Bacillus strains capable of degrading inorganic nitrogen compounds

Abstract

The present invention relates to novel Bacillus strains that are capable of effectively degrading inorganic nitrogen compounds and also inhibiting the growth of aquatic animal pathogens.

Description

[0001] The present invention relates to novel Bacillus strains that are capable of effectively degrading inorganic nitrogen compounds and also inhibiting the growth of aquatic animal pathogens.

[0002] The biological removal of inorganic nitrogen compounds, such as ammonium and nitrate, from aquatic systems is a subject of particular interest, since inorganic nitrogen compounds promote eutrophication and are toxic to many aquatic organisms. Therefore, it is undesirable for the levels of these substances to increase in aquatic systems, particularly in rearing waters. In the past, combinations of autotrophic nitrifying and denitrifying bacteria, which convert ammonium into nitrogen with nitrate as an intermediate, have been described for achieving remediation. At the same time, alternative methods have also been described, for example in US 2016 / 0326034, in which a mixture of Bacillus and lactic acid bacteria for reducing nitrate levels is described. In the methods disclosed in the prior art, a consortia of different microorganisms is usually used to remove unwanted nitrogen compounds, wherein the different microorganisms perform different functions and only the consortia as a whole can achieve satisfactory results.

[0003] According to the present invention, it has surprisingly been found that specific bacteria of the genus Bacillus are able to effectively degrade different inorganic nitrogen compounds, in particular selected from ammonium, nitrite and nitrate, even without the help of other microorganisms. "Degradation" in the present invention preferably refers to the conversion of these compounds into molecular nitrogen.

[0004] It was further surprisingly demonstrated that the identified bacteria are not only able to degrade inorganic nitrogen compounds, but they also possess other beneficial properties, in particular inhibitory activity against pathogens of aquatic animals, which makes them probiotics (also known as "direct fed microorganisms" or "DFMs").

[0005] Therefore, based on the characteristics of this strain, the subject of the present invention is in particular the use of the strain of the present invention for the degradation of inorganic nitrogen compounds, in particular in aquaculture systems, and their use as probiotics, in particular in the feed industry, and very preferably in aquaculture. The different subjects of the present invention will be explained in more detail below.

[0006] Bacillus strains DSM 33349, DSM 33350, DSM 33351 and DSM 33352 have been identified by targeted screening of naturally occurring isolates and are classified as Bacillus subtilis strains. They have been deposited on December 3, 2019 in the name of applicant Evonik Operations GmbH under the deposit number as previously mentioned at DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7B, 38124 Braunschweig, Germany) in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure.

[0007] Therefore, a first subject of the present invention is a Bacillus strain, in particular a Bacillus subtilis strain, or a preparation thereof, wherein the strain or the preparation is capable of degrading at least one inorganic nitrogen compound and is also capable of inhibiting the growth of at least one pathogen.

[0008] According to the invention, the inorganic nitrogen compounds degradable by the strains and preparations of the invention are preferably selected from ammonium, nitrite and nitrate.

[0009] The Bacillus strain according to the present invention, in particular the Bacillus subtilis strain, or a preparation thereof is preferably selected from the following group:

[0010] a) Bacillus strains deposited with DSMZ under one of the following numbers: DSM 33349, DSM 33350, DSM 33351, DSM 33352;

[0011] b) a mutant of a Bacillus strain listed in (a), which has at least 95%, preferably at least 98%, 99 or 99.5% sequence identity with the strain listed in (a);

[0012] c) a preparation of a strain according to (a) or (b);

[0013] d) A preparation comprising an effective mixture of compounds contained in a strain as listed in (a) or (b) or an effective mixture of compounds contained in a preparation as listed in (c).

[0014] The Bacillus strains of the present invention preferably have a 16S rDNA sequence that has at least 99%, preferably at least 99.5% or 99.8%, in particular 100% sequence identity to the sequence according to SEQ ID NO: 1 (in particular strain DSM 33349 or a mutant thereof), SEQ ID NO: 6 (in particular strain DSM 33350 or a mutant thereof), SEQ ID NO: 11 (in particular strain DSM 33351 or a mutant thereof) or SEQ ID NO: 16 (in particular strain DSM 33352 or a mutant thereof).

[0015] In a preferred embodiment of the present invention, the Bacillus strain, in particular strain DSM 33349 or a mutant thereof, exhibits at least one, in particular at least 2, 3 or 4, preferably all, of the following characteristics:

[0016] a) a 16S rDNA sequence which has at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 1 and / or with the 16S rDNA sequence of strain DSM 33349 deposited with DSMZ;

[0017] b) a yqfD sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 2 and / or with the yqfD sequence of strain DSM 33349 deposited with the DSMZ;

[0018] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 3 and / or with the gyrB sequence of strain DSM 33349 deposited with the DSMZ;

[0019] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 4 and / or with the rpoB sequence of strain DSM 33349 deposited with DSMZ;

[0020] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 5 and / or with the groEL sequence of strain DSM 33349 deposited with the DSMZ;

[0021] f) a recT sequence having at least 95%, preferably at least 98%, 99% or 99.5%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 21.

[0022] In another preferred embodiment of the present invention, the Bacillus strain, in particular strain DSM 33350 or a mutant thereof, exhibits at least one, in particular at least 2, 3 or 4, preferably all, of the following characteristics:

[0023] a) a 16S rDNA sequence which has at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 6 and / or with the 16S rDNA sequence of strain DSM 33350 deposited with DSMZ;

[0024] b) a yqfD sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 7 and / or with the yqfD sequence of strain DSM 33350 deposited with the DSMZ;

[0025] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 8 and / or with the gyrB sequence of strain DSM 33350 deposited with the DSMZ;

[0026] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 9 and / or with the rpoB sequence of strain DSM 33350 deposited with DSMZ;

[0027] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 10 and / or with the groEL sequence of strain DSM 33350 deposited with the DSMZ.

[0028] In another preferred embodiment of the present invention, the Bacillus strain, in particular strain DSM 33351 or a mutant thereof, exhibits at least one, in particular at least 2, 3 or 4, preferably all, of the following characteristics:

[0029] a) a 16S rDNA sequence which has at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 11 and / or with the 16S rDNA sequence of strain DSM 33351 deposited with DSMZ;

[0030] b) a yqfD sequence which has at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 12 and / or with the yqfD sequence of strain DSM 33351 deposited with DSMZ;

[0031] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 13 and / or with the gyrB sequence of strain DSM 33351 deposited with the DSMZ;

[0032] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, in particular at least 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 14 and / or with the rpoB sequence of strain DSM 33351 deposited with DSMZ;

[0033] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 15 and / or with the groEL sequence of strain DSM 33351 deposited with the DSMZ;

[0034] f) a sequence having at least 95%, preferably at least 98%, 99% or 99.5%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 22 (encoding the putative gamma-polyglutamate hydrolase PghZ);

[0035] g) a sequence having at least 95%, preferably at least 98%, 99% or 99.5%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 23 (encoding a hypothetical protein).

[0036] In another preferred embodiment of the present invention, the Bacillus strain, in particular strain DSM 33352 or a mutant thereof, exhibits at least one, in particular at least 2, 3 or 4, preferably all, of the following characteristics:

[0037] a) a 16S rDNA sequence which has at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 16 and / or with the 16S rDNA sequence of strain DSM 33352 deposited with DSMZ;

[0038] b) a yqfD sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 17 and / or with the yqfD sequence of strain DSM 33352 deposited with the DSMZ;

[0039] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 18 and / or with the gyrB sequence of strain DSM 33352 deposited with the DSMZ;

[0040] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 19 and / or with the rpoB sequence of strain DSM 33352 deposited with DSMZ;

[0041] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 20 and / or with the groEL sequence of strain DSM 33352 deposited with the DSMZ;

[0042] f) a sequence having at least 95%, preferably at least 98%, 99% or 99.5%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 24 (encoding a putative phage tail tape measure protein);

[0043] g) a sequence having at least 95%, preferably at least 98%, 99% or 99.5%, in particular 100% sequence identity with the polynucleotide sequence according to SEQ ID NO: 25 (encoding a hypothetical protein).

[0044] Therefore, a particular subject of the present invention is also a Bacillus strain, preferably a Bacillus subtilis strain, in particular the strain DSM 33349 or a mutant thereof, which exhibits the following characteristics:

[0045] a) the 16S rDNA sequence according to SEQ ID NO: 1 and / or the 16S rDNA sequence of strain DSM 33349 deposited at DSMZ;

[0046] Preferably, the Bacillus strain exhibits at least one, two or three, preferably all, of the following further characteristics:

[0047] b) the yqfD sequence according to SEQ ID NO: 2 and / or the yqfD sequence of strain DSM 33349 deposited at DSMZ;

[0048] c) the gyrB sequence according to SEQ ID NO: 3 and / or the gyrB sequence of strain DSM 33349 deposited at DSMZ;

[0049] d) the rpoB sequence according to SEQ ID NO: 4 and / or the rpoB sequence of strain DSM 33349 deposited at DSMZ;

[0050] e) the groEL sequence according to SEQ ID NO: 5 and / or the groEL sequence of strain DSM 33349 deposited at DSMZ;

[0051] f) recT sequence according to SEQ ID NO:21.

[0052] Therefore, a particular subject of the present invention is also a Bacillus strain, preferably a Bacillus subtilis strain, in particular the strain DSM 33350 or a mutant thereof, which exhibits the following characteristics:

[0053] a) the 16S rDNA sequence according to SEQ ID NO: 6 and / or the 16S rDNA sequence of strain DSM 33350 deposited with DSMZ;

[0054] Preferably, the Bacillus strain exhibits at least one, two or three, preferably all, of the following further characteristics:

[0055] b) the yqfD sequence according to SEQ ID NO: 7 and / or the yqfD sequence of strain DSM 33350 deposited at DSMZ;

[0056] c) the gyrB sequence according to SEQ ID NO: 8 and / or the gyrB sequence of strain DSM 33350 deposited at DSMZ;

[0057] d) the rpoB sequence according to SEQ ID NO: 9 and / or the rpoB sequence of strain DSM 33350 deposited at DSMZ;

[0058] e) the groEL sequence according to SEQ ID NO: 10 and / or the groEL sequence of strain DSM 33350 deposited at DSMZ.

[0059] Therefore, a particular subject of the present invention is also a Bacillus strain, preferably a Bacillus subtilis strain, in particular the strain DSM 33351 or a mutant thereof, which exhibits the following characteristics:

[0060] a) the 16S rDNA sequence according to SEQ ID NO: 11 and / or the 16S rDNA sequence of strain DSM 33351 deposited at DSMZ;

[0061] Preferably, the Bacillus strain exhibits at least one, two or three, preferably all, of the following further characteristics:

[0062] b) the yqfD sequence according to SEQ ID NO: 12 and / or the yqfD sequence of strain DSM 33351 deposited at DSMZ;

[0063] c) the gyrB sequence according to SEQ ID NO: 13 and / or the gyrB sequence of strain DSM 33351 deposited at DSMZ;

[0064] d) the rpoB sequence according to SEQ ID NO: 14 and / or the rpoB sequence of strain DSM 33351 deposited at DSMZ;

[0065] e) the groEL sequence according to SEQ ID NO: 15 and / or the groEL sequence of strain DSM 33351 deposited with DSMZ;

[0066] f) a sequence according to SEQ ID NO: 22;

[0067] e) The sequence according to SEQ ID NO:23.

[0068] Therefore, a particular subject of the present invention is also a Bacillus strain, preferably a Bacillus subtilis strain, in particular the strain DSM 33352 or a mutant thereof, which exhibits the following characteristics:

[0069] a) the 16S rDNA sequence according to SEQ ID NO: 16 and / or the 16S rDNA sequence of strain DSM 33352 deposited with DSMZ;

[0070] Preferably, the Bacillus strain exhibits at least one, two or three, preferably all, of the following further characteristics:

[0071] b) the yqfD sequence according to SEQ ID NO: 17 and / or the yqfD sequence of strain DSM 33352 deposited at DSMZ;

[0072] c) the gyrB sequence according to SEQ ID NO: 18 and / or the gyrB sequence of strain DSM 33352 deposited at DSMZ;

[0073] d) the rpoB sequence according to SEQ ID NO: 19 and / or the rpoB sequence of strain DSM 33352 deposited at DSMZ;

[0074] e) the groEL sequence according to SEQ ID NO: 20 and / or the groEL sequence of strain DSM 33352 deposited with DSMZ;

[0075] f) a sequence according to SEQ ID NO: 24;

[0076] g) The sequence according to SEQ ID NO:25.

[0077] If not explicitly described otherwise, the term "mutant" or "variant" according to the present invention relates to a strain having a sequence identity of at least 95%, 96% or 97%, preferably at least 98%, 99% or 99.5%, more preferably at least 99.8% or 99.9% relative to the genomic DNA of the parent strain. That is, in this context, if not explicitly mentioned otherwise, the term "sequence identity" is always related to the complete genomic DNA.

[0078] Another subject of the present invention is a composition comprising at least two Bacillus strains according to the invention or preparations thereof, wherein the Bacillus strains and preparations thereof are preferably selected from the group consisting of:

[0079] a) Bacillus strains deposited with DSMZ under one of the following numbers: DSM 33349, DSM 33350, DSM 33351 and DSM 33352;

[0080] b) a mutant of a Bacillus strain listed in (a), which has at least 95%, preferably at least 98%, 99% or 99.5% sequence identity with the strain listed in (a);

[0081] c) a preparation of a strain according to (a) or (b);

[0082] d) A preparation comprising an effective mixture of compounds contained in a strain as listed in (a) or (b) or an effective mixture of compounds contained in a preparation as listed in (c).

[0083] Very preferred according to the invention are compositions comprising a combination of strains DSM 33351 and DSM 33352.

[0084] The bacteria of the present invention are capable of degrading inorganic nitrogen compounds, preferably at least one, more preferably at least two inorganic nitrogen compounds selected from the group consisting of ammonium, nitrite and nitrate.

[0085] Therefore, another subject of the present invention is a method for reducing the amount of inorganic nitrogen compounds in an aqueous system and / or controlling the amount of inorganic nitrogen compounds in an aqueous system, wherein the inorganic nitrogen compound is preferably selected from ammonium, nitrite and nitrate, very preferably from ammonium and nitrite, the method comprising supplying to the aqueous system at least one Bacillus strain according to the invention or a preparation thereof, in particular at least one Bacillus subtilis strain according to the invention or a preparation thereof, or a composition according to the invention, wherein the Bacillus strain and / or its preparation and / or the composition is capable of degrading at least one inorganic nitrogen compound and also of inhibiting the growth of at least one pathogen, in particular at least one pathogen of aquatic animals, wherein the Bacillus strains and preparations, in particular the Bacillus strains and preparations of the composition are preferably selected from the group consisting of:

[0086] a) Bacillus strains, in particular Bacillus subtilis strains, deposited with the DSMZ under one of the following numbers: DSM 33349, DSM 33350, DSM 33351, DSM 33352;

[0087] b) a mutant of a Bacillus strain listed in (a), which has at least 95%, preferably at least 98%, 99% or 99.5% sequence identity with the strain listed in (a);

[0088] c) a preparation of a strain according to (a) or (b);

[0089] d) A preparation comprising an effective mixture of compounds contained in a strain as listed in (a) or (b) or an effective mixture of compounds contained in a preparation as listed in (c).

[0090] The water system of the present invention can be any kind of water system, in particular a natural or artificial water system, such as a lake, pond, basin, ornamental pond, aquarium, aquaculture facility or an isolated area in an ocean, lake or river. Preferably, the water system is a reservoir for raising animals, in particular for raising aquatic animals. Therefore, the water system is very preferably drinking water or breeding water, in particular for raising aquatic animals. Depending on the type of animal to be raised, the water can be salt water as well as fresh water.

[0091] Since bacteria capable of degrading inorganic nitrogen compounds and at the same time suitable as probiotics have not been disclosed in the prior art in this field, another subject of the present invention is a bacterium capable of degrading inorganic nitrogen compounds and at the same time exhibiting probiotic activity (especially against aquatic animals), in particular a bacterium of the genus Bacillus, preferably a bacterium of the species Bacillus subtilis, wherein the bacterium preferably comprises the further characterizing features as disclosed in the present application, in particular the characterizing DNA sequences as described above.

[0092] Being able to degrade inorganic nitrogen compounds according to the present invention preferably means that under at least one test condition, preferably under all test conditions, as described in working example 1, the bacteria are able to remove at least 50%, more preferably at least 80% or 90%, in particular greater than 95% of ammonia and / or nitrate.

[0093] The bacteria according to the present invention are preferably also able to effectively degrade inorganic nitrogen compounds in environments where the C:N ratio is very low, in particular in C minimal medium. In particular, they are preferably able to effectively degrade inorganic nitrogen compounds in environments where the C:N ratio is below 100, in particular 20 to 100, 30 to 90 or 40 to 80.

[0094] The bacteria according to the present invention are preferably capable of growing for at least one day under high salt conditions, in particular in the presence of 1 wt. % NaCl, more preferably in the presence of 1.5 wt. % or 2 wt. % NaCl, in particular in the presence of 2.5 wt. % or 3 wt. % NaCl. In addition, the bacteria according to the present invention are preferably capable of effectively degrading inorganic nitrogen compounds, in particular inorganic nitrogen compounds selected from ammonium, nitrite and nitrate, in an aqueous environment having such a large amount of sodium chloride.

[0095] The spores of the bacteria of the present invention are preferably able to germinate effectively in C minimal medium (i.e. in an environment where the amount of carbon source is relatively low). Preferably, C minimal medium is a medium in which the amount of carbon source is below 50 g / kg, more preferably below 25 g / kg, in particular within the range of 10 to 50 g or 15 to 25 g carbon source / kg medium. Preferably, the spores of the present invention outgrow in this minimal medium within no more than 30 hours, more preferably within no more than 25 hours.

[0096] The bacteria according to the invention are also preferably capable of inhibiting at least one pathogenic microorganism, in particular at least one pathogenic bacterium, wherein the at least one pathogenic microorganism or bacterium is preferably pathogenic to aquatic animals.

[0097] Very preferably, the bacterium according to the invention is capable of inhibiting at least one bacterium selected from the group consisting of Vibrio harveyi, Vibrio parahaemolyticus, Aeromonas hydrophila and / or Streptococcus agalactiae, preferably inhibiting Streptococcus agalactiae.

[0098] Capable of inhibiting a pathogen according to the present invention preferably means that the strain is capable of causing a clearance of the pathogen of at least 3 mm, preferably at least 5 or 10 mm, relative to said pathogen in the well diffusion antagonism assay according to Parente et al. (1995).

[0099] It is particularly preferred according to the invention that the bacteria according to the invention are very effective in inhibiting the growth of Streptococcus agalactiae, in particular the growth of Streptococcus agalactiae DSM 2134. In particular, they are preferably characterized by a pathogen clearance of at least 10 mm, more preferably at least 15 mm, in a well diffusion antagonism test against Streptococcus agalactiae DSM 2134 on LB Kelly agar plates.

[0100] The bacteria of the present invention are preferably characterized by at least one, more preferably at least two, three or four of the following further characteristics:

[0101] They are preferably capable of growing under anaerobic conditions. In addition, they are preferably capable of degrading water-insoluble cellulose and / or protein under such anaerobic conditions.

[0102] Furthermore, the bacteria of the present invention are preferably also capable of degrading water-insoluble cellulose and / or protein under aerobic conditions, in particular in the presence of 2 mM bile.

[0103] The bacterium according to the invention is preferably further characterized by exhibiting at least one, two or three, preferably all, of the following enzyme activities: cellulase activity; xylanase activity; protease activity; catalase activity; superoxide dismutase activity.

[0104] The combined presence of proteolytic activity and the ability to degrade inorganic nitrogen compounds is a very preferred embodiment of the present invention, since such combined activities allow for the complete removal of undigested feed proteins from the rearing water and thus exhibit the desired synergistic effect.

[0105] The bacteria of the present invention preferably also produce lactate, and they are preferably also capable of degrading mycotoxins.

[0106] The bacteria according to the invention are preferably further characterized by being able to grow in the presence of 2 mM bile, more preferably in the presence of 4 mM bile, and / or in the presence of 0.3 wt. % porcine bile and / or in the presence of 0.3 wt. % chicken bile. In particular, they are preferably characterized by being able to proliferate rapidly at such high bile concentrations.

[0107] Furthermore, the bacteria according to the invention, i.e. a significant part (in particular at least 80%) of the respective sample, preferably survive the high temperatures required for pelleting animal feed, in particular they preferably survive a temperature of 80° C., more preferably 95 or 99° C. for at least 20 minutes.

[0108] Without wishing to be bound by any theory, it is believed that the Bacillus strains according to the present invention are able to enhance animal health, in particular intestinal health and / or the health of aquatic animals, through a multifaceted mode of action, including the production of antimicrobial metabolites with selective efficacy by better consumption of available nutrients and competition with pathogenic bacteria, thereby inhibiting the effective establishment of pathogenic bacteria, in particular in the intestine, gills or on the skin. Thus, the enzymes produced by the Bacillus can help establish a balanced microbiota, in particular in aquatic animals, by providing pre-digested nutrients.

[0109] A major advantage of probiotics over antibiotics is that they neither attack bacteria indiscriminately nor cause antibiotic-resistant strains of pathogenic bacteria. Typically, they are able to selectively compete with pathogenic bacteria by producing antimicrobial substances with specific efficacy and, ideally, are able to simultaneously enhance the growth and vitality of beneficial microbial communities, particularly in the intestine or gills. In addition, they are preferably able to stimulate the systemic immune response of the treated animal.

[0110] Mutants or variants according to the present invention are preferably spontaneous mutants. The term "spontaneous mutant" refers to a mutant or variant produced by a parent strain without the intentional use of a mutagen. That is, they are considered to be non-GMO. Such spontaneous mutants can be obtained by classical methods, such as cultivating a subtilis strain in the presence of UV light and / or by applying high temperature or protoplast formation and / or in the presence of certain antibiotics to which the parent strain is sensitive, and testing any resistant mutant for the ability to increase biological activity or improve one or more indicators of enhancing animal health (particularly intestinal health). Other methods for identifying spontaneous mutants are known to those of ordinary skill in the art. But in addition to these preferred spontaneous mutants, all other types of mutants or variants, such as mutants obtained by genetic engineering, are also included in the present invention.

[0111] A specific embodiment of the present invention is a naturally occurring mutant, in particular a spontaneous mutant as defined above, of the bacterium according to the invention, in particular strain DSM 33349, DSM 33350, DSM 33351 or DSM 33352, characterized by the above mentioned features.

[0112] In a preferred embodiment of the invention, mutants and variants of strain DSM 33349, DSM 33350, DSM 33351 or DSM 33352 have the same identifying characteristics as the parent strain from which they were derived.

[0113] In a preferred embodiment of the present invention, the bacteria, preparations and compositions of the present invention are administered orally to animals or humans.

[0114] Therefore, another subject of the present invention is a composition, such as feed, food, drinking water and breeding water and a pharmaceutical composition, in particular a therapeutic composition, which contains at least one bacterium according to the invention, in particular at least one Bacillus strain, preferably a Bacillus subtilis strain, and / or at least one preparation according to the invention and / or at least one composition according to the invention, as described above.

[0115] Another subject of the present invention is also the use of at least one bacterium, in particular at least one Bacillus strain, preferably a Bacillus subtilis strain, and / or at least one preparation and / or at least one composition according to the invention as a probiotic ingredient (DFM) in feed or food.

[0116] In particular, mixtures or combinations of at least two or three strains of the invention may be used, very preferably mixtures or combinations of at least two or at least three strains selected from the group consisting of strains DSM 33349, DSM 33350, DSM 33351 and DSM 33352.

[0117] Preferred foods according to the invention are dairy products, in particular yoghurt, cheese, milk, butter and quark.

[0118] The cells of the bacteria of the present invention may be present as spores (dormant), vegetative cells (growing), transitional cells (transitioning from a growth phase to a spore formation phase) or a combination of at least two, in particular all of these types of cells, in particular in the composition of the present invention. In a preferred embodiment, the composition of the present invention comprises mainly spores, wherein preferably at least 80% or at least 90% of the Bacillus cells are spores, or comprises only spores, i.e. all Bacillus cells contained in the composition are spores.

[0119] Additionally or alternatively, the bacterial cells may also be used in a non-viable, killed form, since non-viable cells are also expected to still have a probiotic effect. Methods for killing cells are known to those skilled in the art.

[0120] The bacteria of the present invention, when administered to animals, preferably enhance the health of such animals and / or improve the overall physical condition of such animals and / or increase the feed conversion rate of such animals and / or reduce the mortality rate of such animals and / or increase the survival rate of such animals and / or improve the weight gain of such animals and / or increase the productivity of such animals and / or increase the disease resistance of such animals and / or modulate the immune response of such animals and / or establish or maintain a healthy intestinal microflora in such animals and / or improve the meat quality of such animals (particularly the elasticity of the meat and / or the hardness of the meat) and / or reduce the excretion of pathogens through the feces of such animals. In particular, the strains and compositions of the present invention can be used to help reestablish a healthy balance of intestinal or gill microflora after the administration of antibiotics for therapeutic purposes.

[0121] Therefore, another subject of the present invention is a method for enhancing the health of animals and / or improving the overall physical condition of animals and / or increasing the feed conversion rate of animals and / or reducing the mortality rate of animals and / or increasing the survival rate of animals and / or improving the weight gain of animals and / or increasing the productivity of animals and / or increasing the disease resistance of animals and / or modulating the immune response of animals and / or establishing or maintaining a healthy intestinal microflora in animals and / or improving the meat quality of animals and / or reducing the excretion of pathogens through animal feces, wherein at least one strain and / or at least one preparation and / or at least one composition according to the invention is administered to animals, in particular aquatic animals, preferably in aquaculture.

[0122] Therefore, another subject of the present invention is also the use of at least one strain and / or at least one preparation and / or at least one composition according to the invention for enhancing the health of animals and / or improving the general physical condition of animals and / or increasing the feed conversion rate of animals and / or reducing the mortality rate of animals and / or increasing the survival rate of animals and / or improving the weight gain of animals and / or increasing the productivity of animals and / or increasing the disease resistance of animals and / or modulating the immune response of animals and / or establishing or maintaining a healthy intestinal microflora in animals and / or improving the meat quality of animals and / or reducing the excretion of pathogens through animal feces, wherein at least one strain and / or at least one preparation and / or at least one composition according to the invention is administered to animals, in particular aquatic animals, preferably in aquaculture.

[0123] Therefore, another subject of the present invention is also at least one strain and / or at least one preparation and / or at least one composition according to the present invention for enhancing the health of an animal and / or improving the overall physical condition of an animal and / or increasing the feed conversion rate of an animal and / or reducing the mortality rate of an animal and / or increasing the survival rate of an animal and / or improving the weight gain of an animal and / or increasing the productivity of an animal and / or increasing the disease resistance of an animal and / or modulating the immune response of an animal and / or establishing or maintaining a healthy intestinal microflora in an animal and / or improving the meat quality of an animal and / or reducing the excretion of pathogens through the feces of an animal, wherein the animal is preferably an aquatic animal, in particular an aquatic animal kept in aquaculture.

[0124] "Increasing the productivity of animals" means in particular any of the following: production of more or higher quality eggs, milk or meat or increasing the yield of weaned offspring.

[0125] The methods and uses of the strains, preparations and compositions of the invention may be therapeutic or non-therapeutic. In a particularly preferred embodiment of the invention, the methods and uses are non-pharmaceutical, in particular feeding applications.

[0126] Due to pathogenic bacteria and other components, untreated animal manure may have harmful environmental effects, in particular for the animals themselves and / or for humans who come into contact with the manure, which can be avoided by feeding the animals with the bacteria, compositions or preparations according to the invention or by directly treating the manure or animal bedding. Therefore, another subject of the present invention is a method for controlling and / or avoiding harmful environmental effects of manure or contaminated liquids, which comprises the step of applying at least one strain, at least one preparation and / or at least one composition according to the invention to manure, contaminated liquid, litter, pit or manure pond. Preferably, the composition is applied in liquid form (e.g. by spraying) or as a powder (e.g. by broadcasting).

[0127] Since harmful bacteria may have a negative impact on the consistency of litter, and may in particular affect fairly fluid or highly fluid litter, which may lead to foot pad lesions in poultry, which can be avoided by feeding the animals with the strains, compositions or preparations according to the invention, another subject of the present invention is a method for controlling and / or improving the consistency of litter, in particular a method for ensuring a solid consistency of litter and / or a method for avoiding foot pad lesions, the method comprising the step of feeding animals, in particular poultry, with at least one strain, at least one preparation and / or at least one composition according to the invention.

[0128] The bacteria, preparations and compositions of the present invention are very preferably used to improve the quality of water or aqueous solutions. Therefore, another subject of the present invention is also a method for controlling and / or improving the quality of water or aqueous solutions (especially drinking water and / or feeding water and / or circulating water and / or waste water), which comprises the step of applying at least one bacterial strain and / or at least one preparation and / or at least one composition of the present invention to water or aqueous solutions.

[0129] Inorganic nitrogen compounds are not only a problem in water and aqueous solutions of aquaculture facilities, but also, for example, in circulating water and wastewater of different kinds, in particular in wastewater treatment facilities. The treatment of soils often leads to eutrophication of waters rich in inorganic nitrogen compounds. The treatment of circulating water or wastewater with the strains and / or preparations and / or compositions according to the invention can help to solve this problem.

[0130] In addition, bacterium, preparation and composition of the present invention can also be used for treating the microbial diseases of plants. Therefore, another theme of the present invention is also a method for treating and / or preventing the microbial diseases of plants (particularly cultivated plants), which comprises the step of applying at least one bacterial strain of the present invention and / or at least one preparation and / or at least one composition to the plants. The application can be carried out in liquid form (such as by spraying) or in solid form (particularly as powder, preferably as a prepared powder).

[0131] By using the strains, preparations and compositions of the present invention, it is preferred to achieve an improvement in at least one of the aforementioned characteristics, wherein the achievement of this characteristic preferably means an improvement of at least 1%, more preferably at least 3% or at least 5% compared to an appropriate negative control. The mean values ​​of negative controls known in the field of animal husbandry can be used, but it is preferred to use negative control animals that are treated the same as the animals tested but are not administered the strains and / or preparations and / or compositions of the present invention.

[0132] In particular, the strains, formulations and compositions of the present invention can be administered or fed to animals in an amount effective to inhibit and / or reduce the growth of pathogenic bacteria, viruses and protozoa, particularly in the intestinal tract of animals or the gills of aquatic animals. Such pathogens include Clostridia, Listeria, Salmonella, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Escherichia coli, Shigella, Haemophilus, Brachyspira, Flavobacterium, Serratia, Yersinia, Edwardsiella, Rennibacterium, Pasteurella, and Vibrio. Relatedly, the methods of the invention can be used to reduce the amount of pathogenic bacteria, viruses, and protozoa discharged in animal feces. The method of the present invention can also be used to maintain or increase the growth of beneficial bacteria (such as lactic acid bacteria), particularly in the intestinal tract of animals or in the gills of aquatic animals. By reducing pathogenic bacteria and / or increasing or maintaining beneficial bacteria, the composition of the present invention can maintain an overall healthy microbial community, particularly in the intestinal tract, on the skin or in the gills of aquatic animals.

[0133] Therefore, another subject of the present invention is also a method for inhibiting and / or reducing the growth of pathogenic bacteria and / or maintaining and / or increasing the growth of beneficial bacteria, in particular in the intestinal tract of animals, on the skin of animals or in the gills of aquatic animals, wherein the strains, preparations and compositions according to the invention are used and wherein the pathogenic bacteria are preferably selected from the genus Clostridium, in particular Clostridium perfringens, C. difficile, C. novyi, C. septicum and C. colinu m), Listeria spp., in particular Listeria monocytogenes, L. seeligeri and L. welshimeri, Salmonella spp., in particular Salmonella enterica, including subspecies enterica, Salmonella arizonae, Salmonella bongori, and in particular serovars, Salmonella gallinarum, llinarum), Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella heidelberg and Salmonella infantis, Enterococcus spp., especially Enterococcus faecalis, Enterococcus faecium and Enterococcus cecorum, Staphylococcus, in particular Staphylococcus aureus (S. aureus), Aeromonas, in particular Aeromonas hydrophila and Aeromonas salmonicida (A. salmonocida), Streptococcus, in particular Streptococcus suis (S. suis), Streptococcus gallinaceus (S. gallinaceus) and Streptococcus agalactiae (S. agalactiae), Campylobacter, in particular Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), Escherichia coli, Haemophilus, in particular Haemophilus parasuis (Haemophilus parasuis, Brachyspira, in particular Brachyspira hyodysenteriae, Flavobacterium, in particular Flavobacterium columnare, Serratia, in particular Serratia liquefaciens, Yersinia, in particular Y. ruckeri, Edwardsiella, in particular E. tarda and E.ictaluria), Nephrobacterium, in particular salmon Nephrobacterium (R. salmoninarum), Pasteurella, in particular Pasteurella piscicida (P. piscicida), Vibrio, in particular Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum (V. anguillarum), Vibrio ordalii (V. ordalii), Vibrio alginolyticus (V. alginolyticus), Vibrio fischeri (V. fischeri) and Vibrio salmonicida (V. salmonicida), and the beneficial bacteria are preferably selected from lactic acid bacteria, in particular Lactobacilli (lactobacilli) and Bifidobacteria (bifidobacteria). Very preferably, the strains to be inhibited are selected from pathogenic strains of aquatic animals, in particular Streptococcus, in particular Alactobacillus, Vibrio, in particular Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio ostreatus, Vibrio alginolyticus, Vibrio fischeri and Vibrio salmonicida, Flavobacterium, in particular Flavobacterium columnaris, and Aeromonas, in particular Aeromonas hydrophila and Aeromonas salmonicida.

[0134] In a preferred embodiment of the present invention, the amount of at least one pathogenic bacteria in aquatic animals, in particular Streptococcus agalactiae (in particular Streptococcus agalactiae DSM 2134), Vibrio harveyi (in particular Vibrio harveyi DSM 19623), Vibrio parahaemolyticus (in particular Vibrio parahaemolyticus DSM 10027) and / or Aeromonas hydrophila (in particular Aeromonas hydrophila DSM 30187), is significantly reduced, preferably by at least 0.5 log, more preferably by at least 1 log, 2 log or 3 log, wherein a reduction in Streptococcus agalactiae is particularly preferred.

[0135] Therefore, another subject of the present invention is also the strains, preparations and compositions according to the invention for inhibiting and / or reducing the growth of pathogenic bacteria and / or for maintaining and / or increasing the growth of beneficial bacteria in the intestinal tract of animals or on the skin of animals or in the gills of aquatic animals, wherein the pathogenic bacteria are preferably selected from the group consisting of Clostridium, in particular Clostridium perfringens, Clostridium difficile, Clostridium novyi, Clostridium septicum and Clostridium entericum, Listeria, in particular Listeria monocytogenes, Listeria sylvi and Listeria welshii, Salmonella, in particular Salmonella enterica, including subspecies enterica, Salmonella arizona, Salmonella bongore, and in particular serotypes, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Choleraesuis, Salmonella Heidelberg and Salmonella Infantis, Enterococcus, in particular Enterococcus faecalis, Enterococcus faecium and Enterococcus caecum, Staphylococcus, especially Staphylococcus aureus, Aeromonas, especially Aeromonas hydrophila and Aeromonas salmonicida, Streptococcus, especially Streptococcus suis, Streptococcus gallinarum and Streptococcus agalactiae, Campylobacter, especially Campylobacter jejuni and Campylobacter coli, Escherichia coli, Haemophilus, especially Haemophilus parasuis, Brachyspira, especially Brachyspira hyodysenteriae, Flavobacterium, especially Flavobacterium columnaris, Serratia, especially Serratia liquefaciens, Yersinia, especially Yersinia ruckeri, Edwardsiella, especially Edwardsiella tarda and Edwardsiella ictaluri, Nephrobacterium, especially Nephrobacter salmonicida, Pasteurella, especially Pasteurella piscicida, and Vibrio, especially Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio ostreatus, Vibrio alginolyticus, Vibrio fischeri and Vibrio salmonicida, and the beneficial bacteria are preferably selected from lactic acid bacteria, especially Lactobacillus and Bifidobacterium.

[0136] Including or in addition to pathogenic bacteria, other pathogens, in particular viruses and algae, can also be inhibited.

[0137] In a specific embodiment of the present invention, the strain and / or preparation and / or composition of the present invention is capable of inhibiting at least one virus selected from the group consisting of: White Spot Syndrome Virus (WSSv), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV), Infectious Hypodermic and Hematopoietic Necrosis Virus (IHHN) and IHHNV, Virus causing Chronic Dwarfism Syndrome or RDS in Penaeus vannamei, Baculovirus-like Virus, Infectious Pancreatic Necrosis Virus (IPNV), Hirame rhabdovirus (HIRRV), Yellowtail Ascites Virus (YAV), Striped Jack Nervous Necrosis Virus (Strip ... Virus, SJNNV), iridovirus, infectious salmon anemia (USA) virus, viruses causing pancreas disease (PD) or viral hemorrhagic septicemia (VHS), viruses causing viral hemorrhagic septicemia, carp infectious pancreatic necrosis or viremia, channel catfish virus, grass carp hemorrhagic virus, nodaviridae such as neuronecrosis virus, infectious salmon anemia virus.

[0138] In another specific embodiment of the present invention, the strain and / or preparation and / or composition of the present invention is capable of inhibiting at least one microalgae, protozoa or toxin selected from the parasites Ceratomyxa shasta, Ichthyophthirius multifillius, Cryptobia salmositica, Lepeophtherius salmonis, Tetrahymena, Trichodina, Epistylus, and dinoflagellates toxins, including toxins that cause Diaarhetic Shellfish Poisoning (DSP), Paralytic Shellfish Poisoning (PSP), Neurotoxin poisoning (NSP) and Ciguatera.

[0139] The presence and / or increased growth of pathogens does or may lead to outbreaks of certain diseases.

[0140] Vibrio is known to be associated with disease and high mortality in shrimp, but can also infect finfish. Tilapia infections can be associated with Streptococcus agalactiae, a widespread bacterium, which can cause, for example, bleeding or erratic swimming. Columnaris disease is caused by the infection of freshwater fish with Flavobacterium columnaris. For example, salmon and trout farms report high annual losses.

[0141] In addition, the presence and / or increased growth of C. perfringens can lead to outbreaks of enteric diseases, particularly necrotic enteritis in pigs and poultry. The presence and / or increased growth of C. perfringens can also lead to outbreaks of other diseases, such as bacterial enteritis, gangrenous dermatitis and cholangiohepatitis. Even the mildest forms of C. perfringens infection may be accompanied by diarrhea, which leads to damp litter and may lead to secondary diseases such as footpad dermatitis. While type C C. perfringens is generally considered to be the main cause of necrotic enteritis and necrotizing hemorrhagic enteritis in piglets, type A is associated with mild necrotizing enterocolitis and villous atrophy in the intestinal diseases of suckling and feeding pigs.

[0142] Clostridium difficile is an important emerging pathogen that causes diarrhea primarily in newborn pigs. Affected piglets may have difficulty breathing, abdominal distension, and scrotal edema.

[0143] Cecal Enterococci are known to cause lameness, arthritis and osteomyelitis in broiler chickens, usually caused by inflammation of the joints and / or bone tissue. In addition, cecal Enterococci can cause pericardial inflammation.

[0144] S. gallinarum can cause septicaemia in poultry. Gross lesions include splenomegaly, hepatomegaly, nephromegaly, and congestion. Multiple areas of necrosis and / or infarction in the liver and spleen associated with valvular endocarditis have also been observed.

[0145] Campylobacter coli is a foodborne bacterium that most people become infected with by eating pork that has been contaminated with the bacteria. It causes gastroenteritis and acute enterocolitis in humans, as well as acute diarrheal illness. Pigs are the primary reservoir, but it can also infect humans, birds, and a variety of other animals.

[0146] Campylobacter jejuni infection is one of the most common causes of gastroenteritis worldwide [Acheson et al. 2001]. Infection is caused by ingestion of contaminated food or water. Campylobacter jejuni is commonly associated with poultry and naturally colonizes the digestive tract of many birds [Colles et al. 2009] (20-100% of retail chickens are contaminated) as well as cattle (especially calves). Some strains of Campylobacter jejuni have been reported to cause enteritis and mortality in newly hatched chicks and poultry.

[0147] Streptococcus suis is an important pathogen of pigs and one of the most important causes of bacterial mortality in post-weaned piglets, causing septicemia, meningitis and a variety of other infections.

[0148] The pathogen can cause other diseases such as polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhoea and oedema disease, and swine dysentery.

[0149] Therefore, another subject of the present invention is also a therapeutic composition comprising at least one strain and / or at least one preparation and / or at least one composition according to the invention.

[0150] Therefore, a very preferred subject of the present invention is a therapeutic composition for the treatment and / or prevention of diseases in aquatic animals, in particular finfish, preferably tilapia, or crustaceans, preferably shrimp, comprising at least one strain and / or at least one preparation and / or at least one composition according to the invention, the diseases being in particular selected from: hemorrhages, swimming instability, Columnaris disease, White Spot Syndrome and / or diseases of the gills.

[0151] Therefore, a further preferred subject matter herein is a therapeutic composition for the treatment and / or prevention of necrotic enteritis and necrotic hemorrhagic enteritis (in particular subclinical necrotic enteritis and necrotic hemorrhagic enteritis) in animals (preferably pigs or poultry), comprising at least one strain and / or at least one preparation and / or at least one composition according to the invention.

[0152] Therefore, another preferred subject matter herein is a therapeutic composition for the treatment and / or prevention of bacterial enteritis, gangrenous dermatitis, cholangiohepatitis, clostridial disease, diarrhea, dyspnea, abdominal distension, scrotal edema, poultry foot inflammation, footpad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhea and edema disease, dysentery, osteomyelitis, inflammation of joints and / or bone tissue, pericardial inflammation, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis, liver or splenic infarction, valvular endocarditis, sepsis and / or meningitis in animals, preferably pigs or poultry.

[0153] Therefore, another preferred subject matter of the present invention is also the treatment and / or prevention of diseases in aquatic animals, in particular finfish, preferably tilapia, or crustaceans, preferably shrimp, and the disease is in particular selected from hemorrhages, swimming instability and columnar disease, wherein at least one strain and / or at least one preparation and / or at least one composition according to the invention is administered to an aquatic animal in need thereof.

[0154] Therefore, another subject of the present invention is also the treatment and / or prevention of diseases in pigs or poultry, in particular intestinal diseases, preferably necrotic enteritis or necrotic hemorrhagic enteritis, in particular subclinical necrotic enteritis or subclinical necrotic hemorrhagic enteritis, wherein at least one strain and / or at least one preparation and / or at least one composition according to the invention is administered to an animal in need thereof.

[0155] Therefore, another subject of the present invention is also the treatment and / or prevention of diseases, preferably diseases of pigs or poultry, which are selected from the group consisting of bacterial enteritis, gangrenous dermatitis, cholangiohepatitis, clostridial disease, diarrhea, dyspnea, abdominal distension, scrotal edema, poultry foot inflammation, foot pad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhea and edema disease, dysentery, osteomyelitis, inflammation of joints and / or bone tissue, pericardial inflammation, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis, liver or spleen infarction, valvular endocarditis, sepsis and / or meningitis, wherein at least one strain and / or at least one preparation and / or at least one composition according to the invention is administered to an animal in need thereof.

[0156] The strains and / or preparations and / or compositions of the invention can be administered to animals in feed and / or drinking water and / or feeding water over multiple days throughout the animal's life or during a specific stage or part of the animal's life. For example, the strains and / or preparations and / or compositions can be administered only in a starter diet or only in a finisher diet of a farm animal.

[0157] A particular subject of the present invention is also a method for enhancing human health and / or improving the overall physical condition of a human and / or increasing the resistance of a human to disease and / or modulating the immune response of a human and / or establishing or maintaining a healthy intestinal microflora in a human, wherein the strain and / or the preparation according to the invention or the composition according to the invention (which comprises such a strain) is administered to a human.

[0158] Therefore, another subject of the present invention is also the use of the strains and / or preparations and / or compositions according to the invention for enhancing the health of humans and / or for improving the general physical condition of humans and / or for increasing the resistance of humans to diseases and / or for modulating the immune response of humans and / or for establishing or maintaining a healthy intestinal microflora in humans, wherein the strains and / or preparations according to the invention or the compositions according to the invention (which comprise such strains) are administered to humans.

[0159] The composition of the present invention, in particular feed, food and pharmaceutical composition, as well as drinking water, feeding water, circulating water or wastewater preferably comprises the strain of the present invention and is present in an amount of about 1×10 3 About 2×10 12 The ratio of CFU / g feed or ml water is, in particular, about 1×10 3 or about 1×10 4 or about 1×10 5 or about 1×10 6 or about 1×10 7 or about 1×10 8 or about 1×10 9 or about 1×10 10 or about 1×10 11 or about 1×10 12 The ratio of CFU / g feed or ml water is preferably about 1×10 4 About 1×10 10 The amount of CFU / g feed or ml water is preferably 1×10 4 Up to 1×10 7 The amount of CFU / g feed or ml water was administered to the animals.

[0160] Accordingly, the preferred amount of the strain and / or preparation of the invention in the feed, food and water compositions of the invention is 0.1 to 10% by weight, more preferably 0.2 to 5% by weight, in particular 0.3 to 3% by weight.

[0161] The method of the present invention can be used for all kinds of animals, in particular all kinds of non-human and non-insect animals, more preferably all kinds of vertebrates such as mammals, aquatic animals and birds, and particularly preferably aquatic animals.

[0162] Animals that may benefit from the present invention include, but are not limited to, farm animals, pets, exotic animals, zoo animals, animals used for sport, recreation, or work.

[0163] The pet is preferably selected from the group consisting of dogs, cats, poultry and domesticated exotic animals.

[0164] The aquatic animal according to the invention is preferably a finfish, in particular an Actinopterygii, or a crustacean. The Actinopterygii include, in particular, tilapia and other cichlids, carp and other cyprinids, salmon and other salmonids, catfish, in particular African catfish and pangasius, tuna, perch, cod, smelt, milkfish, gourami, seabass, in particular barramundi, sea bream, grouper and snakehead.

[0165] Preferred types of salmon and salmonids herein are Atlantic salmon, sockeye salmon, mountain trout, Chinook salmon, dog salmon, silver salmon, Danube taimen, Pacific salmon, pink salmon and trout. Aquatic animals according to the invention are very preferably tilapia.

[0166] Crustaceans include, inter alia, shrimps, lobsters, crabs, prawns and crayfish.

[0167] Preferred shrimp types herein are of the genera Litopenaeus, Farfantepenaeus and Penaeus, in particular Penaeus stylirostris, Penaeus vannamei, Penaeus monodon, Penaeus chinensis, Penaeus occidentalis, Penaeus californiensis, Penaeus semisulcatus, Penaeus esculentu, Penaeus setiferus, Penaeus japonicus, Penaeus aztecus, Penaeus duorarum, Penaeus indicus and Penaeus merguiensis. A highly preferred shrimp according to the present invention is the Litopenaeus vannamei, also known as whiteleg shrimp.

[0168] The aquatic animals, in particular shrimps, treated or fed with the strains and / or formulations and / or compositions according to the invention may in particular be larvae, postlarvae or juvenile shrimps.

[0169] Aquatic animals can also be fish which are subsequently processed into fishmeal or fish oil. In this respect, the fish are preferably herring, pollock, cod or small pelagic fish such as anchovies, blue whiting, capelin, pomfret, jack, mackerel, herring, sardine or horse mackerel. The fishmeal or fish oil thus obtained can in turn be used in aquaculture for the cultivation of edible fish or crustaceans.

[0170] The aquatic animal may also be an oyster, a clam, a cockle, an ark shell, a bivalve, a mussel or a scallop.

[0171] However, aquatic animals may also be small organisms used as feed in aquaculture. These small organisms may be in the form of nematodes, small crustaceans or rotifers, for example.

[0172] The farming of aquatic animals can be carried out in ponds, tanks, basins or in isolated areas in the sea or lakes or rivers, especially in this case in cages or pens. Farming can be used to raise finished food fish, but it can also be used to raise fry that are then released to restock wild fish stocks.

[0173] In salmon farming, the fish are preferably first raised as smolts in freshwater ponds or artificial raceways and then grown in cages or pens floating in the ocean, ponds or rivers and preferably anchored in bays or fjords.

[0174] Therefore, the feed of the present invention is preferably a feed for breeding the above-mentioned animals.

[0175] According to the present invention, bacterial strain, preparation and composition of the present invention can be used with wide dosage range.Daily dosage is, for example, about 1mg to about 500mg per kilogram of live body weight, particularly about 5mg to about 200mg, about 10mg to about 100mg or about 20mg to about 60mg in the scope.

[0176] In another embodiment of the invention, the animal is a farm animal, which is raised for consumption or as a food producer, such as poultry, pigs, and ruminants.

[0177] Poultry can be selected from productive poultry or domestic poultry, but can also be selected from ornamental poultry or wild birds. Preferred productive poultry in this context are chickens, turkeys, ducks and geese. In this context, productive livestock are preferably poultry optimized for the production of young stock or poultry optimized for meat production. Preferred ornamental poultry or wild birds are peacocks, pheasants, partridges, chukkars, guinea fowls, quails, capercaillies, grouses, pigeons and swans, particularly preferably quails. Further preferred poultry are ratites, in particular ostriches and emus, and parrots.

[0178] The ruminant according to the present invention is preferably selected from cattle, goats and sheep. In one embodiment, the composition of the present invention can be fed to preruminants to enhance their health, in particular to reduce the incidence of diarrhea in these animals. Preruminants are ruminants ranging in age from birth to about 12 weeks, including calves.

[0179] The composition of the present invention may comprise at least one carrier or typical feed ingredient or a combination thereof.

[0180] Suitable carriers are inert formulation ingredients that are added to improve recovery, efficacy or physical properties and / or to aid packaging and administration. These carriers may be added alone or in combination. These carriers may be selected from anti-caking agents, antioxidants, bulking agents, binders, structurants, coatings and / or protective agents. Examples of useful carriers include polysaccharides (particularly starch, maltodextrin, methylcellulose, gums, chitosan and / or inulin), protein sources (particularly skimmed milk powder and / or sweet whey powder), peptides, sugars (particularly lactose, trehalose, sucrose and / or glucose), lipids (particularly lecithin, vegetable oils and / or mineral oils), salts (particularly sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and / or sodium citrate) and silicates (particularly clays, particularly beolite clay, amorphous silica, pyrogenic / precipitated silica, zeolites, bleaching earth, Suitable carriers for animal feed additives are described in American Feed Control Officials, Inc.'s Official Publication (which is published annually). See, for example, Official Publication of American Feed Control Officials, Sharon Krebs, ed., 2006 edition, ISBN 1-878341-18-9. The carrier may be added after the fermentation broth is concentrated and / or during and / or after drying. Preferred carriers of the present invention are selected from calcium carbonate, diatomaceous earth and vegetable oils.

[0181] A preferred embodiment of the present invention is a concentrate composition, in particular a feed additive composition, i.e. a composition suitable for the preparation of a feed composition, comprising at least one strain according to the present invention and at least one carrier as described above, wherein the content of the at least one strain is preferably in an amount of 0.1-10 wt.-%, more preferably in an amount of 0.2-5 wt.-%, in particular in an amount of 0.3-3 wt.-%, especially in an amount of 0.4-2.2 wt.-%, and the content of the at least one carrier is preferably in an amount of at least 90 wt.-%, preferably in an amount of 90-99.9 wt.-%, more preferably in an amount of 95-99.8 wt.-%, in particular in an amount of 97-99.7 wt.-%, especially in an amount of 97.8-99.6 wt.-%, and wherein the carrier preferably consists essentially of limestone, in particular limestone with a minor portion of diatomaceous earth and / or vegetable oil.

[0182] These preferred compositions of the present invention (which contain stable strains) can be used to prepare feed and pharmaceutical compositions as well as drinking water and feeding water, which preferably contain the strains of the present invention in an amount as described in the above description. In a preferred embodiment, 50-1000 grams of this concentrated composition, in particular 50, 100, 250, 500 or 1000 grams of this concentrated composition are used per ton of feed, drinking water or feeding water to provide a composition that can be used to feed animals. These concentrated compositions preferably contain at least one strain of the present invention in an amount of 1×10 9 -2×10 11 CFU, especially 2×10 9 -1×10 11 CFU.

[0183] Starting from these concentrate compositions, feed and food compositions can be prepared by mixing the concentrate compositions with typical feed or food ingredients, respectively.

[0184] Suitable typical animal feed ingredients that may be contained in the composition according to the invention and / or used for preparing a feed composition starting from the concentrate composition according to the invention include one or more of the following: proteins, carbohydrates, fats, other probiotics and / or other inorganic nitrogen compound degrading microorganisms, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, carriers, coccidiostats, acid-based products and / or drugs, such as antibiotics.

[0185] Carbohydrate-containing components which can be used according to the invention are, for example, grass, roughage, wheat meal, corn meal, sunflower meal or soya meal and mixtures thereof.

[0186] Protein-containing components that can be used according to the invention are, for example, soy protein, pea protein, wheat protein, corn protein, rice, canola meal, marine animal meal (especially fish meal), terrestrial animal meal and mixtures thereof. "Marine animal meal" includes meat meal, meat and bone meal, blood meal, liver meal, poultry meal, silkworm meal, silkworm pupa meal and combinations thereof.

[0187] Fat is usually provided as marine animal oil, vegetable oil or microbial oil, particularly microalgae oil or its combination.The example of vegetable oil is soybean oil, rapeseed oil, sunflower seed oil, canola oil (canola oil), cottonseed oil, linseed oil and palm oil.Marine animal oil includes the oil of fish oil and krill, bivalve shellfish, squid or shrimp, and other fatty oils from the fish of Engraulidae (Engraulidae), Carangidae (Carangidae), Clupeidae (Clupeidae), Osmeridae (Osmeridae), Scombridae (Scombridae) and / or Ammodytidae (Ammodytidae).The example of the oil of microalgae is particularly the oil of Labyrinthulea (Labyrinthulea), preferably the oil of Schizochytria or Thraustochytria. In addition to the separated oil, defatted biomass itself can also be used as fat source, ie in particular marine animal meal (preferably fish meal) or vegetable meal (in particular soya meal, rapeseed meal, sunflower meal, canola meal, cottonseed meal and / or linseed meal).

[0188] Protein-containing components which can be used according to the invention, which additionally contain fat, are for example fish meal, krill meal, bivalve mollusc meal, squid meal or shrimp shells and combinations thereof.

[0189] The feed according to the invention preferably has a total protein content of 20 to 50% by weight and / or a total fat content of 1 to 15% by weight and / or a total carbohydrate content of 20 to 60% by weight.

[0190] The further probiotics (DFM) and / or microorganisms which can be used according to the invention in combination with the strains and formulations of the invention are preferably bacteria selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, Bacillus coagulans, Bacillus alevi, Bacillus cereus, Bacillus badius, Bacillus thurigiensis, Enterococcus faecium and Pediococcus acidilactici. Preferred examples are Bacillus pumilus DSM 32539 and Bacillus subtilis DSM 32540 (both deposited with the DSMZ on June 14, 2017 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and their derivative strains, Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 32315 (both deposited with the DSMZ on May 12, 2016 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and their derivative strains, Bacillus subtilis PB6 (as described in U.S. Pat. No. 7,247,299 and deposited under ATCC Accession No. PTA-6737), which is registered by Kemin under the trademark subtilis C-3102 (described in U.S. Pat. No. 4,919,936 and deposited as FERM BP-1096 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan) is marketed by Calpis as Bacillus subtilis DSM 17299 is sold under the trademark Bacillus licheniformis DSM 17236 is sold under the trademark Sold as a mixture of spores of Bacillus licheniformis DSMZ 5749 and Bacillus subtilis DSMZ 5750, by Chr. Hansen under the trademark YC sells Bacillus subtilis DSM 29784, sold by Adisseo / Novozymes under the trademark Bacillus subtilis, sold by Chr. Hansen under the trademark The invention also provides a probiotic composition comprising ...

[0191] Prebiotics that can be used according to the invention are preferably oligosaccharides, in particular selected from galacto-oligosaccharides, sialic acid oligosaccharides, lactulose, lactosucrose, fructo-oligosaccharides, palatinose or isomalto-oligosaccharides, saccharosylsucrose, malto-oligosaccharides, isomalto-oligosaccharides, cyclodextrins, gentio-oligosaccharides, soy-oligosaccharides, xylo-oligosaccharides, glucans, pectins, polygalacturonic acid, rhamnogalacturonic acid, mannans, hemicelluloses, arabinogalactans, arabinans, arabinoxylans, resistant starch, mehbiose, chitosan, agarose, inulin, tagatose, polydextrose and alginate.

[0192] The enzymes that can be used in the feed composition according to the invention and that can contribute to the digestion of the feed are preferably selected from the group consisting of phytases (EC 3.1.3.8 or 3.1.3.26), xylanases (EC 3.2.1.8), galactanases (EC 3.2.1.89), galactosidases, in particular α-galactosidases (EC 3.2.1.22), proteases (EC 3.4), phospholipases, in particular phospholipases A1 (EC 3.1.1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3) and D (EC 3.1.4.4), lysophospholipases (EC 3.1.1.5), amylases, in particular α-amylases (EC 3.2.1.1); lysozymes (EC 3.2.1.17), glucanases, in particular β-glucanases (EC 3.2.1.4 or EC 3.2.1.23). 3.2.1.6), glucoamylase, cellulase, pectinase or any mixture thereof.

[0193] Examples of commercially available phytases include Bio-Feed TM Phytase (Novozymes), P and HiPhos TM (DSM Nutritional Products), NatuphosTM (BASF), and Blue(AB Enzymes), XP (Verenium / DuPont) and PHY (DuPont). Other preferred phytases include those described in, for example, WO 98 / 28408, WO 00 / 43503 and WO 03 / 066847.

[0194] Examples of commercially available xylanases include WX and G2 (DSM NutritionalProducts), XT and Barley (AB Vista), (Verenium) and XB (xylanase / β-glucanase, DuPont). Examples of commercially available proteases include ProAct (DSM Nutritional Products).

[0195] Vitamins that can be used according to the invention are, for example, vitamin A, vitamin D3, vitamin E, vitamin K, for example vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenic acid, for example Ca-D-pantothenic acid, or combinations thereof.

[0196] Immunomodulators that may be used are, for example, antibodies, cytokines, spray-dried plasma, interleukins or interferons, or combinations thereof.

[0197] Minerals that can be used according to the invention are, for example, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, phosphorus, magnesium, potassium or sodium, or combinations thereof.

[0198] Amino acids which can be used according to the invention are, for example, lysine, alanine, threonine, methionine or tryptophan, or a combination thereof.

[0199] The feed of the present invention may further comprise betaine and / or choline and / or other physiologically effective methyl donors. The feed may further comprise polyunsaturated fatty acids, in particular DHA and / or EPA.

[0200] Therefore, another embodiment of the present invention is a method for preparing an animal feed composition, which comprises mixing at least one strain and / or at least one preparation and / or at least one concentrate composition of the present invention, in particular in an amount effective to enhance animal health (in particular intestinal health), with feed ingredients (such as proteins, lipids and / or carbohydrates), and optionally other beneficial substances (preferably as described above), to obtain a feed product. The method may also comprise, for example, a pelleting step.

[0201] Standard granulation methods known to those skilled in the art may be used, including extrusion processing of dry or semi-wet feeds.Preferred granulation temperatures are from about 65°C to about 120°C.

[0202] The bacterial strain and composition of the present invention can be obtained by culturing the bacterial strain of the present invention according to methods well known in the art, including by using, for example, the culture medium and other methods described in US 6,060,051, EP0287699 or US2014 / 0010792. Conventional large-scale microbial culture methods include deep fermentation, solid-state fermentation or liquid surface culture. When the fermentation is near the end, as nutrients are exhausted, the cells of the bacterial strain begin to transition from the growth phase to the sporulation phase, so that the final product of the fermentation is mostly spores, metabolites and residual fermentation medium. Spore formation is part of the natural life cycle of these bacterial strains, and is usually initiated by cells in response to nutritional limitations. Fermentation is configured to obtain high-level Bacillus subtilis colony-forming units and promote sporulation. The bacterial cells, spores and metabolites in the culture medium produced by fermentation can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, deep filtration and evaporation. The concentrated fermented broth can be washed, for example, via a diafiltration process to remove residual fermented broth and metabolites.

[0203] The fermentation broth or fermentation broth concentrate can be dried using conventional drying techniques or methods (e.g., spray drying, freeze drying, tray drying, fluidized bed drying, drum drying, or evaporation) with or without the addition of a carrier. The resulting dried product can be further processed, for example by grinding or granulation, to obtain a particular particle size or physical form. As described above, a carrier can also be added after drying.

[0204] The preparation of the strain of the present invention may be a cell-free preparation or a preparation containing cell fragments or a preparation containing a mixture of intact cells and cell fragments.

[0205] The cell-free preparation of bacterial strain of the present invention can be obtained, for example, by centrifugation and / or filtration fermentation broth. Depending on the technology used, these cell-free preparations may not be completely free of cells, but may still comprise a relatively small amount of cells. Because the cell secretes compounds such as metabolites, enzymes and / or peptides into the surrounding culture medium, the supernatant of the cell comprises a mixture of such compounds, particularly metabolites, enzymes and / or peptides secreted by the cells. Therefore, in a preferred embodiment of the present invention, the preparation of bacterial strain is the supernatant of fermentation broth. The preparation of bacterial strain, particularly supernatant, can also be used in dry form, wherein drying can be, for example, carried out by spray drying or freeze drying.

[0206] Compositions comprising bacterial strain cell fragments can be obtained by applying technology known to those skilled in the art, for example, by mechanical methods or by applying high pressure to break cells. According to the degree of force applied, obtain compositions comprising only broken cells or compositions comprising a mixture of cell fragments and intact cells. Homogenization of cells can be achieved, for example, by using a French cell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill, gravel mill, bead mill, high pressure grinding roller, vertical shaft impactor, industrial mixer, high shear mixer, paddle mixer and / or polytron homogenizer. Suitable alternatives are enzymatic and / or chemical treatments of cells.

[0207] The cell-free preparations of the invention also encompass preparations obtained by first disrupting cells using the techniques described above and subsequently removing cell debris and remaining intact cells. Removal of cell debris and remaining intact cells can be performed in particular by centrifugation and / or filtration.

[0208] The preparation of the strain of the invention may comprise as active compound at least one metabolite, preferably a mixture of metabolites as further described below, and / or at least one enzyme selected from proteases, in particular subtilisins, xylanases and / or cellulases, and / or at least one peptide, and / or combinations thereof.

[0209] Preparations containing an effective mixture of metabolites contained in the strains and / or cell preparations of the present invention as described above can be obtained, for example, according to the method described in U.S. Pat. No. 6,060,051. In particular, the preparation can be obtained by precipitating the metabolites contained in the aforementioned preparation using an organic solvent such as ethyl acetate and then redissolving the precipitated metabolites in a suitable solvent. The metabolites can then be purified by size exclusion filtration that groups the metabolites into different fractions based on the molecular weight cutoff.

[0210] The preparation containing the effective mixture of metabolites of the present invention preferably comprises at least five, more preferably at least 6, 7, 8, 9, 10 or 12, in particular all metabolites of each strain of the present invention. The metabolites preferably have a molecular weight of 400-5000 Daltons, more preferably 800-4000 Daltons.

[0211] Preferably, according to the present invention, an effective amount of the strain and / or preparation and / or composition of the present invention is always used in the embodiments of the present invention. The term "effective amount" refers to an amount that produces at least one beneficial effect on the animal and / or the environment compared to an animal not administered with the strain and / or preparation and / or composition of the present invention, but administered with the same diet (including feed and other compounds), in particular with respect to the characteristics as described above.

[0212] In the case of therapeutic applications, it is preferred to use a therapeutic amount of the strain and / or formulation and / or composition of the invention. The term "therapeutic amount" refers to an amount sufficient to improve, reverse or prevent a disease state in an animal. The optimal dosage level for various animals can be easily determined by those skilled in the art by evaluating, inter alia, the ability of the composition to: (i) inhibit or reduce pathogenic bacteria in the intestine or gills at different doses, (ii) increase or maintain the level of beneficial bacteria and / or (iii) enhance animal health (particularly intestinal health) at different doses.

[0213] Working Example

[0214] Example 1: Ability of strains to reduce nitrogen

[0215] Bacillus strains were screened for water remediation properties from different environmental samples. In order to obtain excellent strains for aquaculture bioremediation, various experiments were performed to determine the ability to reduce nitrogen compounds under a wide range of conditions. The strains were tested at different salinities (0-3%) and different C:N ratios (20-2600). Four Bacillus subtilis strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 showed very promising properties, as described below.

[0216] All nitrite (NO2-N) measurements were performed using Nitrite cuvette test 0.015-0.6 mg / L NO2-N and Nitrite cuvette test 0.6-6.0 mg / L NO2-N (Hach Company, Loveland, Colorado, USA). All ammonia (NH4+-N) concentrations were measured by flow injection analyzer (FIA, application system: FOSS FIAstar 5000). Within the system, sample aliquots were alkalized to quantitatively generate ammonia (NH3) from solvated ammonium ions (NH4+). Ammonia diffused through a semipermeable membrane (gas diffusion). The pH changes can then be monitored by an indicator solution (photometric detection, indicator based on the application of 5',5"-dibromo-o-cresolsulfonylphthalide ("Bromkresolpurpur", Sigma-Aldrich, St. Louis, Missouri, USA)). The extinction increases nonlinearly with increasing ammonia concentration and requires appropriate calibration. The effective measuring range of this method is 1-13 mg / L (linear range) and 10-130 mg / L (non-linear range).

[0217] At different salinities, the ammonia reduction ability of all strains was tested in C minimal medium containing only glucose as the carbon source and 5ppm ammonia as the sole nitrogen source. For each Bacillus subtilis strain, 50μl of glycerol stock was inoculated into 10ml C minimal medium (0.0352g / l MnSO4×1H2O, 2.46g / l MnSO4×7H2O, 0.2KH2PO4, 0.6g / l K2HPO4, 0.02FeSO4×7H2O, 0.2792g / l (NH4)2SO4, 10g / l glucose and 0 or 15 or 30g / lNaCl) with a C:N ratio of 68 and incubated in a 100ml shake flask at 28°C and 200rpm. After incubation for 48 hours, the concentration of ammonia was measured using the above method. The percentage amount of ammonia reduced by each strain was calculated with reference to the initial concentration, which was determined in the uninoculated control of each salinity (Table 1.1). Strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to effectively remove ammonia in C minimal medium containing only glucose as carbon source and 5 ppm ammonia as nitrogen source, as a maximum of 0.3% of the initial ammonia concentration was still detectable after 48 h under all salinity conditions.

[0218] Table 1.1 Ammonia reduction in C minimal medium containing only glucose as carbon source and 5 ppm ammonia as nitrogen source at different salinities

[0219]

[0220] All strains were further tested for their ability to reduce nitrite in C minimal medium containing only glucose as a carbon source and 5ppm nitrite as a nitrogen source. For each Bacillus subtilis strain, 50μl of glycerol stock was inoculated into 10ml C minimal medium (0.0352g / l MnSO4×1H2O, 2.46g / l MnSO4×7H2O, 0.2KH2PO4, 0.6g / l K2HPO4, 0.02FeSO4×7H2O, 0.0075g / l NaNO2, 10g / l glucose and 0 or 15 or 30g / l NaCl) with a C:N ratio of 2600 and incubated in a 100ml shake flask at 28°C and 200rpm. After incubation for 48 hours, the concentration of nitrite was measured using the above method. The percentage of nitrite reduced by each strain was calculated with reference to the initial concentration, which was determined in the uninoculated control of each salinity (Table 1.2). At 0 and 15% salinity, strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to effectively remove all nitrite within 48 hours. At 3% salinity, strains DSM 33351 and DSM 33350 reduced nitrite to 0 mg / ml, while strains DSM 33352 and DSM 33349 removed 67% and 87%, respectively, with a reference initial concentration of 5.15 mg / l.

[0221] Table 1.2 Nitrite reduction in C minimal medium containing only glucose as carbon source and 5 ppm nitrite as nitrogen source at different salinities

[0222]

[0223] All strains were tested for their ability to reduce ammonia and nitrite in C minimal medium containing only glucose as carbon source and both nitrite and ammonia at a final concentration of 2.5 ppm and a C:N ratio of 20. A single colony of strain DSM 33351, DSM 33352, DSM 33349 or DSM 33350 grown on TSA agar at 37°C for 24 h was inoculated in 10 ml of the corresponding medium (0.0352 g / l MnSO4×1H2O, 2.46 g / l MnSO4×7H2O, 0.2 KH2PO4, 0.6 g / l K2HPO4, 0.02 FeSO4×7H2O, 0.1396 g / l (NH4)2SO4, 0.00375 g / l NaNO2, 1.517 g / l glucose and 0 or 15 or 30 g / l NaCl). The culture was incubated at 28°C with 200rpm. After 48 hours, the concentration of ammonia and nitrite was measured by the above method. The amount of ammonia and nitrite that each bacterial strain reduced was calculated with reference to the initial concentration, which was measured in the uninoculated control of each salinity (Table 1.3). Bacteria strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to remove 99.7% of the initial ammonia and 100% of the initial nitrite simultaneously after 48 hours at all salinities in the C minimal medium containing only glucose as a carbon source and a final concentration of 2.5ppm nitrite and ammonia as a nitrogen source.

[0224] Table 1.2 Reduction of ammonia and nitrite in C minimal medium containing only glucose as carbon source and nitrite and ammonia at a final concentration of 2.5 ppm as nitrogen source at different salinities

[0225]

[0226] Overall, these data indicate that Bacillus subtilis strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 can use both ammonia and nitrite as sole nitrogen sources and effectively remove both compounds within 48 hours.

[0227] Example 2: Outgrowth and Nitrogen Reduction of Spores in C Minimal Medium

[0228] The ability of Bacillus strains to produce spores is a major advantage in obtaining long-term stable products.

[0229] To produce spores of Bacillus subtilis DSM 33351, DSM 33352, DSM 33349 and DSM 33350, the strains were pre-grown for 14 hours at 30°C in 50 g of medium containing 43 g / kg soy flour and 22 g / kg sugar solution (320 g / kg glucose, 90 g / kg fructose, 350 g / kg maltose and 100 g / kg maltotriose) and shaking at 200 rpm. Next, 14 g of the previous culture was inoculated into 200 g of spore-forming medium and incubated at 30°C while shaking at 200 rpm. The sporulation medium consisted of 21 g / kg of the above sugar solution, 43 g / kg soy flour, 0.29 g / kg MgSO4×7H2O, 0.032 g / kg MnSO4×H2O, 0.023 g / kg FeSO4×7H2O, and 0.003 g / kg ZnSO4×7H2O. After 48 hours of incubation, spores were harvested by centrifugation and resuspended in PBS buffer containing 20% ​​glycerol and stored at -80°C.

[0230] Spores DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were tested for their potential to grow out in C minimal medium with ammonia and nitrite as nitrogen sources and their ability to effectively reduce ammonia and nitrite.

[0231] To test the outgrowth in a microtiter plate-based assay, 10 of the strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were cultured in PBS. 6 CFU / ml spores were inoculated into the corresponding C minimal medium in a final volume of 150 μl ml, with glucose as the sole carbon source and ammonia and nitrite as nitrogen sources (0.0352 g / l MnSO4×1H2O, 2.46 g / l MnSO4×7H2O, 0.2 KH2PO4, 0.6 g / l K2HPO4, 0.02 FeSO4×7H2O, 0.1396 g / l (NH4)2SO4, 0.00375 g / l NaNO2 and 10 g / l glucose (C:N ratio=20)) and supplemented with 10% PrestoBlue TM (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The spores were plated in a microtiter plate reader (TECAN M1000 Pro) at 37°C with 350 rpm orbital shaking for 30 h. Spore outgrowth was detected by the metabolic activity of growing cells, which would convert PrestoBlue TMThe non-fluorescent resazurin is reduced to bright red fluorescent resorufin. Fluorescence is measured in real time during the culture (excitation filter: 550-12 nm; emission filter: 590 nm). The resulting kinetics are used to calculate the time point of spore growth halo based on a defined threshold. The number of hours until growth halo for strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 are described in Table 2.1. All strains were able to grow in C minimal medium containing only glucose as a C source and a final concentration of 2.5 ppm nitrite and ammonia within 23.35 h, while DSM 33349 grew the fastest at 18.89 h.

[0232] Table 2.1 Growth of spores in C minimal medium containing only glucose as carbon source and nitrite and ammonia at a final concentration of 2.5 ppm as nitrogen sources.

[0233] strain Time until growth halo [h] DSM 33351 23.35 DSM 33352 21.14 DSM 33349 18.89 DSM 33350 20.00

[0234] The ability of spores of strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 to remove ammonia and nitrite in C minimal medium containing only glucose as carbon source and nitrite and ammonia at a final concentration of 2.5 ppm each was also tested. In a 100 ml shake flask, 10 ml of the corresponding medium (0.0352 g / l MnSO4×1H2O, 2.46 g / l MnSO4×7H2O, 0.2 KH2PO4, 0.6 g / l K2HPO4, 0.02 FeSO4×7H2O, 0.1396 g / l (NH4)2SO4, 0.00375 g / l NaNO2 and 10 g / l glucose (C:N ratio=20)) was inoculated with 10 spores of DSM 33351, DSM 33352, DSM 33349 and DSM 33350. 7 CFU / ml of spore stock. The culture was incubated at 28°C and 200rpm. After 52 hours, the concentrations of ammonia and nitrite were determined using the above method. The amount of ammonia and nitrite reduced by each strain was calculated with reference to the initial concentration measured in the uninoculated control (Table 2.2). Ammonia and nitrite were effectively removed from the flask inoculated with the spores of strains DSM 33351, DSM 33352, DSM33349 and DSM 33350. These strains were able to simultaneously remove at least 99.6% of ammonia and 100% of nitrite within 52 hours.

[0235] Table 2.2 Reduction of ammonia and nitrite in C minimal medium inoculated with spores containing only glucose as C source and nitrite and ammonia as nitrogen sources at a final concentration of 2.5 ppm

[0236]

[0237] These data indicate that spores of strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 are not only able to germinate and grow in C minimal medium containing 2.5 ppm nitrite and ammonia as the sole nitrogen sources, but they are also able to remove 2.5 ppm each of nitrite and ammonia within 52 hours.

[0238] Example 3: Ability of strains to inhibit pathogens

[0239] The ability to inhibit different pathogens important in aquaculture was analyzed using the well diffusion antagonism test (Parente et al., 1995). The pathogens tested in these assays were Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnaris DSM 25092.

[0240] Vibrio are known to be associated with disease and high mortality in shrimp, but can also infect fish (Chatterjee and Haldar, 2012). Tilapia infections can be associated with Streptococcus agalactiae, a widespread bacterium, which can cause, for example, bleeding or swimming instability (Mishra et al., 2018). Columnaris disease is caused by the infection of freshwater fish with Flavobacterium columnaris. For example, salmon and trout farms report high annual losses (Pulkkinen et al., 2010).

[0241] Pathogen grows in liquid medium under appropriate conditions until the optical density of OD600 reaches at least 1.0. With sterile spatula, pathogen is inoculated on the surface of Caso Yeast agar plate. Cut out holes with a diameter of 9mm in dry plates. Bacterial strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were grown for 16 hours at 37°C and 200rpm in 100ml shake flasks in LB-Kelly (LB medium supplemented with the trace element solution of DSMZ medium 1032). The culture of bacterial strain DSM 33351, DSM 33352, DSM 33349 and DSM33350 adjusted as control and 100 μl OD5.0 was used to fill the cut-out holes. Plates were incubated under appropriate conditions, and the clearance zone (in mm) from the edge of the cut-out hole to the border of the bacterial lawn removed was measured. The area was measured horizontally and vertically, and averaged. Low (+), medium (++), high (+++) and very high (++++) inhibition scores were applied according to Table 3.1.

[0242] Table 3.1 Pathogen inhibition scores based on pore size and clearance zone in pore diffusion antagonism assays

[0243] score Aperture + clearance area [mm] Low (+) ≤9.5mm Medium (++) >9.5≤15 High (+++) >15≤25 Very high (++++) >25≤30

[0244] The results of the inhibition of pathogens by strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 are shown in Tables 3.2 to 3.6, respectively.

[0245] Table 3.2 Inhibitory ability of Bacillus subtilis DSM 33351 against different pathogens in the well diffusion antagonism test on Caso Yeast medium. The inhibitory strength was scored according to Table 4.1.

[0246] Pathogens Inhibition strength Vibrio harveyi DSM 19623 + Vibrio parahaemolyticus DSM 10027 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++

[0247] The data showed that Bacillus subtilis DSM 33351 could inhibit the growth of Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187 and Streptococcus agalactiae DSM 2134.

[0248] Table 3.3 Inhibitory ability of Bacillus subtilis DSM 33352 against different pathogens in the well diffusion antagonism test on Caso Yeast medium. The inhibitory strength was scored according to Table 5.1.

[0249] Pathogens Inhibition strength Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 +++

[0250] The data showed that Bacillus subtilis DSM 33352 could inhibit the growth of Aeromonas hydrophila DSM 30187 and Streptococcus agalactiae DSM2134.

[0251] Table 3.4 Inhibitory ability of Bacillus subtilis DSM 33349 against different pathogens in the well diffusion antagonism test on Caso Yeast medium. The inhibitory strength was scored according to Table 6.1.

[0252] Pathogens Inhibition strength Vibrio harveyi DSM 19623 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++ Flavobacterium columnaris DSM 25092 +

[0253] The data showed that Bacillus subtilis DSM 33349 could inhibit the growth of Vibrio harveyi DSM 19623, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnaris DSM 25092.

[0254] Table 3.4 Inhibitory ability of Bacillus subtilis DSM 33350 against different pathogens in the well diffusion antagonism test on Caso Yeast medium. The inhibition strength was scored according to Table 7.1.

[0255] Pathogens Inhibition strength Vibrio harveyi DSM 19623 + Vibrio parahaemolyticus DSM 10027 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++ Flavobacterium columnaris DSM 25092 +

[0256] The data showed that Bacillus subtilis DSM 33350 could inhibit the growth of Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnaris DSM 25092.

[0257] literature

[0258] Parente,E.,Brienza,C.,Moles,M.,&Ricciardi,A.(1995).A comparison ofmethods for the measurement of bacteriocin activity.Journal ofmicrobiological methods,22(1),95-108.

[0259] Chatterjee,S.and Haldar,S.(2012).Vibrio Related Diseases inAquaculture and Development of Rapid and Accurate IdentificationMethods.J.Marine Sci.Res.Dev.S1:002.doi:10.4172 / 2155-9910.S1-002.

[0260] Pulkkinen,K.,Suomalainen,L.-R.,Read,AF,Ebert,D., P.andValtonen,ET(2010).Intensive fish farming and the evolution of pathogenvirulence: the case of columnaris disease in Finland.Proc.R.Soc.B.277,593–600.

[0261] Mishra,A.,Nam,G.H.,Gim,J.A.,Lee,H.E.,Jo,A.and Kim,H.S.(2018).CurrentChallenges of Streptococcus Infection and Effective Molecular,Cellular,andEnvironmental Control Methods in Aquaculture.Mol.Cells.41(6):495-505

Claims

1. A Bacillus subtilis strain or a preparation thereof, wherein the Bacillus subtilis strain or the preparation thereof is selected from the following group: a) the Bacillus strain deposited with DSMZ under the number DSM 33349; b) the Bacillus strain deposited with DSMZ under the number DSM 33350; c) the Bacillus strain deposited with DSMZ under the number DSM 33351; d) Bacillus strains deposited with DSMZ under number DSM 33352; e) A preparation of a strain according to any one of a) to d).

2. A composition comprising the Bacillus strain or its preparation according to claim 1, wherein the Bacillus strain and its preparation are selected from the group consisting of: a) Bacillus strains deposited with DSMZ under one of the following numbers: DSM 33349, DSM 33350, DSM 33351 and DSM 33352; b) Preparation of the strain according to a).

3. Use of the Bacillus strain or preparation according to claim 1 or the composition according to claim 2 for reducing the amount of inorganic nitrogen compounds in a water system, wherein the inorganic nitrogen compound is selected from ammonium, nitrite and nitrate and combinations thereof.

4. Use of the Bacillus strain or preparation according to claim 1 or the composition according to claim 2 as a probiotic ingredient (DFM) in a feed or food product.

5. A feed composition or food composition comprising the Bacillus strain or preparation thereof according to claim 1 or the composition according to claim 2 and at least one additional feed or food ingredient selected from the group consisting of proteins, carbohydrates, fats, other probiotics, prebiotics, enzymes, vitamins, milk replacers, minerals, amino acids, and combinations thereof.

6. A pharmaceutical composition comprising the Bacillus strain or its preparation according to claim 1 or the composition according to claim 2 and a pharmaceutically acceptable carrier.

7. A method for reducing the amount of inorganic nitrogen compounds in a water system and / or controlling the amount of inorganic nitrogen compounds in a water system, the method comprising supplying the water system with the strain and / or preparation according to claim 1 and / or the composition according to any one of claims 2, 5 or 6, wherein the inorganic nitrogen compound is selected from ammonium, nitrite and nitrate, and wherein the water system is breeding water.

8. Use of the Bacillus strain or the preparation thereof according to claim 1 and / or the composition according to claim 2 and / or the feed composition according to claim 5 and / or the pharmaceutical composition according to claim 6 for preparing feed for feeding aquatic animals.

9. The use according to claim 8, wherein the aquatic animal is a crustacean or a finfish.

10. Use of the strain and / or preparation according to claim 1 and / or the composition according to any one of claims 2, 5 or 6 in the preparation of feed, wherein the feed is used to enhance the health of animals and / or improve the overall physical condition of animals and / or increase the feed conversion rate of animals and / or reduce the mortality rate of animals and / or increase the survival rate of animals and / or improve the weight gain of animals and / or improve the disease resistance of animals and / or regulate the immune response of animals and / or establish or maintain a healthy intestinal microbial community in animals and / or improve the meat quality of animals and / or reduce the excretion of pathogens through animal feces; and, wherein the animal is an aquatic animal.

11. The use according to claim 10, wherein the aquatic animal is a finfish or a crustacean.

12. The use according to claim 10, wherein the aquatic animal is tilapia or shrimp.

13. A method for controlling and / or improving the quality of water or an aqueous solution, the method comprising the step of applying the strain and / or the preparation according to claim 1 and / or the composition according to any one of claims 2, 5 or 6 to water or an aqueous solution.

14. The method according to claim 13, wherein the water is drinking water, breeding water, circulating water or waste water.

15. A method for controlling and / or avoiding harmful environmental effects of manure or contaminated liquids, the method comprising the step of applying the strain according to claim 1 and / or its preparation and / or the composition according to any one of claims 2, 5 or 6 to manure or contaminated liquids.

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