Bacillus subtilis strains with probiotic activity

Abstract

The invention relates to a novel bacillus subtilis (B.subtilis) with a strong inhibition effect on clostridium perfringens (C. perfringens), and an application of the bacillus subtilis as a probiotic.

Description

[0001] This application is a divisional application of the patent application filed on May 31, 2017, with application number 201710398521.0 and titled "Bacillus subtilis strain with probiotic activity".

[0002] This invention relates to a novel strain of Bacillus subtilis with a strong inhibitory effect on Clostridium perfringens and its use as a probiotic.

[0003] The use of Bacillus subtilis strains as a probiotic ingredient in the feed industry is well known in the art. Probiotics (also known as "direct feed microbes" or "DFM") function by positively influencing the gut microbiota by supporting the growth of beneficial bacteria and / or inhibiting the growth of pathogenic bacteria. Ideally, the use of antibiotic growth promoters (APGs) becomes redundant through the use of probiotics. In addition, it is desirable for probiotics to fulfill additional functions, such as aiding in the digestion of specific feed ingredients.

[0004] Therefore, given the current state of the technology, there is a need for probiotics that can positively influence the gut microbiota and ideally fulfill at least one other function.

[0005] Surprisingly, the bacteria according to the invention have been found to exhibit many advantageous characteristics. In addition to the ability to inhibit the growth of Clostridium perfringens (a major commercially relevant pathogen in poultry), they have shown a particularly high reproduction rate in the presence of bile and aid in the digestion of cellulose in a very efficient manner.

[0006] Bacillus subtilis DSM 32315 has been identified by screening naturally occurring isolates. It has been deposited in DSMZ on May 12, 2016, in the name of Evonik Degussa GmbH under the aforementioned accession number, in accordance with the Budapest Convention on the International Recognition of the Preservation of Microorganisms for Patent Proceedings.

[0007] Therefore, the first subject of this invention is Bacillus subtilis strains selected from the group consisting of and / or preparations of said Bacillus subtilis strains:

[0008] a) Bacillus subtilis strain preserved in DSMZ as DSM 32315;

[0009] b) A mutant strain of Bacillus subtilis preserved in DSM 32315, having all the identification characteristics of strain DSM 32315, wherein the mutant strain preferably has at least 95%, preferably at least 96, 97 or 98%, more preferably at least 99 or 99.5% DNA sequence identity with strain DSM 32315.

[0010] c)(a) or (b) preparations;

[0011] d) Preparations containing an effective mixture of metabolites as described in (a), (b) or (c).

[0012] The Bacillus subtilis strain DSM 32315 deposited in DSMZ exhibits the following characteristic sequence:

[0013] a) A 16S rDNA sequence having at least 99.5%, especially 100%, sequence identity with the polynucleotide sequence according to SEQ ID NO:1;

[0014] b) The yqfD sequence has at least 99.5%, especially 100%, sequence identity with the polynucleotide sequence according to SEQ ID NO:2;

[0015] c) The gyrB sequence has at least 99.5%, especially 100%, sequence identity with the polynucleotide sequence according to SEQ ID NO:3;

[0016] d) The rpoB sequence has at least 99.5%, especially 100%, sequence identity with the polynucleotide sequence according to SEQ ID NO:4;

[0017] e) The groEL sequence has at least 99.5%, especially 100%, sequence identity with the polynucleotide sequence according to SEQ ID NO:5.

[0018] Therefore, another subject of the present invention is Bacillus subtilis strains or preparations thereof, particularly Bacillus subtilis strains having the above-described characteristics, exhibiting at least one, preferably all, of the following characteristics:

[0019] a) The 16S rDNA sequence has at least 99%, preferably at least 99.5%, more preferably at least 99.8% or 99.9%, especially 100% sequence identity with the polynucleotide sequence according to SEQ ID NO:1;

[0020] b) The yqfD sequence has at least 99%, preferably at least 99.5%, more preferably at least 99.8% or 99.9%, and especially 100% sequence identity with the polynucleotide sequence according to SEQ ID NO:2;

[0021] c) The gyrB sequence has at least 99%, preferably at least 99.5%, more preferably at least 99.8% or 99.9%, and especially 100% sequence identity with the polynucleotide sequence according to SEQ ID NO:3.

[0022] Preferably, this Bacillus subtilis strain exhibits at least one, more preferably all of the following further characteristics:

[0023] d) The rpoB sequence has at least 99%, preferably at least 99.5%, more preferably at least 99.8% or 99.9%, and especially 100% sequence identity with the polynucleotide sequence according to SEQ ID NO:4;

[0024] e) The groEL sequence has at least 99%, preferably at least 99.5%, more preferably at least 99.8% or 99.9%, especially 100% sequence identity with the polynucleotide sequence according to SEQ ID NO:5.

[0025] Therefore, a particular subject of this invention is still Bacillus subtilis strains, which exhibit the following characteristics:

[0026] a) Based on the 16S rDNA sequence of SEQ ID NO:1;

[0027] b) Based on the yqfD sequence of SEQ ID NO:2;

[0028] c) Based on the gyrB sequence of SEQ ID NO:3.

[0029] Preferably, this Bacillus subtilis strain exhibits the following further characteristics:

[0030] d) Based on the rpoB sequence of SEQ ID NO:4;

[0031] e) Based on the groEL sequence of SEQ ID NO:5.

[0032] Preferably, the strain of the present invention is characterized by at least one, more preferably all of the following further features:

[0033] They are preferably capable of growing under anaerobic conditions. Furthermore, they are preferably capable of degrading water-insoluble cellulose under such anaerobic conditions.

[0034] They are preferably highly effective at inhibiting infectious bacteria, particularly Clostridium perfringens. In particular, they are preferably characterized by pathogen clearance of at least 10 mm, more preferably at least 13 mm, in a well diffusion antagonism test of Clostridium perfringens strain ATCC 13124 on LB Kelly agar plates.

[0035] The spores are preferably viable at low pH levels, and are more preferably viable when exposed to pH levels as low as 4.0, particularly as low as 3.0, and more preferably as low as 2.0 for at least one hour.

[0036] Preferably, the strain of the present invention is further characterized in that it is capable of growing in the presence of 0.05 wt.-% acetic acid, 0.05 wt.-% propionic acid and / or 0.2 wt.-% lactic acid.

[0037] Preferably, it is further characterized by a cellulase activity of at least 200 mU / mL, more preferably at least 300 mU / mL, particularly at least 350 mU / mL, especially about 369 mU / mL, and a xylanase activity of at least 15 mU / mL, more preferably at least 20 mU / mL, especially at least 25 mU / mL, especially about 33 mU / mL.

[0038] Preferably, the Bacillus subtilis strain of the present invention is further characterized in that it is capable of growing in the presence of 2 mM bile, preferably in the presence of 4 mM bile. In particular, it is characterized by, preferably, achieving an AUC5 performance value of at least 0.5, preferably at least 0.65, especially at least 0.8, particularly about 0.88, and an AUC10 performance value of at least 1.2, preferably at least 1.4, especially at least 1.6, particularly about 1.7, in the presence of 2 mM bile.

[0039] Furthermore, the strain is preferably capable of growing under high-salt conditions, particularly in the presence of 5 wt.% NaCl, for at least one day.

[0040] Furthermore, the strains of the present invention preferably survive at the high temperatures required for animal feed pelleting, particularly preferably at a temperature of 80°C for at least 20 minutes. Without being bound by theory, it is believed that the Bacillus subtilis strains according to the present invention enhance animal health through multiple mechanisms, including the production of selectively effective antibacterial metabolites and the inhibition of the effective establishment of pathogenic bacteria in the gut by better consuming available nutrients and competing with pathogenic bacteria.

[0041] Compared to antibiotics, a key advantage of probiotics is that they do not indiscriminately destroy bacteria, nor do they lead to antibiotic-resistant strains of pathogenic bacteria. Typically, they selectively compete with pathogenic bacteria by producing antimicrobial substances with specific efficacy, and ideally, they simultaneously enhance the growth and survival of beneficial gut microbiota. Furthermore, they preferably stimulate a systemic immune response in treated animals.

[0042] The DSM 32315 mutant strains of the present invention are preferably spontaneous mutant strains. The term "spontaneous mutant strain" refers to a mutant strain derived from DSM 32315 without the intentional use of a mutagen. Such spontaneous mutant strains can be obtained by conventional methods, such as growing Bacillus subtilis strains in the presence of UV light or in the presence of a specific antibiotic to which the parent is susceptible, and testing any resistant mutant strain for enhanced bioactivity or ability to enhance one or more animal health markers. Other methods for identifying spontaneous mutant strains are known to those skilled in the art. However, in addition to these preferred spontaneous mutant strains, all other types of DSM 32315 mutant strains, such as those obtained through genetic engineering, are also included in this invention.

[0043] A specific embodiment of the present invention is a naturally occurring mutant strain of strain DSM 32315 characterized by the above-described features.

[0044] In a preferred embodiment of the invention, the strains and preparations of the invention are preferably administered orally to animals or humans.

[0045] Therefore, another subject of the present invention is compositions containing Bacillus subtilis and / or preparations of the present invention, such as feed, food, beverages and water for feeding, as well as therapeutic compositions.

[0046] Another subject of the present invention is the use of Bacillus subtilis and / or its preparations as a probiotic component (DFM) in feed or food.

[0047] Preferred foods according to the invention are dairy products, particularly yogurt, cheese, milk, butter, and low-fat soft cheese (quark).

[0048] The cells of the strains of the present invention may exist as spores (which are dormant), vegetative cells (which are growing), transitional cells (which are transitioning from the growth phase to the spore-forming phase), or at least two of these types, particularly combinations of all these cell types, especially in the compositions of the present invention. In a preferred embodiment, the compositions of the present invention comprise primarily or only spores.

[0049] Preferably, when administered to animals, the Bacillus subtilis strains of the present invention and compositions containing them enhance the health of these animals and / or improve their general physical condition and / or improve their feed conversion ratio and / or reduce their mortality and / or improve their survival rate and / or improve their weight gain and / or improve their productivity and / or improve their disease resistance and / or improve their immune response and / or establish or maintain a healthy gut microbiota and / or reduce pathogen shedding through their feces. In particular, the strains and compositions of the present invention can be used to help restore a healthy balance to the gut microbiota after antibiotic administration for therapeutic purposes.

[0050] Therefore, another subject of the present invention is a method for enhancing animal health and / or improving the general physical condition of animals and / or improving feed conversion ratio and / or reducing animal mortality and / or improving animal survival rate and / or improving animal weight gain and / or improving animal productivity and / or improving animal disease resistance and / or improving animal immune response and / or establishing or maintaining a healthy gut microbiota and / or reducing pathogen shedding through animal feces, wherein the animals are administered the strains and / or preparations of the present invention or compositions of the present invention containing such strains.

[0051] Therefore, another subject of the present invention is the use of the strains and / or preparations and / or compositions of the present invention for enhancing animal health and / or improving the general physical condition of animals and / or improving feed conversion ratios and / or reducing animal mortality and / or improving animal survival and / or improving animal weight gain and / or improving animal productivity and / or improving animal disease resistance and / or improving animal immune response and / or establishing or maintaining a healthy gut microbiota and / or reducing pathogen shedding through animal feces, wherein the strains and / or preparations of the present invention or compositions of the present invention comprising these strains are administered to the animals.

[0052] Another subject of the present invention is, therefore, the strains and preparations of the present invention as described above, and compositions of the present invention containing those strains, for enhancing animal health and / or for improving the general physical condition of animals and / or for improving animal feed conversion ratio and / or for reducing animal mortality and / or for improving animal survival rate and / or for improving animal weight gain and / or for improving animal productivity and / or for improving animal disease resistance and / or for improving animal immune response and / or for establishing or maintaining a healthy gut microbiota of animals and / or for reducing pathogen shedding through animal feces.

[0053] "Increasing animal productivity" specifically refers to any of the following: producing more or higher quality eggs, milk or meat, or increasing the production of weaned offspring.

[0054] The strains, preparations, and compositions of the present invention can be used in a therapeutic or non-therapeutic manner. In a particularly preferred embodiment of the invention, the methods and uses are non-therapeutic, particularly for animal husbandry applications.

[0055] Because untreated animal manure has harmful environmental effects due to pathogenic bacteria and other components, particularly concerning the animals themselves and / or the people exposed to it, this can be avoided by feeding animals or directly treating animal manure or bedding with the strains, compositions, or preparations of the present invention. Therefore, another subject of the present invention is a method for controlling and / or avoiding the harmful environmental effects of manure or contaminated liquids, comprising the step of applying at least one strain, preparation, and / or composition according to the present invention to manure, contaminated liquids, bedding, pits, or manure pools. Preferably, the composition is applied in liquid form, for example, by spraying, or as a powder, for example, by spreading.

[0056] Because harmful bacteria can negatively affect the consistency of bedding, particularly affecting rather non-consistent or highly non-consistent bedding, which can lead to footpad damage in poultry, and can be avoided by feeding animals with the strains, compositions, or preparations of the present invention, another subject of the present invention is a method for controlling and / or improving bedding consistency, particularly a method for ensuring consistent bedding consistency and / or avoiding footpad damage, said method comprising the step of feeding animals, particularly poultry, with at least one strain, preparation, and / or composition of the present invention.

[0057] The strains and preparations according to the invention can also be used to improve water quality. Another subject of the invention is therefore a method for controlling and / or improving the quality of water or aqueous solutions, particularly drinking water and / or aquatic water, comprising the step of applying at least one strain and / or at least one preparation and / or at least one composition according to the invention to water or aqueous solutions.

[0058] Furthermore, the strains and preparations according to the invention can also be used to treat microbial diseases of plants. Another object of the invention is therefore a method for treating and / or preventing microbial diseases of plants, particularly cultivated plants, comprising the step of applying at least one strain and / or at least one preparation and / or at least one composition according to the invention to the plant. This application can be in liquid form, such as by spraying, or in solid form, particularly as a powder.

[0059] By using the strains, preparations, and compositions of the present invention, it is preferable to achieve an improvement in at least one of the above-described characteristics, wherein the achievement of the characteristic preferably represents an improvement of at least 1%, more preferably at least 3% or at least 5%, compared to a suitable negative control. The average value of negative controls known in the animal husbandry field can be used, but it is preferable to use negative control animals that received the same treatment as the test animals but were not treated with the strains and / or preparations of the present invention.

[0060] Specifically, the strains, preparations, and compositions of the present invention can be administered or fed to animals to effectively inhibit and / or reduce the content of pathogenic bacteria in the animal gut. Such pathogenic bacteria include Clostridia, Listeria, Salmonella, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Escherichia coli, and Vibrio. Relatedly, the methods of the present invention can be used to reduce the content of pathogenic bacteria shed in animal feces. The methods of the present invention can also be used to maintain or enhance the growth of beneficial bacteria in the animal gut, such as lactic acid bacteria. By reducing pathogenic bacteria and / or enhancing or maintaining beneficial bacteria, the compositions of the present invention are able to maintain an overall healthy intestinal microbiota.

[0061] Therefore, another object of the present invention is a method for inhibiting and / or reducing the growth of harmful or pathogenic bacteria in the animal gut and / or maintaining and / or increasing the growth of beneficial bacteria in the animal gut, wherein the animal is given the strains and / or preparations and / or compositions of the present invention, and wherein the pathogenic bacteria are preferably selected from the genus *Clostridium*, particularly *Clostridium perfringens* and *Clostridium difficile*, the genus *Listeria*, particularly *Listeria monocytogenes*, *Listeria seeligeri* and *Listeria welshimeri*, the genus *Salmonella*, particularly *Salmonella enterica*, *Salmonella gallinarum*, *Salmonella pullorum*, *Salmonella Arizonae*, and *Salmonella typhimurium*. Salmonella typhimurium, Salmonella enteritidis, and Salmonella bongori; Enterococcus spp., especially Enterococcus faecalis, Enterococcus faecium, and Enterococcus cecum; Staphylococcus spp., especially Staphylococcus aureus; Aeromonas spp.; Streptococcus spp., especially Streptococcus suis and Streptococcus gallinarum; Campylobacter spp., especially Campylobacter jejuni and Campylobacter coli; Escherichia coli and Vibrio spp., especially Vibrio parahemolyticus and Vibrio harvesti; and beneficial bacteria are preferably selected from lactic acid bacteria, especially Lactobacillus and Bifidobacterium.

[0062] In a preferred embodiment of the invention, the content of at least one pathogenic bacterium, particularly Clostridium perfringens, is reduced by at least 0.5 log, more preferably by at least 1 log, 2 log or 3 log.

[0063] Therefore, another subject of the present invention is the strains, preparations, and compositions of the present invention for inhibiting and / or reducing the growth of pathogenic bacteria in the animal gut and / or for maintaining and / or increasing the growth of beneficial bacteria in the animal gut, wherein the pathogenic bacteria are preferably selected from the genus *Clostridium*, particularly *Clostridium perfringens* and *Clostridium difficile*, the genus *Listeria*, particularly *Listeria monocytogenes*, *Listeria stearothermiae*, and *Listeria wiltii*, and the genus *Salmonella*, particularly *Salmonella enterica*, *Salmonella gallinarum*, *Salmonella pullorum*, and *Salmonella argentea*. Salmonella, Salmonella typhimurium, Salmonella enteritidis and Salmonella chaditidis, Enterococcus spp., especially Enterococcus faecalis, Enterococcus faecium and Enterococcus cecum, Staphylococcus spp., especially Staphylococcus aureus, Aeromonas spp., Streptococcus spp., especially Streptococcus suis and S. gallinaceus, Campylobacter spp., especially Campylobacter jejuni and Campylobacter coli, Escherichia coli and Vibrio spp., especially Vibrio parahaemolyticus and Vibrio harbinii, and beneficial bacteria are preferably selected from lactic acid bacteria, especially Lactobacillus and Bifidobacterium.

[0064] The presence and / or increased growth of pathogenic bacteria can indeed lead to outbreaks of specific diseases. For example, the presence and / or increased growth of *Clostridium perfringens* can lead to outbreaks of intestinal diseases, particularly necrotic enteritis in poultry. The presence and / or increased growth of *Clostridium perfringens* can also lead to outbreaks of other diseases, such as bacterial enteritis, gangrenous dermatitis, and colangiohepatitis. Even the mildest form of *Clostridium perfringens* infection can be accompanied by dysentery, which causes damp bedding and can lead to secondary diseases such as footpad dermatitis.

[0065] Another subject of the invention therefore includes therapeutic compositions comprising the strains and / or compositions of the invention as described above.

[0066] Therefore, the preferred subject matter here is a therapeutic composition for treating and / or preventing necrotic enteritis in animals, preferably poultry, the composition comprising the strains and / or compositions of the present invention as described above.

[0067] Therefore, another preferred subject matter in this content is a therapeutic composition for treating and / or preventing bacterial enteritis, gangrenous dermatitis, biliary hepatitis, clostridial disease, dysentery and / or footpad dermatitis in animals, preferably poultry, said composition comprising the strains and / or compositions of the present invention as described above.

[0068] Therefore, another subject of the present invention is the treatment and / or prevention of diseases, particularly intestinal diseases, preferably necrotic enteritis in poultry, especially subclinical necrotic enteritis, wherein the strains and / or compositions and / or preparations of the present invention are administered to animals in need.

[0069] Therefore, another subject of the present invention is the treatment and / or prevention of diseases, preferably poultry diseases, selected from bacterial enteritis, gangrenous dermatitis, biliary hepatitis, clostridial disease, dysentery and / or footpad dermatitis, wherein the strains and / or compositions and / or preparations of the present invention are administered to animals in need.

[0070] The strains and / or preparations and / or compositions of the present invention can be administered to animals in feed and / or drinking water, said administration being carried out over multiple days throughout the animal's life or during a specific stage or portion of the animal's life. For example, the strains and / or compositions can be administered only in the starter diet of farm animals or only in the finishing diet.

[0071] A particular subject of the present invention is also a method for enhancing human health and / or improving general physical condition and / or enhancing disease resistance and / or enhancing immune response and / or establishing or maintaining a healthy gut microbiota, wherein the strains and / or preparations of the present invention or compositions of the present invention comprising said strains are administered to a human.

[0072] Another subject of the invention is therefore the use of the strains and / or preparations and / or compositions of the invention for enhancing human health and / or improving general physical condition and / or enhancing disease resistance and / or enhancing immune response and / or establishing or maintaining a healthy gut microbiota in humans, wherein the strains and / or preparations of the invention or the compositions of the invention comprising said strains are administered to humans.

[0073] The compositions of the present invention, particularly feed, food, and pharmaceutical compositions, as well as drinking water or aquaculture water, preferably contain the strains of the present invention and are expressed in an amount of about 1 × 10⁻⁶. 3 Approximately 2×10 12 The ratio of CFU / g feed or ml water, especially approximately 1×10⁻⁶. 3 Or approximately 1×10 4 Or approximately 1×10 5 Or approximately 1×10 6 Or approximately 1×10 7 Or approximately 1×10 8 Or approximately 1×10 9 Or approximately 1×10 10 Or approximately 1×10 11 Or approximately 1×10 12 The ratio of CFU / g feed or ml water is preferably about 1×10 4 To approximately 1×10 10 The CFU / g feed or ml water content is preferably 1×10⁻⁶. 4 Up to 1×10 7 The dosage should be CFU / g of feed or ml of water, and administered to the animals.

[0074] Accordingly, the preferred content range of the strains and / or preparations of the present invention in the feed, food and water compositions of the present invention is preferably 0.1 wt.-% to 10 wt.-%, more preferably 0.2 wt.-% to 5 wt.-%, and particularly 0.3 wt.-% to 3 wt.-%.

[0075] The method of the present invention can be used for all kinds of animals, especially all kinds of non-human and non-insect animals, more preferably all kinds of vertebrates, such as mammals, aquatic animals and birds.

[0076] Animals that can benefit from this invention include, but are not limited to, farm animals, pets, exotic animals, zoo animals, aquatic animals, and animals used for exercise, recreation, or work.

[0077] Pets are preferred from dogs, cats, poultry, and domesticated exotic animals.

[0078] The preferred aquatic animals are finned fish and crustaceans, with a preferred purpose for human nutrition. These specifically include carp, tilapia, catfish, tuna, salmon, trout, Australian lungfish, bream, perch, cod, shrimp, lobster, crab, prawn, and crayfish. Preferred salmon types in this context are Atlantic salmon, sockeye salmon, masus salmon, king salmon, keta salmon, coho salmon, Danube salmon, Pacific salmon, and pink salmon.

[0079] Other preferred aquatic animals are farmed fish, which are subsequently processed to obtain fish meat or fish oil. In this regard, the fish are preferably herring, sardines, bream, anchovies, capelin, or cod.

[0080] In a further preferred embodiment, the animals are farm animals, raised for consumption or as food producers, such as poultry, pigs, and ruminants.

[0081] Poultry can be selected from commercial or domesticated poultry, but can also come from ornamental poultry or wild birds.

[0082] In this context, the preferred production poultry are chickens, turkeys, ducks, and geese. In this context, the preferred production farm animals are poultry optimized for producing young livestock or poultry optimized for meat production.

[0083] Preferred ornamental poultry or wild birds include peacocks, pheasants, partridges, chukkar (rock partridges), guinea fowl, quails, grouse, ptarmigans (capercaillie), pigeons, and swans, with quails being particularly preferred.

[0084] Other preferred poultry are flat-breasted birds, especially ostriches and emus, as well as parrots.

[0085] The ruminants according to the invention are preferably selected from cattle, goats, and sheep. In one embodiment, the compositions of the invention can be fed to ruminant preruminants to enhance their health and, in particular, reduce the incidence of diarrhea in these animals. Ruminant preruminants are ruminants ranging in age from birth to about twelve weeks, including calves.

[0086] The compositions of the present invention may contain at least one carrier or a common feed ingredient or a combination thereof.

[0087] Suitable carriers are inert formulation ingredients added to improve collection, efficacy, or physical properties and / or facilitate packaging and application. Such carriers can be added alone or in combination. These carriers can be selected from anti-caking agents, antioxidants, fillers, and / or protectants. Examples of useful carriers include polysaccharides (especially starch, maltodextrin, methylcellulose, gum, chitosan, and / or inulin), protein sources (especially skim milk powder and / or sweet whey powder), peptides, sugars (especially lactose, trehalose, sucrose, and / or dextrose), lipids (especially lecithin, vegetable oils, and / or animal oils), salts (especially sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate, and / or sodium citrate), and silicates (especially clays, particularly beolite clay, amorphous silica, calcined / precipitated silica, zeolite, Fuller's clay, baylith, clintpolite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminum silicate, magnesium silicate, and / or calcium silicate). Suitable carriers for animal feed additives are listed in American Feed Control Officials, Inc.'s Official Publication, which is published annually. See, for example, Official Publication of American Feed Control Officials, edited by Sharon Krebs, 2006 edition, ISBN 1-878341-18-9. The carrier may be added after the fermentation broth has been concentrated and / or during and / or after drying. Preferred carriers according to the invention are selected from calcium carbonate, diatomaceous earth, and vegetable oils.

[0088] Preferred embodiments of the present invention are concentrated compositions, particularly feed additive compositions, i.e., compositions suitable for preparing feed compositions, comprising at least one strain of the present invention and at least one carrier as described above, wherein the at least one strain is contained in an amount of 0.1 to 10 wt.%, more preferably in an amount of 0.2 to 5 wt.%, particularly in an amount of 0.3 to 3 wt.%, especially in an amount of 0.4 to 2.2 wt.%, and the at least one carrier is preferably contained in an amount of at least 90 wt.%, preferably in an amount of 90 to 99.9 wt.%, more preferably in an amount of 95 to 99.8 wt.%, particularly in an amount of 97 to 99.7 wt.%, especially in an amount of 97.8 to 99.6 wt.%, and wherein the carrier is preferably composed substantially of limestone, particularly limestone having a small portion of diatomaceous earth and / or vegetable oil.

[0089] These preferred compositions of the present invention, containing stable strains, can be used in the preparation of feed and pharmaceutical compositions, as well as drinking water and animal husbandry water, and preferably contain the strains according to the invention in the amounts described above. In a preferred embodiment, 200 to 1000 grams of such concentrated compositions, particularly 250, 500, or 1000 grams, are used per ton of feed, drinking water, or animal husbandry water to provide compositions suitable for animal husbandry. These concentrated compositions preferably contain a content of 1 × 10⁻⁶. 9 Up to 2×10 11 CFU, especially 2×10 9 Up to 1×10 11 At least one strain of the present invention in the CFU / g concentrated composition.

[0090] Starting with these concentrated compositions, feed and food compositions can be prepared by mixing the concentrated compositions separately with common feed or food ingredients.

[0091] Suitable common animal feed ingredients that may be included in the compositions according to the invention and / or used in the preparation of feed compositions from the concentrated compositions according to the invention include one or more of the following: proteins, carbohydrates, fats, other probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, coccidiostats, acid-based products and / or drugs, such as antibiotics.

[0092] The carbohydrate-containing components that can be used according to the present invention are, for example, forage, coarse grains, wheat flour, sunflower flour or soybean flour, and mixtures thereof.

[0093] The protein-containing components that can be used according to the present invention are, for example, soy protein, pea protein, wheat gluten or corn gluten, and mixtures thereof.

[0094] The fatty components that can be used according to the invention are, in particular, oils, of animal and plant origin, such as vegetable oils, for example, soybean oil, rapeseed oil, sunflower oil, flaxseed oil or palm oil, fish oil, and mixtures thereof.

[0095] Other protein-containing components containing fat that can be used according to the present invention include, for example, fish meal, krill meal, bivalve meal, squid meal, or shrimp meal, and combinations thereof.

[0096] The additional probiotics (DFM) that can be used in conjunction with the strains and preparations of the present invention are preferably selected from Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, Bacillus scoagulans, Bacillus alevis, Bacillus cereus, Bacillus badius, Bacillus thurigiensis, Enterococcus faecium, and Pediococcus acidilactici. Preferred examples are Bacillus licheniformis DSM 32314 (deposited in DSMZ on May 12, 2016, pursuant to the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Proceedings) and its derivatives, and Bacillus subtilis PB6 (as described in U.S. Patent No. 7,247,299 and deposited as ATCC Accession No. PTA-6737), which was obtained by Kemin under a trademark. Sales of Bacillus subtilis C-3102 (as described in U.S. Patent No. 4,919,936 and deposited as FERM BP-1096 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan), by Calpis as Sold, Bacillus subtilis DSM 17299, by Chr. Hansen under trademark. The sale of a mixture of Bacillus subtilis DSM 17299 and Bacillus licheniformis DSM 17236 by Chr. Hansen under trademark. Sales, by Chr. Hansen in accordance with trademarks A mixture of commercially available Bacillus licheniformis and Bacillus subtilis spores, or the Bacillus rennet strain described in U.S. Patent No. 6,849,256. Other non-spore-forming probiotics, such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, or Hansenula, may also be used in the compositions of the present invention. In particular, in food compositions, additional probiotics known for their health benefits, such as lactic acid-producing bacteria, particularly Lactobacillus or Bifidobacterium, may be used. If these additional probiotics are not formulated as part of the compositions of the present invention, they may be administered together with the compositions of the present invention (simultaneously or at different times).

[0097] The prebiotics that can be used according to the present invention are preferably oligosaccharides, particularly selected from galacto-oligosaccharides, silayloligosaccharides, lactulose, lactosucrose, fructooligosaccharides, paraginose or isomaltooligosaccharides, sucrose, maltodextrin, isomaltooligosaccharides, cyclodextrin, gentiosaccharides, soybean oligosaccharides, xylooligosaccharides, dextran, pectin, polygalacturonic acid, rhamnose galacturonic acid, mannan, hemicellulose, arabinogalactan, arabinogalactan, arabinoxylan, resistant starch, mehbiose, chitosan, agarose, inulin, tagatose, polydextrose, and alginate.

[0098] Enzymes that can be used in the feed compositions according to the invention and that contribute to feed digestion are preferably selected from phytase (EC 3.1.3.8 or 3.1.3.26), xylanase (EC 3.2.1.8), galactanase (EC 3.2.1.89), galactosidase, especially α-galactosidase (EC 3.2.1.22), protease (EC 3.4), phospholipase, especially phospholipase 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), lysophospholipase (EC 3.1.1.5), amylase, especially α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17), and glucanase, especially β-glucanase (EC 3.1.1.5). 3.2.1.4 or EC 3.2.1.6), glucosylamylase, cellulase, pectinase, or any mixture thereof.

[0099] Examples of commercially available phytases include Bio-Feed TM Phytase (Novozymes) P and HiPhos TM (DSM Nutritional Products), Natuphos TM (BASF) and Blue (AB Enzymes), XP (Verenium / DuPont) and PHY (DuPont). Other preferred phytases include, for example, those described in WO 98 / 28408, WO 00 / 43503 and WO 03 / 066847.

[0100] 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).

[0101] The vitamins that can be used according to the present invention are, for example, vitamin A, vitamin D3, vitamin E, vitamin K, such as vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as calcium D-pantothenate, or combinations thereof.

[0102] Immunomodulators that can be used include, for example, antibiotics, cytokines, spray-dried plasma, interleukins or interferons, or combinations thereof.

[0103] The minerals that can be used according to the present invention are, for example, boron, cobalt, chlorine, chromium, copper, chlorine, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, magnesium, potassium or sodium, or combinations thereof.

[0104] The amino acids that can be used according to the present invention are, for example, lysine, alanine, threonine, methionine, or tryptophan, or combinations thereof.

[0105] Therefore, another aspect of the invention is a method for preparing an animal feed composition, comprising mixing at least one strain and / or at least one preparation and / or at least one concentrated composition of the invention, particularly at a level that effectively enhances animal health, with feed ingredients such as proteins, lipids, and / or carbohydrates, and optionally more beneficial substances, preferably as described above, to provide a feed product. The method may also include, for example, a pelleting step.

[0106] Standard pelleting methods known to those skilled in the art can be used, including extrusion processing of dry or semi-wet feed. Preferred pelleting temperatures are from about 65°C to about 120°C.

[0107] The strains and compositions of the present invention can be obtained by culturing the strains of the present invention according to methods known in the art, including using the culture media described in, for example, US 6,060,051, EP0287699, or US2014 / 0010792, and other methods. Conventional large-scale microbial culture methods include submerged fermentation, solid-state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, the cells of the strain begin to transition from the growth phase to the sporulation phase, such that the final products of fermentation are mostly sporulations, metabolites, and residual fermentation medium. Sporulation is part of the natural life cycle of these strains and is usually initiated by cellular responses to nutrient limitations. Fermentation is programmed to obtain high levels of colony-forming units of Bacillus subtilis cells and to promote sporulation. The bacterial cells, sporulations, and metabolites in the culture medium obtained from fermentation can be used directly or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, depth filtration, and evaporation. The concentrated fermentation broth can be washed, for example by percolation, to remove residual fermentation broth and metabolites.

[0108] Fermentation broth or concentrate can be dried using conventional drying processes or methods, such as 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, such as by grinding or granulation, to obtain a specific particle size or physical form. A carrier, as described above, can also be added after drying.

[0109] The preparations of the strains of the present invention can be cell-free preparations, preparations containing cell debris, or preparations containing a mixture of intact cells and cell debris. For example, cell-free preparations of the strains of the present invention can be obtained by centrifugation and / or filtration of the fermentation broth. Depending on the technique used, these cell-free preparations may not be completely cell-free, but may still contain a small amount of cells. Because cells secrete compounds, such as metabolites, enzymes, and / or peptides, into the surrounding environment, the cell supernatant contains a mixture of these compounds, particularly metabolites, enzymes, and / or peptides, as secreted by the cells. Therefore, in a preferred embodiment of the invention, the preparation of the strain is the supernatant of the fermentation broth.

[0110] Compositions containing cell fragments of the bacterial strain can be obtained by applying techniques known to those skilled in the art, such as mechanically or by applying high pressure, to rupture cells. Depending on the applied pressure, compositions containing only ruptured cells or compositions containing a mixture of cell fragments and intact cells are obtained. For example, cell homogenization can be achieved using a French cell press, ultrasonic apparatus, homogenizer, microfluidic apparatus, ball mill, rod mill, pebble 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 the cells.

[0111] The cell-free preparation of the present invention further includes obtaining the preparation by first rupturing cells using the technique described above and then removing cell debris and remaining intact cells. In particular, the removal of cell debris and remaining intact cells can be performed by centrifugation and / or filtration.

[0112] The preparations of the strains of the present invention may contain at least one metabolite as an active compound, preferably a mixture of metabolites, as further described below, and / or at least one enzyme selected from proteases (especially subtilisin), xylanase and / or cellulase, and / or at least one peptide, and / or a combination thereof.

[0113] For example, a preparation containing a mixture of potent metabolites contained in the strains of the present invention and / or in the cell preparations described above can be obtained according to the methods set forth in U.S. Patent No. 6,060,051. Specifically, the preparation can be obtained by precipitating the metabolites contained in the preparations described above using an organic solvent, such as ethyl acetate, and subsequently redissolving the precipitated metabolites in a suitable solvent. The metabolites can then be purified by size exclusion filtration, which separates the metabolites into different fractions based on molecular weight cutoff.

[0114] Preparations containing a mixture of effective metabolites of the present invention preferably contain at least five, more preferably at least six, seven, eight, nine, ten, or twelve, particularly all, metabolites of the strain of the present invention. The metabolites of strain DSM 32315 are listed in Table 5.1. The metabolites preferably have a molecular weight of 400 to 4000 Daltons, more preferably 500 to 3500 Daltons.

[0115] Preferably, according to the invention, an effective amount of the strains and / or preparations and / or compositions of the invention is always used in the embodiments of the invention. The term "effective amount" refers to the content that achieves at least one beneficial effect on animals and / or the environment compared to animals that are not given the strains and / or preparations and / or compositions of the invention but otherwise given the same diet (including feed and other compounds), particularly with respect to the characteristics already described above.

[0116] In therapeutic applications, therapeutic amounts of the strains and / or preparations and / or compositions of the present invention are preferred. The term "therapeutic amount" refers to a concentration sufficient to improve, reverse, or prevent disease in animals. The optimal dosage level for various animals can be readily determined by those skilled in the art by evaluating, in particular, the ability of the composition to (i) inhibit or reduce pathogenic bacteria in the gut at different dosages, (ii) increase or maintain the level of beneficial bacteria, and / or (iii) enhance animal health at different dosages. Example

[0117] Example 1. Strains with characteristics associated with survival in the gastrointestinal tract

[0118] Bacillus subtilis strains were screened from various environmental samples to obtain superior strains suitable for direct animal feeding as microorganisms / probiotics. Because the aim was to allow the strains to reach their full potential in the gut of the target animals, strains were pre-screened to withstand various environmental and gut-related conditions. Spores were generated (Nicholson and Setlow 1990), washed, and incubated at 80°C for 20 minutes (pasteurization), followed by calf infusion agar (VI, Difco). TM(Becton Dickinson GmbH, Heidelberg, Germany) was titrated with a logarithmic / 1:10 dilution. The second highest dilution before growth was stored at -80°C and used as the starting point for normalization for all other evaluations from the spore state. To simulate gastric passage (Argenzio 2004a; Trampel and Duke 2004), survival under acid exposure was evaluated based on Larsen et al. (2014). Growth of vegetative cells at low pH was also evaluated, showing growth under gastric / foregut and gizzard conditions, as well as at pH 7 in the presence of up to 4 mM bile (B8631, CAS 8008-63-8, Sigma-Aldrich), to confirm the growth of the strain in the proximal part of the small intestine immediately after gastric or gizzard clearance (Argenzio 2004b; Trampel and Duke 2004). By using VI medium supplemented with 2.5 mM KNO3 under anaerobic conditions (Anaerobic Pak) TM The fitness of strains in the anaerobic gut was evaluated by inoculating standardized spore solutions (Argenzio 2004b; Trampel and Duke 2004) (Glaser et al., 1995). Furthermore, the anaerobic proteolytic and cellulose-hydrolyzing activities of the strains were evaluated on VI agar plates supplemented with 1% skim milk powder (70166, Sigma-Aldrich) or 0.1% water-insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland). Osmotic stress, also found in the gut, was assessed by measuring growth on VI agar supplemented with 5% NaCl (Argenzio 2004b; Trampel and Duke 2004). Finally, granulation stability was determined by exposing spores to 99°C for 20 min (Palop et al., 1996) and subsequently inoculating them onto VI agar.

[0119] Bacillus subtilis strain DSM 32315 survived in a simulated gastric passage, and growth of strain DSM 32315 was observed starting at pH 6. Strain DSM 32315 grows anaerobically and is able to degrade water-insoluble cellulose and protein under anaerobic conditions. Strain DSM 32315 is able to grow in the presence of 2 and 4 mM bile, as well as in the presence of 5% NaCl. Strain DSM 32315 reached a growth rate of 8.42 × 10⁸. 8 The average number of spores was CFU / mL, and the spores of strain DSM 32315 were viable after exposure to 99°C for 20 min.

[0120] References:

[0121] Argenzio, R. A. (2004a). Secretion of the Stomach and Accessory Glands, p. 405 - 418. In: Reece, W. O. (ed.), Duke’s Physiology of Domestic Animals; 12th ed., Chapter 25; Cornell University Press, Ithaca, New York, USA.

[0122] Argenzio, R. A. (2004b). Digestive and Absorptive Function of the Intestines, p. 419 - 437. In: Reece, W. O. (ed.), Duke’s Physiology of Domestic Animals; 12th ed., Chapter 26; Cornell University Press, Ithaca, New York, USA.

[0123] Dawson, R. M. C.; Elliot, D. C.; Elliot, W. H.; Jones, K. M. (1986), Data for Biochemical Research; 3rd ed., Oxford Science Publishing, United Kingdom.

[0124] Den Besten HMW, Mols M, Moezelaar R, Zwietering MH, Abee T. (2009). Phenotypic and transcriptomic analyses of mildly and severely salt - stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol. 75: 4111 - 9.

[0125] Glaser, P., A. Danchin, F. Kunst, P. Zuber and MMNakano. (1995). Identification and isolation of a gene required for nitrate assimilation and anaerobicgrowth of Bacillus subtilis. J. Bacteriol. 177:1112-1115.

[0126] Nicholson WL, Setlow P. Sporulation, germination and outgrowth. In: Harwood CR, Cutting SM editors, Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons Ltd.; 1990. pp. 27-74.

[0127] Palop,A.,Raso,J.,Pagan,R.,Condon,S. and Sala,FJ(1996).Influence of pHon heat resistance of Bacillus licheniformis in buffer and homogenizedfoods.International Journal of Food Microbiology 29,1-10.

[0128] Trampel, DW and Duke, GE (2004). Avian Digestion, pp. 488-500. In: Reece, WO (ed.), Duke's Physiology of Domestic Animals; 12th ed., Chapter 29; Cornell University Press, Ithaca, New York, USA.

[0129] Example 2. Quantitative evaluation of the performance-bile tolerance of comparative strains of direct feeding microorganisms (DFM) / probiotics used in animal nutrition relative to existing technologies.

[0130] To evaluate the competitiveness of Bacillus subtilis strain DSM 32315 selected from Example 1, benchmark analyses were performed using commercially available Bacillus subtilis strains DSM 17299 and DSM 5750. Since the two strains were identical for each test performed, strain DSM 17299 was used as a reference for data in this and all subsequent examples. The suitability of the strain for testing in the proximal small intestine in the presence of bile at neutral pH after gastric passage was determined by growing the strain in VIB medium supplemented with 2 mM bile (Argenzio 2004b; Trampel and Duke 2004). An overnight culture containing 50 μL of candidate strain cell suspension and 10 mL of VIB in a 100 mL Erlenmeyer flask was incubated at 37 °C and 200 rpm. Approximately 50 μL of the overnight culture was then transferred to a 100-well honeycomb plate containing 1 mL of pH 7 VIB with 2 mM bile (Oy Growth Curves Ab Ltd, formerly Thermo Labsystems, Helsinki, Finland) to obtain an OD of 0.2 / mL. Strain-specific growth was observed at 37 °C and 200 rpm for 48 h, with OD measured every 15 min using a Bioscreen CMBR (Oy Growth Curves Ab Ltd) equipped with the BioLink software package. Before calculating the area under the curve (AUC), the average three blank OD readings of the choline-only culture medium (blank) were subtracted from each culture at each time point. The quantitative evaluation of each strain was compared based on the area under the curve (AUC5, time in OD × h) for 0-5 h, the area under the curve (AUC10, time in OD × h) for 0-10 h, and the time until the strain reached its maximum optical density (Tmax in h). Statistical analysis was performed using a one-way ANOVA procedure in Statistical software (Minitab Inc., State College, PA, USA). The results can be found in Table 2.1.

[0131] Table 2.1. Growth of Bacillus subtilis strain DSM 32315 and the baseline strain in the presence of 2 mM bile.

[0132] strain ID AUC5 AUC10 Tmax DSM 32315 <![CDATA[0.884 A ]]> <![CDATA[1.730 A ]]> <![CDATA[17.5 B ]]> DSM 17299 <![CDATA[0.371 B ]]> <![CDATA[1.381 B ]]> <![CDATA[27.4 A ]]> P-value P<0.001 P＝0.001 P<0.001 SEM 0.013 0.020 0.2

[0133] AUC5, Area under the curve of optical density × h between time points 0 and 5h; AUC10, Area under the curve of optical density × h between time points 0 and 10h; Tmax, Time in h until the maximum optical density is reached; SEM, Pooled mean standard deviation. A,B The statement indicating that there are no identical letters is a significant difference.

[0134] In direct comparisons, in the presence of 2 mM bile, strain DSM 32315 reached its maximum OD 10 h faster than the baseline Bacillus subtilis strain DSM17299. Furthermore, compared to the baseline strain DSM 17299, strain DSM 32315 grew 2.4 times faster in the first 5 hours and 1.3 times faster in the first 10 hours of testing.

[0135] References:

[0136] Argenzio, RA (2004b). Digestive and Absorptive Function of the Intestines, p. 419-437. In: Reece, WO (editor), Duke's Physiology of Domestic Animals; 12th Edition, Chapter 26; Cornell University Press, Ithaca, New York, USA.

[0137] Trampel, DW and Duke, GE (2004). Avian Digestion, pp. 488-500. In: Reece, WO (ed.), Duke's Physiology of Domestic Animals; 12th ed., Chapter 29; Cornell University Press, Ithaca, New York, USA.

[0138] Example 3. Performance of comparative strains of direct-feed microorganisms (DFM) / probiotics relative to existing technologies for animal nutrition – growth in the presence of short-chain fatty acids (SCFA).

[0139] Comparative growth of strains DSM 32142 and DSM 17299 was evaluated in the presence of short-chain fatty acids, as these short-chain fatty acids were observed to have increasing importance for the large intestine (Argenzio 2004b; Trampel and Duke 2004). The test was initiated using a standardized spore solution as described in Example 1, testing aerobic growth in VI medium at 37°C and pH 6, with readouts for growth versus no growth. For this test, VI medium was adjusted to pH 6 using McIlvaine buffer (Palop et al., 1996) and subsequently supplemented with 0.05% acetic acid (HA, 537020, CAS 64-19-7, Sigma-Aldrich), 0.05% propionic acid (HP, P1386, CAS 79-09-4, Sigma-Aldrich), or 0.2% lactic acid (HL, W261106, CAS 50-21-5, Sigma-Aldrich). The results can be found in Table 3.1.

[0140] Table 3.1. Growth evaluation of Bacillus subtilis strain DSM 32315 and reference strain DSM 17299 at pH 6 in the presence of short-chain fatty acids.

[0141] strain ID Acetic acid propionic acid lactic acid DSM 32315 yes yes yes DSM 17299 No growth No growth No growth

[0142] Bacillus subtilis strain DSM 32142 can grow at pH 6 in the presence of acetic acid, propionic acid and lactic acid, while strain DSM 17299 cannot grow from spores under these conditions.

[0143] References:

[0144] Argenzio, RA (2004b). Digestive and Absorptive Function of the Intestines, p. 419-437. In: Reece, WO (editor), Duke's Physiology of Domestic Animals; 12th Edition, Chapter 26; Cornell University Press, Ithaca, New York, USA.

[0145] Palop,A.,Raso,J.,Pagan,R.,Condon,S. and Sala,FJ(1996).Influence of pHon heatresistance of Bacillus licheniformis in buffer and homogenizedfoods.International Journal ofFood Microbiology 29,1-10.

[0146] Trampel, DW and Duke, GE (2004). Avian Digestion, pp. 488-500. In: Reece, WO (ed.), Duke's Physiology of Domestic Animals; 12th ed., Chapter 29; Cornell University Press, Ithaca, New York, USA.

[0147] Example 4. Quantitative evaluation of the performance-enzyme activity of comparative strains of direct feeding microorganisms (DFM) / probiotics relative to existing technologies for animal nutrition.

[0148] Similar to the tests performed in Example 2, strains DSM 32315 and DSM 17299 were compared to evaluate their respective carbohydrate degradation activities. Cellulase and xylanase activities were determined as described by Larsen et al. (2014). Analysis was performed in three independent runs, and averages were calculated in milli-units per microliter of solution. Statistical analysis was performed using a one-way ANOVA procedure with Statistical software (Minitab Inc., State College, PA, USA). The results are shown in Table 4.1.

[0149] Table 4.1. Cellulase and xylanase activities of strains DSM 32315 and DSM 17299.

[0150]

[0151] SEM, pooled mean standard deviation; A，B Significant differences are indicated by the absence of identical letters. In direct comparisons, strain DSM 32315 demonstrated a 7.5-fold increase in cellulase activity and a 2.1-fold increase in xylanase activity compared to the baseline strain DSM 17299.

[0152] References:

[0153] Larsen, N., Thorsen, L., Kpikpi, EN, Stuer-Lauridsen, B., Cantor, MD, Nielsen, B., Brockmann, E., Derkx, EMF and Jespersen, L. (2014). Characterization of Bacillus spp.strains for use as probiotic additives in pig feed.Appliedmicrobiology and biotechnology,98(3),1105-1118.

[0154] Example 5. Comparative strain performance of direct feeding microorganisms (DFM) / probiotics relative to existing technologies for animal nutrition – expression of metabolites and pathogen inhibition.

[0155] Similar to the tests performed in Example 2, strain DSM 32315 and the baseline strain DSM 17299 were compared to evaluate the quantity of metabolites expressed and the amount of pathogens inhibited in their respective media. For metabolite expression analysis, the starting culture was grown and tested as described by Scholz et al. (2011). 100 μL of the culture was transferred to the master culture from 10 mL of Luria Bertami liquid medium (LB, Thermo Fisher Scientific) grown at 37 °C and 160 rpm for 24 h in a 100 mL flask. The master culture was grown in 10 mL of LB broth containing 0.2 mL / L Kelly T trace metal solution (LB Kelly, Scholz et al., 2011) or in 10 mL of trypsin-enriched soybean liquid medium (Oxoid, Thermo Fisher Scientific) containing 0.6% yeast extract (Y1625, CAS 8013-1-2, Sigma-Aldrich; the resulting liquid medium is abbreviated as TSBYE), both in 100 mL flasks at 37 °C and 160 rpm for 24 h. 4 mL of the master culture was mixed with 2 mL of n-butanol in a 15 mL tube, vortexed for 3 min, and then sonicated for 15 min. After centrifugation at 5000 rpm for 1 min, the organic phase was transferred, vacuum dried, and analyzed using high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS; Chen et al., 2006). Each sample was measured in two different modes (negative and positive modes), and mass spectra were obtained. The peaks obtained, as reported similarly in Teo and Tan (2005), were converted to molecular weights in Da. The comparison results can be found in Table 5.1.

[0156]

[0157] In addition, the inhibition of *Clostridium perfringens* by *Bacillus subtilis* bacteriocin, which is part of the metabolites listed in Table 5.1, was evaluated using a pore diffusion antagonism test (Parente et al., 1995), but no closer studies were available. The four pathogenic *Clostridium perfringens* candidates tested were *Clostridium perfringens* strain ATCC 13124 from Teo and Tan (2005), and three pathogenic *Clostridium perfringens* wild-type isolates from poultry and pigs, obtained from the University of Leipzig, Faculty of Veterinary Medicine, Department of Bacteriology and Mycology, Prof. Dr. Christoph Baums, Potsdam, Germany. The *Clostridium perfringens* strain A- described by Leipzig are as follows: strains 2300-1-17 and 2300-1-18, isolated from the digestive tract of chickens with necrotic enteritis. Both strains produced α-toxins, with strain 2300-1-17 also expressing NetB toxins (Savva et al., 2013; Uzal et al., 2014), while strain 2300-1-18 tested positive for β2-toxins (Allaart et al., 2012). Strain 2300-1-19 (Songer and Uzal 2005) was isolated from the digestive tract of flushed piglets exhibiting symptoms of Clostridium type A enteritis. Growth conditions and media were as described by Teo and Tan (2005). Briefly, Bacillus strains were grown for 24 h in 10 mL TSBYE and LBKelly starting cultures in 100 mL flasks at 37 °C and 160 rpm, respectively, under a 5% CO2 atmosphere. Clostridium perfringens starting cultures were anaerobic in fluid thioglycolate liquid medium (FTB, Becton Dickenson) (AnaeroPak). TMThermo Fischer Scientific culture was incubated in 100 mL flasks at 37°C and 160 rpm for 24 h, and then spread onto agar plates (TSBYE, containing 1% agar, short TSAYE) using sterile swabs. The inoculated TSAYE plates were then anaerobically cultured overnight at 37°C to obtain Clostridium perfringens bacterial growth. After overnight growth, three 9 mm diameter wells were cut from the agar containing the bacterial growth. The first well served as an uninoculated control without culture. The second well was inoculated with 100 μL of a non-Clostridium perfringens inhibitory Bacillus strain (Bacillus cereus toyoi variant, NCIMB 40112). The third well was inoculated with 100 μL of Bacillus subtilis DSM 32315 or DSM 17299 starting culture. After incubation at 37°C for 24 h, the cleared area was measured from the edge of the cut hole to the boundary of the cleared bacterial growth to determine the cleared zone in mm. Measurements were taken twice per colony (horizontal and vertical) and then averaged. Analysis was performed in a double-plate run for each Bacillus subtilis antagonism test and culture medium. Fisher LSD was used for averaging analysis.

[0158] Statistical analysis was performed using a one-way ANOVA program in 16Statistical software (Minitab Inc., State College, PA, USA). Results for pathogen inhibition (strains grown in LBKelly) can be found in Table 5.2, and results for pathogen inhibition (strains grown in TSBYE) can be found in Table 5.3.

[0159] Table 5.2. Comparison of the inhibitory abilities of Bacillus subtilis DSM 32315 and DSM 17299 in the pore diffusion test of pathogenic gas-producing Clostridium subtilis (strains grown in LB Kelly), values ​​in mm of pathogen clearance.

[0160] SEM, pooled mean standard deviation; A,B The statement indicating that there are no identical letters is a significant difference.

[0161] Table 5.3. Comparison of the inhibitory abilities of Bacillus subtilis DSM 32315 and DSM 17299 in the pathogenic gas-producing Clostridium perfringens well diffusion test (strains grown in TSBYE), values ​​in mm of pathogen clearance.

[0162]

[0163]

[0164] SEM, pooled mean standard deviation; A,B The statement indicating that there are no identical letters is a significant difference.

[0165] References

[0166] Allaart, J.G., de Bruijn, N.D., van Asten, A.J., Fabri, T.H., and A. (2012). NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands. Avian Pathol., 41:541-546 Chen, X.H., Vater, J., Piel, J., Franke, P., Scholz, R., Schneider, K., Koumoutsi, A., Hiteroth, G., Grammel, N., Strittmatter, A.W., Gottschalk, G., Süssmuth, R. and Borriss, R. (2006). Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB42. Journal of bacteriology, 188(11), 4024-4036.

[0167] Parente, E., Brienza, C., Moles, M., & Ricciardi, A. (1995). A comparison of methods for the measurement of bacteriocin activity. Journal of microbiological methods, 22(1), 95 - 108. Savva, G. S., Fernandes da Costa, S. P., Bokori - Brown, M., Naylor, C. E., Cole, A. R., Moss, D. S., Titball, R - W., and Basak, A. 2013. Molecular architecture and functional analysis of NetB, a pore - forming toxin from Clostridium perfringens. J Biol.Chem., 288:3512 - 3522.

[0168] Scholz, R., Molohon, K. J., Nachtigall, J., Vater, J., Markley, A. L., Süssmuth, R. D., Mitchell, D. A., and Borriss, R. (2011). Plantazolicin, a novel microcin B17 / streptolysin S - like natural product from Bacillus amyloliquefaciens FZB42. Journal of bacteriology, 193(1), 215 - 224. Songer, J. G., and Uzal, F. A. (2005). Clostridial enteric infections in pigs. Journal of veterinary diagnostic investigation, 17(6), 528 - 536.

[0169] Teo, AY-L. and Tan, H.-M. (2005). Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis from the gastrointerstinal tracts of healthy chickens. Appl. Environm. Microbiol., 71: 4185-90.

[0170] Uzal, FA, Fredman, JC, Shrestha, A., Theoret, JR, Garcia, J., Awad, MM, Adams, V., Moore, RJ, Rood, JI, and McClane, BA (2014). Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol., 9:361-377. Example 6. Comparison of the performance of broiler chickens raised in India, fed with novel Bacillus subtilis, competing Bacillus subtilis products, or antibiotic growth promoters.

[0171] The growth performance of broiler chickens was studied in ground enclosures with used bedding straw, using Vencobb 400 chickens (Venkateshwara Hatcheries Pvt. Ltd., India) at hatching day size. Four dietary treatments were randomly assigned, with twelve replicates per treatment and 25 birds per enclosure. The birds were fed one of the dietary treatments for three phases: starter (days 1–14), growing (days 15–28), and finishing (days 29–42). The basal diet was primarily based on corn-soybean meal (Table 6.1) containing 500 g / MT dinotolmide to control coccidiosis. The basal diet also included 4% meat and bone meal (MBM) as an additional challenge, as MBM is a predisposing factor for necrotizing enterocolitis caused by Clostridium perfringens in broilers (M'Sadeqa et al., 2015). The four dietary treatments were primarily based on corn-soybean meal (Table 6.1) and included: 1. Basal control (control), 2. Control + 50 g Bacitracin Methylene Disalicyclate / MT feed (BMD), 3. Control + 500 g of a competing product / MT feed containing Bacillus subtilis strains, containing 1.6*10 9CFU / g (DSM 17299), 4. Control +250 g / MT Bacillus subtilis strain DSM32315, containing 2.0*10 cfu / g (DSM 17299), 9 cfu / g (DSM 32315). Experimental treatments involved infants aged 1–42 days fed a controlled diet in a mashed form with free access. Statistical analysis was performed using SAS vs9.4 (SAS Institute Inc., USA) with a one-way ANOVA program and LSD assay. Results regarding body weight, feed conversion ratio, and mortality are recorded in Table 6.2.

[0172] Table 6.1. Composition and nutritional profile of a basic diet

[0173] Element,% Opening (1-14d) Growth (15-28 days) Fattening (29-42 days) corn 53.98 62.27 64.54 Soy flour, 48% CP 36.09 27.64 24.63 meat and bone meal 4.00 4.00 4.00 soybean oil 2.46 2.60 3.60 dicalcium phosphate 22 1.48 1.51 1.53 Calcium carbonate 0.39 0.45 0.16 Premixes (including vitamin-min mixtures) 0.65 0.65 0.65 Sodium chloride 0.28 0.29 0.29 Sodium bicarbonate 0.10 0.10 0.10 Choline chloride 50 0.10 0.10 0.10 DL-methionine 0.29 0.23 0.22 L-Lysine HCl 0.12 0.13 0.14 L-threonine 0.05 0.04 0.05 Nutritional composition ME, kcal / kg 2950 3050 3150 CP, % 23.50 20.08 18.83 Ca 1.00 1.00 1.00 Available P 0.45 0.45 0.45 Lys 1.36 1.14 1.06 Met 0.63 0.53 0.50 M+C 0.99 0.85 0.80 Thr 0.92 0.78 0.74 Trp 0.27 0.22 0.20 Arg 1.59 1.33 1.23 Ile 0.97 0.81 0.75 Leu 1.93 1.70 1.61 Val 1.08 0.92 0.86

[0174] Table 6.2. Animal performance from day 0 to day 42 with or without dietary supplementation containing Bacillus subtilis-based feed additives or antibiotic growth promoters.

[0175] BW, average bird body weight over a specific time period; FCR, feed conversion ratio calculated based on feed and weight gain over a specific time period; Control, basal diet without additives; BMD, treatment with addition of bacitracin methylene disalicyclate to the basal diet; DSM 17299, treatment with addition of DSM17299 to the basal diet; DSM32315, treatment with addition of strain DSM 32315 to the basal diet; Difference, the numerical difference observed when comparing DSM 32315 with the control; Relative %, the difference between DSM 32315 and the control as a percentage change from the control.

[0176] During the start-up and growth phase from 1 to 21 days, DSM 32315 increased the free broiler rate (FCR) of broilers by 0.7% compared to the control. Feeding broilers with DSM 32315 also resulted in a 2.1% increase in body weight on day 21 compared to the control. While these improvements were not as significant as those observed with the highly effective antibiotic growth promoter BMD, DSM 32315 did improve broiler performance at this stage to a greater extent than DSM 17299.

[0177] Throughout the broiler's growth period, DSM 32315 improved body weight and feed conversion ratio by 1.4% and -1.7%, respectively. These improvements were again higher than those observed when using the competing product DSM 17299. Although body weight at day 42 was still better in the antibiotic growth promoter group (BMD), the DSM 32315 treatment reduced FCR by an additional 0.01 (0.6%) compared to the BMD-supplemented treatment group.

[0178] In summary, these results indicate that birds fed DSM 32315 outperformed those fed the control or DSM 17299, and in at least some parameters, showed similar performance to highly effective antibiotic growth promoters.

[0179] References

[0180] M'Sadeqa, S., Wua S., Swicka. RA and M. Chocta (2015). Towards the control of necrotic enteritis in broiler chickens with in-feed antibiotics phasing-outworldwide. Animal Nutrition. 1:1-11.

[0181] Example 7. Comparison of the performance of broiler chickens raised in Thailand with those fed a novel Bacillus subtilis, a competing Bacillus subtilis product, or an antibiotic growth promoter.

[0182] A study of broiler growth performance was conducted using 1-day-old male Ross 308 (Aviagen Asia, Thailand) chickens placed in ground enclosures with used straw bedding. Four dietary treatments were administered, with 16 replicates per treatment and 12 birds per enclosure. Birds were fed one of the dietary treatments for three phases: starter (days 1–14), growing (days 15–28), and finishing (days 29–42). The basal diet was primarily based on corn-soybean meal (Table 7.1) and contained 60 g of salinomycin / MT feed to control coccidiosis. The basal diet also included 5% dried corn distillers grains and solubles (DDGS), as the addition of DDGS, especially in the starter diet, can reduce broiler growth rate (Lumpkins et al., 2004) and is a predisposing factor for necrotic enteritis caused by Clostridium perfringens in broilers (Macklin et al., 2011). The four dietary treatments included: 1. Basal control (control), 2. Control + 20g of bacitracin zinc / MT feed (ZnB), and 3. Control + 500g of a competing product / MT feed containing Bacillus subtilis strains, containing 1.6*109 cfu / g (DSM 17299), 4. Control + 250g Bacillus subtilis strain DSM 32315 / MT, containing 2.0*10 9 cfu / g (DSM 32315). Basal diet. Statistical analysis was performed using SAS vs9.4 (SAS Institute Inc., USA) with a one-way ANOVA procedure and LSD post-test analysis. Animal growth performance from 0 to 42 days can be found in Table 7.2.

[0183] Table 7.1. Composition and Nutritional Components of a Basic Diet

[0184]

[0185]

[0186] Table 7.2. Animal performance from day 0 to day 42 with or without dietary supplementation containing Bacillus subtilis-based feed additives or antibiotic growth promoters.

[0187]

[0188] BW, average bird body weight over a specific time period; FCR, feed conversion ratio calculated based on feed and weight gain over a specific time period and adjusted for mortality; Control, basal diet without additives; ZnB, treatment with added bacitracin zinc; DSM17299, treatment with added DSM17299 to the basal diet; DSM 32315, treatment with added strain DSM 32315 to the basal diet; Difference, the numerical difference observed when comparing DSM 32315 with the control; Relative %, the difference between DSM 32315 and the control as a percentage change from the control.

[0189] Throughout the growth period, from day 1 to day 42, treatment with DSM 32315 reduced the free circulatory rate (FCR) by 2.7%. While this effect was also observed in other treatment groups, BMD and DSM 17299, the DSM 32315 treatment also resulted in a final body weight increase of 6.9% from the control. Furthermore, DSM 17299 reduced bird mortality by 2.25% compared to the untreated control, and by 0.64% compared to DSM 17299.

[0190] These results indicate that while all treatments improved feed conversion throughout the phase, supplementation with DSM 32315 resulted in the heaviest birds achieving their final weight and a notable reduction in mortality.

[0191] References

[0192] Lmupkins, BS, Batal, AB, & Dale, NM (2004). Evaluation of distillersdried grains with solubles as a feed ingredient for broilers. Poultry Science, 83(11), 1891-1896.

[0193] Macklin, KS, Rose, LN, and Dozier III, WA (2001). The effects of different levels of DDGS on necrotic enteritis development in broilerchickens. In Western Poultry Disease Conference, p.123.

[0194] Example 8. Comparison of the performance of broiler chickens with and without a diet containing new Bacillus subtilis when subjected to the challenge of induced necrotizing enteritis.

[0195] A necrotizing enteritis challenge study was conducted using 1-day-old male Ross 308 (Aviagen) chickens in ground enclosures. Birds were randomly assigned to three dietary treatments, with eleven replicate enclosures per treatment and 14 birds per enclosure. Birds were fed one of the dietary treatments for three phases: starter (days 1–14), growing (days 15–28), and finishing (days 29–42). The basal diet was primarily based on corn-soybean meal (Table 8.1). The starter diet was prepared in 2mm broken pieces, while the growing and finishing diets were in 4mm pieces. The three treatments included: 1. Basal control (control), 2. Positive control + 650g napromycin coccidioidomycin ( G100 (Elanco USA) / MT feed (napramide), 3. Control + 250g Bacillus subtilis strain DSM 32315 / MT, containing 2.0×10 9 cfu / g (DSM 32315). Experimental treatments were performed from 1 to 35 days of age with free access to feed.

[0196] Necrotic enteritis is a disease in chickens caused by causative infection of *Eimeria maxima* in conjunction with *Clostridium perfringens* infection, typically occurring in 3–4 week old broilers (Timbermont et al. 2011). The necrotizing enteritis challenge was induced in two parts: at 12 days of age, each bird was orally inoculated with 5000 *Eimeria maxima* oocytes, and at 16 days of age, each bird was given an overnight culture of 300 μl of a wild-type *Clostridium perfringens* strain originally isolated from the ileal contents of broilers suffering from necrotizing enteritis. Results regarding body weight, feed conversion ratio, and mortality are reported in Table 8.2. Mortality was reported between days 11 and 35 to control for early bird losses due to chicken quality unrelated to the induced disease challenge. Because footpad dermatitis is associated with damp bedding (Taira et al. 2014), and intestinal health problems such as necrotizing enteritis can lead to damp bedding (Timbermont et al. 2011), footpad damage scores were also measured. Based on Welfare Quality 2009 (Rushen et al. 2011), the footpads of sampled birds were scored for hoof dermatitis (i.e., footpad disease), with values ​​ranging from 0 (representing no signs of hoof dermatitis) to 4 (severe hoof dermatitis). The results of processing the footpad damage scores are reported in Table 8.3. Specific bacterial groups (Bacillus and Clostridium perfringens) were calculated from ileal and cecal digestive matter from days 11, 18, and 35. DNA extracted from the digestive matter samples was used for quantitative PCR using species-specific primers of interest. Results of molecular calculations on the bacterial groups (Bacillus and Clostridium perfringens) at days 11, 18, and 35 are reported in Tables 8.4, 8.5, and 8.6, respectively.

[0197] Table 8.1. Composition and nutritional profile of a basic diet

[0198]

[0199]

[0200] d, day; CP, crude protein; ME, metabolizable energy; kcal, kilocalorie; kg, kilogram

[0201] Table 8.2. Animal performance on days 0 to 35, with and without supplementation with feed additives based on Bacillus subtilis or napramide.

[0202]

[0203] BW, average bird body weight; FCR, feed conversion ratio, calculated as feed to weight gain for a specific period; aFCR, feed conversion ratio, calculated as feed to weight gain for a specific period and adjusted for mortality; mortality rate, mortality rate on days 11–35, percentage of the enclosed population; control, no additives in the basal diet; Narasin, treatment with narasin coccidioidomycin added to the basal diet; DSM 32315, treatment with a new strain of Bacillus subtilis DSM 32315 added to the basal diet; difference, numerical difference observed when DSM32315 is compared to the control; relative %, difference between DSM 32315 and the control, expressed as a percentage change in the control.

[0204] Table 8.3. Foot pad injury scoring, with and without supplementation with feed additives based on Bacillus subtilis or napromycin.

[0205]

[0206] Control: No additives in the basal diet; Narasin: Treatment with narasin as a coccidioidomycin in the basal diet; DSM 32315: Treatment with a novel Bacillus subtilis strain, DSM 32315, in the basal diet; Difference: The numerical difference observed when DSM 32315 is compared to the control; Relative %, The difference between DSM 32315 and the control, expressed as a percentage change in the control.

[0207] Table 8.4. Molecular calculation of Bacillus subtilis and Clostridium perfringens in the ileum and cecum of broiler chickens at 11 days of age, with and without supplementation with feed additives based on Bacillus subtilis or napromycin.

[0208]

[0209] Control: No additives in the basal diet; Narasin: Treatment with narasin as a coccidioidomycin in the basal diet; DSM 32315: Treatment with a novel Bacillus subtilis strain, DSM 32315, in the basal diet; Difference: Numerical differences observed when DSM 32315 is compared to the control.

[0210] Table 8.5. Molecular calculation of Bacillus subtilis and Clostridium perfringens in the ileum and cecum of broiler chickens at 18 days of age, with and without supplementation with feed additives based on Bacillus subtilis or napramide.

[0211]

[0212] Control: No additives in the basal diet; Narasin: Treatment with narasin as a coccidioidomycin in the basal diet; DSM 32315: Treatment with a novel Bacillus subtilis strain, DSM 32315, in the basal diet; Difference: Numerical differences observed when DSM 32315 is compared to the control.

[0213] Table 8.6. Molecular calculation of Bacillus subtilis and Clostridium perfringens in the ileum and cecum of broiler chickens at 35 days of age, with and without supplementation with feed additives based on Bacillus subtilis or napramide.

[0214]

[0215] Control: No additives in the basal diet; Narasin: Treatment with narasin as a coccidioidomycin in the basal diet; DSM 32315: Treatment with a novel Bacillus subtilis strain, DSM 32315, in the basal diet; Difference: Numerical differences observed when DSM 32315 is compared to the control.

[0216] This study provides clear evidence supporting the beneficial effects of the probiotic strain DSM 32315 on the performance of broiler chickens facing the health challenge of necrotizing enterocolitis. Compared to baseline controls, broilers fed DSM 32315 showed increased body weight, while feed conversion ratio and mortality-calibrated feed conversion ratio were decreased. These findings regarding the probiotic strain are similar to those of naramycin, a positive control in this model and an anticoccidial additive that prevents necrotizing enterocolitis outbreaks. Necrotic enterocolitis-related footpad damage was also reduced in broilers fed a diet including the DSM 32315 strain compared to baseline controls. This could be due to reduced moisture and pathogens in the bedding, but this was not tested in this study.

[0217] Bacterial populations in the ileum and cecum of the birds were counted on days 11, 18, and 35. In the treatment groups fed a diet containing Bacillus subtilis strain DSM 32315, consistently higher levels of Bacillus were counted at all time points and tissue sites. However, changes in Bacillus were observed in the napramide-treated groups compared to the control; these findings were subtle and not consistent with those observed in the DSM 32315-treated groups at all tissues and time points.

[0218] When counting Clostridium perfringens, the causative agent of necrotizing enterocolitis, the DSM 32315 treatment group consistently showed reduced counts of these pathogens compared to the control group. This is similar to the positive control group in which napramide was added to the feed to prevent necrotizing enterocolitis outbreaks.

[0219] The data presented in this study provide clear evidence of the ability of DSM 32315 to suppress necrotizing enterocolitis and prevent subsequent losses due to performance loss, death or footpad damage, or adverse slaughter conditions.

[0220] References

[0221] Rushen J.,A.Butterworth and JCSwanson(2011).Animal Behavior andWell-Being Symposium:Farm animal welfare assurance:Science and application.Journal of Animal Science 89:1219-1228.

[0222] Taira K.,T.Nagai,T.Obi and K.Takase(2014).Effect of Litter Moistureon the Development of Footpad Dermatitis in Broiler Chickens.J Vet Med Sci.76(4):583–586.

[0223] Timbermont L., F. Haesebrouck, R. Ducatelle and F. Van Immerseel (2011). Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathol. 40(4): 341-347.

[0224] Example 9. Evaluation of the strain's tolerance to feed pellet production and feed storage.

[0225] To test the tolerance of Bacillus subtilis DSM 32315 spores to feed pelleting, the spores were added to a broiler growing diet (Table 9.1), which was then pelleted at 85°C. Prior to pelleting, ten replicates of the mixed feed were isolated for spore counting. The feed was then pelleted at 85°C, yielding five additional replicates. Viable spores from both pre- and post-pelleting samples were serially diluted, counted, and their feed concentrations were calculated. The spore count results are shown in Table 9.2.

[0226] Table 9.1. Composition of broiler diets used for granulation tolerance assessment.

[0227]

[0228] Table 9.2. Spore counts in feed before and after pelleting

[0229] DSM 32315 spore count Feed before pelleting Pelleted feed Spore recovery % 100.0％ 79.9％

[0230] Spore recovery % is calculated as the percentage of counted spores relative to the spores before granulation.

[0231] The spore count after pelleting remained within 30% of the expected range from the feed before pelleting, indicating that there was no significant loss of Bacillus subtilis DSM 32315 spores during pelleting.

[0232] To test the time stability of Bacillus subtilis DSM 32315 spores in the feed, the spores were added to the broiler growing diet (the same formulation as described previously in Table 9.1). The feed was pelleted at 85°C, and three replicates of the diet were stored in a climate-controlled room at 40°C and 85% humidity. Spore counts were performed after pelleting and at 2, 4, 8, and 12 weeks after storage. The spore count results are shown in Table 9.3.

[0233] 9.3. Resuscitation of DSM 32315 spores in pelleted feed after storage in a climate chamber.

[0234] Spore recovery % is calculated as the percentage of the counted spores relative to the spores directly counted after granulation and storage for 0 weeks.

[0235] The spore count in the pelleted feed fluctuated over time but remained within 30% of the expected spore count obtained directly after pelleting. Therefore, it can be concluded that no significant loss of Bacillus subtilis DSM 32315 spores was observed after 12 weeks of storage.

[0236] Example 10. Well diffusion antagonism test for different pathogenic strains

[0237] Pore ​​diffusion antagonism was tested against three different pathogens: Enterococcus cecorum DSM 20683, Streptococcus gallinaceus DSM 15349, and Streptococcus suis ATCC 43765.

[0238] Cecal enterococci are known to cause lameness, arthritis, and osteomyelitis in broilers, typically caused by joint and / or bone tissue involvement. Other cecal enterococci can cause pericarditis [Kense et al. 2011]. DSM 20683 was isolated from the chicken cecum.

[0239] S. gallinaceus can cause septicemia in poultry. Total losses include splenomegaly, hepatomegaly, renal enlargement, and congestion. Necrosis or infarction in multiple areas of the liver has also been observed in association with valvular endocarditis [Collins et al. 2002].

[0240] Streptococcus suis is a major pathogen in pigs and one of the leading causes of bacterial mortality in piglets after weaning, causing septicemia, meningitis, and many other infections [Goyette-Desjardins et al. 2014]. ATCC 43765 belongs to serological group: R; serotype 2, and was isolated from pigs.

[0241] Bacillus strains were grown for 16 h at 37°C and 200 rpm in 10 mL TSBYE (30 g / L TSB + 6 g / L yeast extract) or LB-Kelly (LB medium supplemented with DSMZ media 1032 micronutrient solution) in 100 mL shake flasks. The pathogenic strains were grown to a density of at least 1 at 595 nm under conditions suitable for liquid culture, and then 100 μL were spread on an agar surface using a sterile spatula. BHI agar plates were used for *S. gallinaceus*, and TSBYE agar plates were used for *Enterococcus cecum* and *Streptococcus suis*. Three 9 mm diameter wells were cut into the dried plates. The first well served as a non-inoculated medium control without culture. The second well was inoculated with 100 μL of a non-inhibitory Bacillus strain (Bacillus cereus var. toyoi, NCIMB 40112). The third well was inoculated with 100 μL of Bacillus subtilis DSM32315 or DSM 17299 culture. After incubation at 37°C for 24 h, the clear area was measured in mm, from the edge of the cut-off empty space to the boundary of the cleared bacterial colony. Each colony was measured twice (horizontally and vertically) and then averaged. The results are shown in Table 10.1.

[0242] Table 10.1. Comparison of the inhibitory effects of Bacillus subtilis DSM 32315 and DSM 17299 on pathogenic strains in the pore diffusion antagonism test, with values ​​expressed as pathogen clearance in mm.

[0243]

[0244] Data shows that DSM 32315, especially compared to DSM17299, can effectively inhibit the growth of Enterococcus cecum, S. gallinaceus, and Streptococcus suis.

[0245] References

[0246] MJ Kense,WJM Landman(2011).Enterococcus cecoruminfections in broilerbreeders and their offspring:molecular epidemiology.Avian Pathology Vol.40,Iss.6.

[0247] MD Collins, RA Hutson, E Falsen, E Ingana, M Bisgaard (2002). Streptococcus gallinaceus sp. nov., from chickens. International Journal of Systematic and Evolutionary Microbiology. 52: 1161–1164.

[0248] G Goyette-Desjardins, JP Auger, J Xu, M Segura, M Gottschalk (2014). Streptococcus suis, an important pig pathogen and emerging zoonotic agent—an update on the worldwide distribution based on serotyping and sequencetyping. Emerg Microbes Infect. 3(6):e45.

Claims

1. Bacillus subtilis strains or preparations thereof, selected from: a) Bacillus subtilis strain preserved in DSMZ as DSM 32315; The preparation of b)(a), characterized in that It is capable of inhibiting Clostridium perfringens and contains an effective mixture of at least 10 metabolites contained in (a); c)(a) preparation, wherein the preparation is the supernatant of the fermentation broth of strain DSM 32315.

2. The Bacillus subtilis strain of claim 1 or its preparation thereof, wherein the Bacillus subtilis strain exhibits the following characteristics: a) Having a yqfD sequence according to the sequence of SEQ ID NO:2; and / or b) A gyrB sequence having the sequence according to SEQ ID NO:

3.

3. The Bacillus subtilis strain or preparation thereof of claim 1 or 2, wherein the Bacillus subtilis strain contains a 16S rDNA sequence having the sequence according to SEQ ID NO:

1.

4. The Bacillus subtilis strain or preparation thereof according to claim 1 or 2, wherein the Bacillus subtilis strain exhibits the following further characteristics: c) An rpoB sequence having the sequence according to SEQ ID NO:4; and / or d) A groEL sequence having the sequence according to SEQ ID NO:

5.

5. The Bacillus subtilis strain or preparation thereof according to claim 1 or 2, wherein the Bacillus subtilis strain is characterized by being capable of anaerobic growth.

6. The Bacillus subtilis strain or its preparation according to claim 5, characterized in that... It can degrade water-insoluble cellulose and protein under anaerobic conditions.

7. The Bacillus subtilis strain or its preparation according to claim 1 or 2, characterized in that... In the well diffusion antagonism assay on LB Kelly agar plates of Clostridium perfringens strain ATCC 13124, pathogen clearance was at least 10 mm.

8. The Bacillus subtilis strain or preparation thereof according to claim 1 or 2, wherein the Bacillus subtilis strain is characterized by being able to grow in the presence of 0.05 wt.-% acetic acid, 0.05 wt.-% propionic acid and / or 0.2 wt.-% lactic acid.

9. The Bacillus subtilis strain or its preparation according to claim 1 or 2, characterized in that... At least 200 mU / mL of cellulase activity and / or at least 15 mU / mL of xylanase activity.

10. The Bacillus subtilis strain or its preparation according to claim 9, characterized in that... At least 350 mU / mL of cellulase activity and / or at least 25 mU / mL of xylanase activity.

11. The Bacillus subtilis strain or preparation thereof according to claim 1 or 2, wherein the Bacillus subtilis strain is characterized by being able to grow in the presence of 2 mM bile.

12. The Bacillus subtilis strain or preparation thereof according to claim 11, wherein the Bacillus subtilis strain is characterized by an AUC 5 performance of at least 0.8 in the presence of 2 mM bile.

13. The Bacillus subtilis strain or preparation thereof according to claim 11, wherein the Bacillus subtilis strain is characterized by an AUC 10 performance of at least 1.6 in the presence of 2 mM bile.

14. The Bacillus subtilis strain or preparation thereof according to claim 11, wherein the Bacillus subtilis strain is characterized by being able to grow in the presence of 4 mM bile.

15. Use of the Bacillus subtilis strain or preparation thereof according to any of the preceding claims as a probiotic component (DFM) in feed or food.

16. A feed or food composition comprising a strain of Bacillus subtilis or a preparation thereof according to any one of claims 1 to 14 and at least one other feed or food ingredient.

17. The feed or food composition according to claim 16, characterized in that... The at least one other feed or food ingredient is selected from proteins, carbohydrates, fats, other probiotics, prebiotics, vitamins, immunomodulators, milk substitutes, minerals, coccidiostats, acid-based products, and combinations thereof.

18. The feed or food composition according to claim 16 or 17, wherein the at least one other feed or food ingredient comprises amino acids.

19. A pharmaceutical composition comprising a) A Bacillus subtilis strain or preparation thereof according to any one of claims 1 to 14, and b) Pharmacologically acceptable carrier.

20. The composition according to claim 16, 17 or 19, wherein the composition is used to improve the health of an animal or human.

21. The composition of claim 20, wherein the composition is used to improve the intestinal health of an animal or human.

22. A method for raising animals, characterized in that... The animal is treated with a Bacillus subtilis strain or preparation according to any one of claims 1 to 14 and / or a feed composition according to any one of claims 16 to 18, wherein the method is a non-therapeutic method.

23. The method of claim 22, wherein the animal is a bird.

24. Use of the Bacillus subtilis strain or preparation according to any one of claims 1 to 14, or the composition according to any one of claims 16 to 21, in the preparation of a medicament for improving the health of animals or humans.

25. The use according to claim 24, wherein the health condition is an intestinal health condition.

26. Use of the Bacillus subtilis strain or preparation according to any one of claims 1 to 14, or the composition according to any one of claims 16 to 21, in the preparation of a medicament for enhancing animal health and / or improving the overall physical condition of animals and / or improving feed conversion ratio and / or reducing animal mortality and / or improving animal survival rate and / or improving animal weight gain and / or improving animal disease resistance and / or improving animal immune response and / or establishing or maintaining a healthy gut microbiota and / or reducing pathogen shedding through animal feces.

27. A method for controlling and / or avoiding the harmful environmental effects of fertilizers or contaminated liquids, the method comprising the step of applying at least one strain and / or at least one preparation and / or at least one composition according to any one of claims 1 to 14 to fertilizers, contaminated liquids, bedding, pits or fertilizer pools.

28. A method for controlling and / or improving the quality of water or an aqueous solution, the method comprising the step of applying at least one strain and / or at least one preparation and / or at least one composition according to any one of claims 1 to 14 to water or an aqueous solution.

29. The method of claim 28, wherein the water or aqueous solution is drinking water and / or aquatic water.

30. A method for treating and / or preventing microbial diseases in cultivated plants, comprising the step of applying at least one strain and / or at least one preparation of any one of claims 1 to 14 or at least one composition of any one of claims 16 to 21 to the cultivated plant.

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