case the addition of the cell free supernatant instead of the common biocide mixture led to very little decrease of defoaming performance which could just be seen in the pour down on the polyvinylchloride foil. Addition of the defoamer decreased the foam height, which was not affected by the addition of the inventive fermentation broth, as shown in table 2.

[0138] Table 2 Defoaming properties of inventive stabilized emulsion against commonly preserved emulsion; both are visual evaluations (1=very bad, 5=foam free)

[0139] Regarding the compatibility a huge increase was observed with both samples containing the cell free supernatant, as shown in table 3. Addition of the defoamer severely decreases the compatibility of the coating. The addition of the inventive fermentation broth however significantly increased the compatibility - as can be seen from the severe reduction of bubble defects (cf. Figs. 6B with Figs. 6C and 6D). 202400023 Foreign Filing 21

[0140] Table 3 Defoaming properties of inventive stabilized emulsion against commonly preserved emulsion; foil drain is a visual evaluation of the defoaming (1=very bad, 5=foam free)

[0141] Example 5: Improved wetting behavior of the fermentation broth

[0142] First experiments showed that the fermentation broth has a positive impact on the wetting behavior. First the wetting of a silicone coated polyethylene foil has been analyzed. Therefore 10 pL droplets of the fermentation broth and water have been applied to the surface and let spread for 1 minute. The diameter of the droplet had been measured by analysis of microscopic images. The images were taken with 2.5x magnitude, top view with scattered light. The measurements have been conducted with Keyence Profilometer VR 4000.

[0143] The fermentation broth showed wider spread droplets which indicates better wetting behavior compared to water (table 4). Consequently, the inventive fermentation broths can act as wetting agents.

[0144] Table 4 Diameter of droplet on silicone coated foil after 1 minute

[0145] Example 6: Improved wetting of organic pigments

[0146] Pigment wetting was evaluated with organic pigments due to their higher difficulty in wetting of aqueous media when compared to inorganic particles. Heliogenblue L 7085 (BASF), Heliogengreen and Hostaperm Violet RL (Heubach Group) were chosen to test wetting behavior. For these tests, demineralized water serves as a negative example. A 0.5 wt.-% solution of the siloxane-based dispersing agent TEGO® Dispers 750 W (Evonik) serves as a comparative example of a state of the art wetting agent. The inventive example utilizes a concentrated fermentation broth cFB, which was obtained as described in the following:

[0147] In the initial step, fermentation broth produced from DSM34979 (as described in Example 2) was centrifuged in six 450 mL centrifuge bottles using a Sigma 6-16KS centrifuge at 10,000 x g and 25 °C for 10 minutes. As the supernatant remained turbid, a second centrifugation was performed under more stringent conditions (14,000 x g, 30 minutes, 25 °C). The resulting supernatant was subsequently sterile- filtered using a Sartorius Sartopore 2 filter under a pressure of <2 bar. The clear filtrate was transferred into a 2 L beaker.

[0148] An acid precipitation was then carried out by adjusting the pH from 6.9 to 2.0 using sulfuric acid at a concentration of 4.0 mol / L. The resulting mixture was stored overnight at 4 °C. On the following day, the 202400023 Foreign Filing 22 suspension was centrifuged again (10,000 x g, 10 minutes, 25 °C). After removal of the supernatant, twice the mass of the obtained precipitate was resuspended in water adjusted to pH 2.0. The suspension was stirred for 10 minutes at 600 rpm using a magnetic stirrer and subsequently centrifuged again under the same conditions. This washing step was repeated once.

[0149] The final precipitate was transferred to a separate beaker, and half the mass of the original fermentation broth was added. The pH of the mixture was then adjusted to 7.0 by the addition of 1 M sodium hydroxide.

[0150] For the pigment wetting tests, 0.2 g of a pigment is placed onto the surface of one of the test solutions described above and wetting is observed visually every 0.5 h for 24h. In the pure water containing samples the pigments remain on the surface for 24h. The samples containing either the fermentation broth cFB or the siloxane based dispersing agent immediately start wetting all three types of pigments. The fermentation broth cFB containing samples reach full wetting of the pigments after 1 .5 h. The dispersing agent is slightly faster where full wetting is reached after 1 h. Results of the visual evaluation are summarized in Table 5. While the siloxane-based dispersing agent leads to a faster wetting than the inventive composition, for a non-siloxane based wetting agent this performance was equally outstanding as well as surprising.

[0151] Table 5 Visual evaluation of pigment wetting overtime (1= no pigment wetting; 5= complete pigment wetting)

[0152] Example 7: Improved wetting of organic pigments

[0153] Pigment concentrates were further evaluated regarding the inhibition of outgrowth of any environmental contamination which occurs from the raw materials themselves or due to the production process. Samples were produced using 35.8 wt.-% demineralized water, 23.1 wt.-% dispersing agent, 1.0 wt.-% TEGO® Foamex 1488 (from Evonik), and 40.1 wt.-% Heliogen Blue L 7085. In a comparative example, the dispersing agent was TEGO® Dispers 750 W was used. In the inventive examples, TEGO® Dispers 750 W was replaced by 10 to 30 wt.-% by the fermentation broth described in Example 6. The pigment concentrates were produced by dispersing the material in a skandex shaker with 1.7-2.1 mm glass beads (paste:glass beads ratio 1 :1) for 30 min to a fineness < 10 pm measured with a grindometer after post mixture of all raw materials in 250 ml glass bottles. Glass beads were separated from the pigment concentrate by filtration over a 240 pm nylon sieve.

[0154] The raw materials were checked by plating on malt broth agar plates. 100 pL for the liquid components and 100 pg of the solid components were evaluated prior to the pigment concentrates to check possible sources of contamination. The main sources were determined to be the demineralized water (mainly bacterial 202400023 Foreign Filing 23 contamination; Fig 7A) and the Heliogen Blue L 7085 pigment (mainly fungal contamination; Fig. 7B). The inhibition testing was conducted only with the pigment concentrate samples. For evaluation of the growth inhibtion, 100pL of each pigment concentrate was plated on malt broth agar plates and then incubated for 7 days at 21 °C. Each sample was tested in triplicate. The results are shown in Figure 7: Fig. 7C only uses the siloxane-based dispersing agent. In Figs. D to F the siloxane-based dispersing agent was replaced by an inventive fermentation broth by 10 to 30 wt.-%, which leads to an increasing inhibition of microbial growth.

Claims

202400023 Foreign Filing 24Claims1 . Bacillus velezensis strain or preparation thereof selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.

2. Coating composition or additive composition therefore, preferably pigment paste, paint, varnish, or additive composition therefore, comprising at least one Bacillus velezensis strain or a preparation thereof.

3. Coating composition according to claim 2, wherein the Bacillus velezensis strain or preparation thereof is selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.

4. Coating composition according to claim 2 or 3, wherein the coating composition contains a cell-free preparation of the Bacillus velezensis strain, wherein the cell-free preparation is preferably an optionally concentrated or dried supernatant of a fermentation broth of the Bacillus velezensis strain.

5. Coating composition according to any of claims 2 to 4, wherein the Bacillus velezensis strain or preparation thereof possesses inhibitory activity against fungi or outgrowth of fungal spores, in particular against the fungi Fusarium solani and / or Penicillium chrysogenum or outgrowth of their spores.202400023 Foreign Filing 256. Coating composition according to any of claims 2 to 5, wherein the Bacillus velezensis strain or preparation thereof is contained in the coating composition in a concentration of from 0.01 to 30 wt.-%, preferably in a concentration of from 0.05 to 15 wt.-%, in particular in a concentration of from 0.1 to 10 wt.-%, above all in a concentration of from 0.2 to 5 wt.-%.

7. Coating composition according to any of claims 2 to 6, wherein the coating composition comprises at least one component selected from of binders, pigments, fillers, dispersant, defoamers, surfactants, complexing agent, and / or pH regulator, preferably at least a binder, more preferably at least a binder and a pigment.

8. Coating composition according to claim 7, wherein the binder is a polymer dispersion, preferably selected from the group consisting of a polyolefin dispersion, acrylic latex, vinyl acrylic latex, epoxy resin dispersion, polyurethane dispersion, alkyd resin dispersion, vinyl acetate dispersion, and ethylene vinyl acetate dispersion, polyester dispersion.

9. Use of a strain of the species Bacillus velezensis or of a preparation thereof as preservative for coating compositions, in particular for suppressing mold formation and / or sporulation in a coating composition or additive composition therefore, preferably pigment paste, paint, varnish, or additive composition therefore.

10. Bacillus velezensis strain or preparation thereof or composition containing a Bacillus velezensis strain or preparation thereof, in particular according to claim 1 , for use in the treatment, prevention or mitigating of the course of a disease associated with fungal infection, in particular associated with fungal infection by Penicillium chrysogenum, preferably selected from allergies and lung infections, or associated with fungal infection by Fusarium solani, preferably selected from mycoses, fusariosis, corneal infections, fungal keratitis, disseminated disease, osteomyelitis, skin infection, fungemia and endophthalmitis.11 . Pharmaceutical composition containing a pharmaceutically acceptable carrier and a Bacillus velezensis strain or preparation thereof selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.202400023 Foreign Filing 2612. Pharmaceutical composition according to claim 8 for use in a method of treating , preventing or mitigating the course of a disease associated with Penicillium chyrosgenum, in particular selected from allergies and lung infections, and / or associated with Fusarium solani, in particular selected from, mycoses, fusariosis, corneal infections, fungal keratitis, disseminated disease, osteomyelitis, skin infection, fungemia and endophthalmitis.

13. Use of a Bacillus velezensis strain or preparation thereof according to claim 10 or of a composition according to claim 11 for treating plant diseases.

14. Cosmetic composition containing a Bacillus velezensis strain or preparation thereof selected from the following group: a) Bacillus velezensis DSM 34979; b) Bacillus velezensis DSM 34982; c) A mutant of the strains as mentioned under (a) or (b) with a sequence identity of at least 95, 97 or 98 %, preferably of at least 99, 99.5 or 99.8 %, more preferably of at least 99.9 or at least 99.95 %, above all of at least 99.98 or 99.99 % to the genomic sequence of any of the strains as mentioned under (a) or (b); d) A preparation of at least one microorganism as mentioned under (a) to (c), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell extract or cell lysate of such at least one microorganism.

15. Cosmetic composition according to claim 14 containing at least one ingredient selected from surfactants, phospholipids, sphingoid bases, free fatty acids, protein hydrolyzates, polymers, light stabilizers, anti-perspirants, deodorants, peptides, amino acids, vitamins, extracts from plants and algae, cosmetic oils, emollients, antioxidants, preservatives, thickeners, viscosity regulators, stabiliziers, hydrotropes, solids, fillers, film formers, conditioners, insect repellents, self-tanning agents, odor absorbers, solvents, perfumes and dyes.

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