Bacillus composition for improving gastrointestinal health and faeces score

Abstract

The present disclosure generally relates to compositions and methods for improving the fecal score of an animal using a new combination of probiotic Bacillus strains and administering the same to the animal. The present disclosure also relates to compositions and methods for improving gastrointestinal health in an animal using a novel combination of probiotic Bacillus strains. The present disclosure further relates to compositions and methods for increasing sugar release and protein solubility of animal feed using a combination of probiotic Bacillus strains.

Description

Technical Field

[0001] This disclosure generally relates to compositions and methods for improving fecal scores, which utilize novel combinations of probiotic Bacillus strains and administer the compositions to animals. This disclosure also relates to compositions and methods for improving gastrointestinal health using novel combinations of probiotic Bacillus strains. This disclosure further relates to compositions and methods for increasing sugar release and protein solubility in animal feed using novel combinations of probiotic Bacillus strains. Background Technology

[0002] Bacillus-based probiotic feed additives are known for their positive health effects in animal feed. These products are relevant to the feed industry because spores are heat-stable and can survive granulation processes at temperatures up to 90-95°C. Endospore-forming bacteria Bacillus subtilis and Bacillus amyloliquefaciens are generally considered safe (GRAS) by the U.S. Food and Drug Administration (FDA) and are acceptable for inclusion in animal diets or water by the Association of American Feed Control Officials (AAFCO).

[0003] Many pet owners are concerned about their animals' overall health, including gastrointestinal health. Dog owners, in particular, are interested in improving the overall health of their pets' gastrointestinal system, including improving their dog's fecal score / stool consistency. While there are attempts in the art to provide solutions that improve fecal scores, further improvements are needed in the art to achieve real benefits for pet owners. It would be highly advantageous to provide solutions that are easy for pet owners to administer and significantly improve their pets' gastrointestinal health and stool consistency. If the digestibility of the animal feed provided to pets can also be improved, that would be an additional benefit. Summary of the Invention

[0004] This disclosure provides strains of Bacillus spp. selected from a) strains deposited as DSM 32324, b) strains deposited as DSM 32325, and c) strains deposited as DSM 25840, which are sensitive to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and exhibit inhibitory activity against Escherichia coli and Clostridium perfringens. Therefore, all strains of this disclosure are considered sensitive to the antibiotics specified in EFSA Journal 2012;10(6):2740.

[0005] Example 1 provides results from an in vitro reducing sugar release assay. The compositions of this disclosure provide improvements in carbohydrate degradation and sugar release from animal feed.

[0006] Example 2 provides results from an in vitro protein solubility assay. The compositions of this disclosure provide an increase in protein solubility. While not wishing to be bound by theory, it is believed that the compositions of this disclosure can improve animal feed utilization by increasing the release of both energy and protein from animal feed. This is thought to lead to an increase in energy and protein intake in animals.

[0007] The compositions disclosed herein can provide improvements in gastrointestinal health and fecal scores in animals that ingest the compositions.

[0008] Example 3 provides results from in vivo studies showing improvements in fecal scores in dogs that ingested the compositions of this disclosure.

[0009] While not wishing to be bound by theory, it is believed that providing animals with the compositions of this disclosure can improve the overall gastrointestinal health of the animals and improve the achieved fecal scores, while also improving the digestibility of animal feed and leading to increased sugar release and increased protein solubility.

[0010] Furthermore, it is expected that by increasing protein absorption, the amount of protein entering the animal's hindgut for microbial fermentation will be reduced, thereby reducing the production of putrefactive compounds and fecal odor.

[0011] definition

[0012] Generally, the terms and phrases used herein have their generally accepted meanings, which can be found with reference to standard textbooks, journal references, and background information known to those skilled in the art. The following definitions are provided to clarify their specific use within the context of this disclosure.

[0013] As used in this article, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0014] As used herein, the term “and / or” means used in combination (“and”) and exclusively (“or”), that is, “A and / or B” means “A alone, or B alone, or A and B together”.

[0015] Animals: The term “animals” includes, but is not limited to, canines (e.g., dogs), felines (e.g., cats), equines (e.g., horses), bovines (e.g., cattle), sheep (e.g., sheep), goats (e.g., goats), suidae (e.g., pigs, such as piglets, weaned piglets, growing pigs, fattening pigs, castrated boars, poults, gilts, sows, pregnant sows) and rabbits, as well as birds (including but not limited to poultry, such as broilers, breeder chickens, laying hens, ostriches, quails, turkeys, ducks, geese), domesticated fur-bearing animals (e.g., ferrets, mink, weasels), ruminants (e.g., cattle, sheep, goats, camels, and giraffes), and fish (e.g., finned fish), shellfish, and other aquatic animals. Finned fishes include all vertebrate fish, which can be bony or cartilaginous fish, such as salmon, trout, tilapia, catfish and carp, as well as shellfish, including but not limited to clams, lobsters, shrimp, crabs, prawns and oysters.

[0016] As used herein, the term "animal feed" means any compound, preparation, or mixture suitable for or intended for ingestion by animals. Animal feed may contain concentrates as well as, for example, vitamins, minerals, enzymes, amino acids, and / or other feed ingredients (e.g., in premixes).

[0017] As used herein, the term "complete feed" is generally used for animal feed for pets, such as dogs and cats, and refers to ready-to-eat animal feed that contains concentrates and, for example, vitamins, minerals, enzymes, amino acids and / or other feed ingredients (e.g., in premixes). An example of a complete feed for dogs is the control diet provided in Example 2.

[0018] As used herein, the term "composition" refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be probiotics, direct-feed microorganisms (DFM), animal feed additives or premixes, or animal feeds, such as complete feeds.

[0019] As used herein, the term "direct-feed microbes" or "DFM" refers to live microbes, including spores, that, when administered in adequate amounts, confer benefits to the host, such as improved gastrointestinal digestion or health.

[0020] As used herein, the term "dry matter content" refers to the amount of dry matter in feces after passing through the animal's gastrointestinal tract, excluding water content.

[0021] As used herein, the terms "effective amount," "effective concentration," or "effective dose" are defined as the quantity, concentration, or dose of a bacterial strain sufficient to improve the physical, mental, health, digestion, or productivity of an animal. The actual effective dose in absolute terms depends on factors including the health status of the animal under discussion. The "effective amount," "effective concentration," or "effective dose" of a bacterial strain can be determined by conventional methods known to those skilled in the art. Examples of effective amounts are described in Example 2.

[0022] As used herein, the terms "feeding animals" or "ingesting animals" mean administering the composition of the invention orally to an animal in an effective amount. Oral administration may be repeated, for example, once or more daily over a specific period of time (e.g., several days, weeks, months, or months). Therefore, the terms "feeding" or "ingesting" mean any type of oral administration, such as administration via animal feed or drinking water, or in some cases via oral force-feeding or aerosol spraying.

[0023] As used herein, the terms “fecal score” or “fecal consistency” are used interchangeably and refer to the scoring scheme described in Example 2. An improved fecal score according to this disclosure is an average fecal score higher than 3 measured over a period of time.

[0024] As used herein, the term "isolated" means that the bacterial strain described herein is in a non-naturally existing form or environment, that is, the strain has at least partially separated from one or more, or all, of its naturally occurring components associated with it in nature.

[0025] As used in this article, the term "carbohydrate" refers to sugars, a group of molecules including sugars, starches, and fiber. Sugars are classified into four chemical categories based on size: monosaccharides (such as glucose), disaccharides, oligosaccharides, and polysaccharides. Carbohydrates, along with proteins and fats, are one of the three major nutrients found in animal feed. The animal body breaks down carbohydrates into glucose. Glucose, or blood sugar, is the primary energy source for the body's cells, tissues, and organs.

[0026] As used herein, the term "pet" refers to companion animals such as domestic cats and dogs, ferrets, rats, rabbits, hamsters, guinea pigs, reptiles, and birds such as parrots, lorikeets, falcons, and eagles. In one embodiment of this disclosure, a pet is a dog.

[0027] As used herein, the term "pet food" includes dry, semi-moist, or wet (moist) products. Wet food includes foods with a moisture content of 70%–90% and are typically sold in containers such as cans, pouches, and / or trays. Dry food includes foods with a similar composition but with 5%–15% moisture content, typically offered as small, biscuit-like coarse grains. Semi-moist food includes foods with a moisture content ranging from over 15% up to 70%. The amount of moisture in any product can affect the types of probiotic bacteria that can be added. Food products of any moisture level can be ready-to-eat. Pet food treats include treat bars, cereal bars, snacks, biscuits, beverages (such as cat milk), and desserts.

[0028] Pet food products can be cooked products that may contain meat or animal-derived materials (e.g., beef, chicken, eggs, turkey, lamb, fish, blood, bone marrow, bone marrow, etc., or their byproducts). Alternatively, the composition may be meatless (preferably including meat substitutes such as soy, corn, gluten, or soy products) to provide protein. The composition may contain additional protein sources such as soy protein concentrate, milk, protein, gluten, etc. The composition may also include starch, such as one or more grains (e.g., wheat, corn, rice, oats, barley, etc.) or may be starch-free. The composition may contain or be a gelled starch matrix. The composition may contain one or more types of fiber, such as sugar beet pulp, chicory pulp, chicory, coconut endosperm fiber, wheat fiber, fructooligosaccharides, galactooligosaccharides, inulin, etc. Fiber may be prebiotics. The combination of probiotics and prebiotics can form a synbiotic combination. The most suitable composition is the pet food described herein, which is sold as pet food, particularly for domestic dogs or cats. Preferably, the pet food is in dry form, such as a dried ready-to-eat cereal product containing a cooked starch matrix, which is produced by extrusion cooking and is called pet grains.

[0029] As used herein, the term "premix" refers to a composition containing at least two strains, which is added to a carrier to prepare a premix, which is then added to animal feed at the desired inclusion rate.

[0030] As used herein, the term "probiotics" is generally described as a dietary supplement containing potentially beneficial bacteria, and, according to the currently adopted definition, as a live microorganism that can confer health benefits to the host. Probiotic microorganisms help the body's naturally occurring gut microbiota rebuild itself. Probiotic microorganisms have been reported and are known to strengthen the immune system and prevent infection, reduce inflammation, improve mineral absorption, and prevent the growth of harmful bacteria. In some parts of the world, probiotics are referred to as direct-feed microorganisms (DFM).

[0031] As used herein, the term "protein" means any compound, preparation, or mixture that includes at least one protein obtained or derived from plant or animal sources, including modified proteins and protein derivatives. Plant proteins can be obtained from plant protein sources such as legumes and cereals, for example from the Leguminosae family, such as soybeans, lupins, peas, or common beans; Brassicaceae and Chenopodiaceae families, such as beets, sugar beets, spinach, or quinoa; and materials from plants of the Poaceae family. Other examples of plant protein sources are cereals such as barley, wheat, rye, oats, maize, rice, and sorghum. Animal proteins are obtained from animal protein sources such as meat sources, such as poultry, lamb, pork, fish, or animal by-products such as fishmeal and bone meal.

[0032] Proteins are complex molecules present in all living organisms and provide many essential functions within the body. These molecules are formed by “building blocks” of 20 amino acids, which can be considered essential or non-essential—essential amino acids cannot be synthesized in the body and must be obtained through diet, while non-essential amino acids can be synthesized by the body through the metabolism of other amino acids. The number of essential amino acids depends on the species in question. Therefore, the dissolution of proteins is necessary to provide essential amino acids for building hair, skin, nails / toenails, muscles, tendons, ligaments, and cartilage. Proteins also play a major role in hormone production and promote immune system function.

[0033] As used herein, the term "reducing sugar" means any sugar having a reactive aldehyde group or capable of forming a reactive aldehyde group to allow the sugar to act as a reducing agent (also known as a simple sugar). The reducing end is formed through the enzymatic cleavage of glycosidic bonds between polymeric carbohydrates. Reducing sugars include glucose, glyceraldehyde, and galactose, as well as disaccharides such as lactose and maltose, and can be measured by the Nelson-Somogyi (NS) or dinitrosalicylic acid (DNS) method. DNS is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. This determination simulates the process when feed is ingested by an animal and digested in the digestive tract. The ability of compositions of this disclosure containing Bacillus strains to degrade carbohydrates to release sugars has been investigated in Example 1.

[0034] As used herein, the term "antibiotic susceptibility" refers to the phenotypic property of a bacterial strain, namely, that its growth is inhibited under conditions where it would otherwise be able to grow. In this context, antibiotic susceptibility is tested according to CLSI guidelines (M07-A8 and M45-A2). For Bacillus strains, a strain is considered sensitive if growth is detected only at or below the breakpoint concentration specified in EFSA Journal 2012;10(6):2740 for vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. Regarding ampicillin, EFSA does not provide a breakpoint concentration for Bacillus strains; 4 mg / L has been selected as the breakpoint concentration for determining susceptibility.

[0035] As used herein, the terms "spore" and "endospore" are used interchangeably and have their conventional meanings well known and understood by those skilled in the art. As used herein, the term spore refers to a microorganism in a dormant, protected, non-reproductive state. Attached Figure Description

[0036] Figure 1

[0037] Figure 1 Results of in vitro assays were shown, examining the release of sugars (labeled "glucose equivalent") after animal feed samples were mixed in a liquid medium containing the composition of the present invention (probiotic sample, labeled "PRO") and in a liquid medium not containing the composition of the present invention (control sample, labeled "Control").

[0038] Figure 2

[0039] Figure 2 Results of in vitro assays were shown, investigating protein solubility in animal feed samples after mixing in a liquid medium containing the compositions of the present invention (probiotic sample, labeled "PRO") and in a liquid medium not containing the compositions of the present invention (control sample, labeled "Control"). Detailed Implementation

[0040] This disclosure relates to an animal feed, animal feed additive, or animal feed premix composition comprising at least two Bacillus strains selected from strains deposited as DSM 32324, DSM 32325, and DSM 25840.

[0041] (i) Sensitive to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

[0042] (ii) It has inhibitory activity against Escherichia coli, Salmonella spp. and Clostridium perfringens.

[0043] (iii) It has the ability to promote carbohydrate degradation in animal feed and thereby increase the amount of available sugars.

[0044] (iv) Possesses the ability to promote protein solubility in animal feed and thereby increase the amount of dissolved protein, and

[0045] (iiii) It has the ability to improve gastrointestinal health and average fecal score in animals.

[0046] The bacterial strains described in this article are isolated, meaning that their form or environment does not exist in nature.

[0047] If the Bacillus strain of this disclosure exhibits an inhibition zone of at least 0.5 mm against Escherichia coli, the strain is considered to show inhibitory activity (low inhibition) against Escherichia coli. Preferably, the inhibition zone is at least 0.5 mm to 2 mm (medium), more preferably, more than 2 mm (high). The inhibition zone may vary for different Escherichia coli strains. For a strain of the present invention considered to exhibit inhibitory activity against Escherichia coli, it should show an inhibition zone of at least 0.5 mm for all tested Escherichia coli strains. Preferably, the inhibition zones for two, three, or four Escherichia coli strains, or even more preferably, for all five Escherichia coli strains, are at least 0.5 mm to 2 mm. Even more preferably, the inhibition zones for two, three, or four Escherichia coli strains, or even more preferably, for all five Escherichia coli strains, are more than 2 mm.

[0048] If a Bacillus strain of this disclosure exhibits an inhibition zone of at least 0.5 mm against Clostridium perfringens, then the Bacillus is considered to have inhibitory activity (low inhibition) against Clostridium perfringens. Preferably, the inhibition zone is at least 0.5 mm to 2 mm (moderate), more preferably, more than 2 mm (high). The inhibition zone may vary for different Clostridium perfringens strains. For a strain of this invention considered to exhibit inhibitory activity against Clostridium perfringens, it should show an inhibition zone of at least 0.5 mm for all tested Clostridium perfringens strains. Preferably, the inhibition zones for two, three, or four Clostridium perfringens strains, or even more preferably, for all five Clostridium perfringens strains, are at least 0.5 mm to 2 mm. Even more preferably, the inhibition zones for two, three, or four Clostridium perfringens strains, or even more preferably, for all five Clostridium perfringens strains, are more than 2 mm.

[0049] If the *Bacillus* strain of this disclosure exhibits an inhibition zone of at least 0.5 mm against *Salmonella*, then the *Bacillus* strain is considered to have inhibitory activity (low inhibition) against *Salmonella*. Preferably, the inhibition zone is at least 0.5 mm to 2 mm (medium), more preferably, more than 2 mm (high). The inhibition zone may vary for different *Salmonella* strains. For a strain of this invention considered to exhibit inhibitory activity against *Salmonella*, it should show an inhibition zone of at least 0.5 mm for all tested *Salmonella* strains. Preferably, the inhibition zones for two, three, or four *Salmonella* strains, or even more preferably, for all five *Salmonella* strains, are at least 0.5 mm to 2 mm. Even more preferably, the inhibition zones for two, three, or four *Salmonella* strains, or even more preferably, for all five *Salmonella* strains, are more than 2 mm.

[0050] Preferably, the Bacillus strains of this disclosure should also be able to increase the sugar content by degrading the crude fiber and / or starch components of animal feed. The ability of the Bacillus strains in the compositions of this disclosure to degrade carbohydrates into sugars has been studied in Example 1, and the results are in Figure 1 Provided in the examples. When tested as described in the examples, if a Bacillus strain can increase the amount of available sugar in the feed to at least 500 kJ / kg of feed, then the strain is considered preferred.

[0051] Those skilled in the art can repeat these measurements to determine whether a particular Bacillus strain meets the requirements for antibiotic susceptibility, inhibitory activity, and degradation of carbohydrates and proteins. In this way, those skilled in the art will be able to perform inhibitory tests on these properties of the strain. Obviously, these measurements can be performed in any order, and some strains that do not meet the stated criteria can be excluded during the testing process.

[0052] This disclosure further provides information containing 10 5 Up to 10 12 A Bacillus composition of at least two Bacillus strains of this disclosure, at a colony forming unit / gram (CFU / g). In one aspect of this disclosure, the Bacillus composition comprises a combination of two Bacillus strains of this disclosure, such as Bacillus subtilis DSM 32324 and Bacillus amyloliquefaciens DSM 25840.

[0053] In another aspect of this disclosure, the Bacillus composition comprises three Bacillus strains of this disclosure, such as a combination of Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324, Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840, and Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325.

[0054] The Bacillus composition according to the present invention may comprise a combination of at least two Bacillus strains of the present invention and at least one other Bacillus strain. The Bacillus composition may comprise at least two strains, for example at least three, at least four, or at least five Bacillus strains, wherein at least two are Bacillus strains of the present invention.

[0055] Bacillus strains can be used in any combination in a Bacillus composition. For example, a Bacillus composition may contain at least one Bacillus strain of the present invention and / or at least one Bacillus licheniformis strain and / or at least one Bacillus amyloliquefaciens strain, such as two Bacillus subtilis strains and at least one Bacillus amyloliquefaciens strain of the present invention.

[0056] The composition may comprise Bacillus subtilis DSM32324 and / or Bacillus subtilis DSM 32325 in combination with Bacillus amyloliquefaciens DSM 25840 and / or Bacillus licheniformis DSM 17236 and / or Bacillus subtilis DSM 19489 and / or Bacillus subtilis DSM 17231. Any other possible combinations of the Bacillus strains of the present invention with other Bacillus strains are also possible.

[0057] As a specific example, the Bacillus composition comprises Bacillus subtilis DSM 32324, Bacillus subtilis DSM 32325, and Bacillus amyloliquefaciens DSM 25840. As another specific example, the Bacillus composition comprises Bacillus subtilis DSM 32324, Bacillus subtilis DSM 32325, and Bacillus licheniformis DSM 17236. In yet another specific example, the Bacillus composition comprises Bacillus subtilis DSM32324, Bacillus subtilis DSM32325, and Bacillus subtilis DSM19489.

[0058] In a specific embodiment, the composition further comprises bacteria from one or more of the following strains of the genus Bacillus: Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus simplex, Bacillus mojavensis, Bacillus safensis, Bacillus simplex, Bacillus atrophaeus, Bacillus methylotrophicus, and Bacillus sicca. Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis, or any combination thereof.

[0059] In a further embodiment, the composition comprises bacteria from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, or any combination thereof.

[0060] Preferred examples of such bacteria are species such as Enterococcus faecium, for example, Enterococcus faecium DSM 22502.

[0061] As a specific example, the Bacillus composition comprises Bacillus subtilis DSM 32324, Bacillus amyloliquefaciens DSM 25840, and Enterococcus faecalis DSM 22502.

[0062] In a specific embodiment, the composition further comprises one or more types of yeast. The one or more types of yeast may be selected from the family Saccharomycetaceae, genera of yeast (e.g., Saccharomyces cerevisiae and / or Saccharomyces boulardii), genera of Kluyveromyces (e.g., Kluyveromyces marxianus and Kluyveromyces lactis), genera of Candida (e.g., Candida utilis, also known as round yeast), genera of Pichia (e.g., Pichia pastoris), genera of Torulaspora (e.g., Torulaspora delbrueckii), genera of Phaffia, and phylum Basidiomycota.

[0063] Based on CFU / g composition, the proportion of each strain in the composition is 1-99% of the total bacterial isolates, for example 20-80%, for example 30-70%, more particularly 20%, 33%, 40% or 50%. The strains may be present in approximately equal or unequal amounts.

[0064] One or more related strains are provided in commercially relevant forms known to those skilled in the art. Therefore, in one embodiment, one or more strains of the composition are present in a dried (e.g., spray-dried) or frozen form. The composition can be provided in any suitable form, such as a liquid like a gel, slurry, powder, or pellet. In a preferred aspect of this disclosure, the composition is in the form of a dry powder, capsule, or oral paste.

[0065] In one embodiment, the composition comprises 10 5 Up to 10 12 CFU / g, more preferably 10 6 Up to 10 12 CFU / g and most preferably 10 7 Up to 10 12 CFU / g, for example 10 8 Up to 10 11 CFU / g, for example 10 9 Up to 10 10 Each bacterial strain (i.e., the Bacillus strains disclosed herein) contains CFU / g. The composition comprises at least 5 x 10⁻⁶ CFU / g of each bacterial strain. 4 Each strain / gram of composition in CFU distinguishes the compositions of the present invention from animal feed, for example, that has naturally occurring strains.

[0066] The term “CFU / g” refers to the gram weight of the composition, which includes a carrier such as calcium carbonate, an anti-caking agent such as aluminum silicate and diatomaceous earth, and other components present in the composition.

[0067] The compositions of this invention comprise at least two Bacillus strains as disclosed herein and at least one carrier, excipients and / or diluents and / or other components that make the composition suitable for feeding animals or as an additive to drinking water. It is non-toxic to animals when administered to them. Many such available agents can be added. Without limitation, examples include standardizing agents, extenders, wetting and lubricants, preservatives, lipids, stabilizers, solubilizers, flow aids, and emulsifiers. Examples particularly suitable for animal administration include ground corn cobs, salt, ground limestone, calcium carbonate, sodium bentonite, zeolite, ground soybean hulls, citrus pomace, dairy by-products, animal protein products, cereal products, plant protein products, processed cereal products and by-products, roughage products, molasses fermentation by-products such as dried distillers' grains and / or solubles, citric acid and glutamic acid fermentation by-products, etc.

[0068] Bacillus bacteria exist as both spores and vegetative cells, the latter capable of dividing to produce more vegetative cells. When Bacillus is referred to herein, it refers to both spores and vegetative cells, unless the context otherwise indicates.

[0069] In the compositions of this disclosure, Bacillus strains are preferably provided as spores. The primary function of spore formation is generally to ensure the survival of bacteria during periods of environmental stress. Therefore, they are resistant to ultraviolet and gamma radiation, dehydration, lysozyme, temperature, starvation, and chemical disinfectants. The spore coat is impermeable to many toxic molecules and may also contain enzymes involved in germination. The core has normal cellular structures, such as DNA and ribosomes, but the spore is metabolically inactive.

[0070] In one aspect, the present invention provides animal feed, animal feed additives or premixes comprising at least two Bacillus strains according to the present disclosure, and further comprising one or more of the following components: concentrates, vitamins, minerals, enzymes, amino acids and / or other feed ingredients, such as flavoring agents, yeast extracts, activated carbon, mineral clay, silica or combinations thereof.

[0071] Animal feeds for carnivores and omnivores, such as pets, cats, and dogs, typically containing starch sources, textured proteins, or semi-wet flakes, can be produced using typical ingredients used in pet food manufacturing, such as grain sources (e.g., corn, rice, wheat, barley), protein sources (e.g., meat sources, such as poultry, beef, lamb, pork, fish) or animal by-products (e.g., fish and bone meal), plant sources (e.g., soybean, corn gluten, casein, whey, egg); fats (e.g., vegetable oils, animal fats, fish oil), plant fibers (beet pulp, soybean hulls, cellulose), optionally vitamins (e.g., vitamins E, C, B1, B2, B6); and minerals (e.g., calcium sources, phosphorus sources, salts, trace minerals) and various flavorings or flavor enhancers, processing aids, and preservatives, to prepare pet food that meets the nutritional requirements of pets and has the necessary aesthetic characteristics.

[0072] In a specific embodiment, the animal feed consists of or contains milk (e.g., from sows, cows, goats, sheep), for example, for feeding newborn animals. In another specific embodiment, the animal feed consists of or contains milk substitutes, for example, for feeding newborn animals.

[0073] In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and / or one or more macrominerals. Typically, fat-soluble and water-soluble vitamins, as well as trace minerals, form part of a so-called premix intended to be added to the feed, while macrominerals are typically added to the feed separately. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as D-calcium pantothenate. Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, and zinc. Non-limiting examples of macrominerals include calcium, magnesium, potassium, and sodium.

[0074] The animal feed, animal feed additive, or premix of the present invention may further contain at least one enzyme 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); α-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase, for example, α-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and β-glucanase (EC 3.1.1.1). 3.2.1.4 or EC3.2.1.6), or any mixture thereof.

[0075] The animal feeds, such as complete feeds, animal feed additives, or premixes of the present invention, may further comprise one or more added amino acids. Examples of amino acids used in animal feeds are lysine, alanine, β-alanine, threonine, methionine, and tryptophan. The animal feeds, animal feed additives, or premixes of the present invention may further comprise colorants, stabilizers, growth-improving additives, and flavor compounds / flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen species (ROS) generating substances, antimicrobial peptides, and antifungal peptides. Examples of colorants are carotenoids, such as β-carotene, astaxanthin, and lutein. Examples of flavor compounds / flavorings are methoxycresol, anethole, decanolactone, undecylactone, and / or dodecalactone, ionone, irisone, gingerol, piperidine, propylene oxide benzofuranone, butylene oxide benzofuranone, capsaicin, and tannic acid. Examples of polyunsaturated fatty acids are C18, C20, and C22 polyunsaturated fatty acids, such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, and γ-linolenic acid. Examples of reactive oxygen species (ROS) generators are chemical substances, such as perborates, persulfates, or percarbonates; and enzymes, such as oxidases, oxygenases, or synthases.

[0076] In one embodiment, the animal feed, such as complete feed, animal feed additive, or premix, contains one or more anticoccidial drugs.

[0077] The animal feed, such as a complete feed, animal feed additive, or premix, further comprises a carrier. The carrier may comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1,2-propanediol or 1,3-propanediol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, whey, whey permeate, wheat flour, wheat bran, corn gluten meal, starch, and cellulose.

[0078] In one embodiment, the one or more bacterial strains are stable when subjected to pressure applied / achieved during the granulation extrusion process. In a specific embodiment, the one or more bacterial strains are stable at pressures ranging from 1 bar to 40 bar.

[0079] In specific embodiments, the one or more bacterial strains are stable at high temperatures. Specifically, the bacterial strains are stable when subjected to temperatures achieved during the granulation extrusion process. In even more specific embodiments, the one or more bacterial strains are stable at temperatures ranging from 70°C to 120°C.

[0080] Animal diets can be manufactured, for example, as paste feed (non-granulated), pellet feed, or extruded feed. Typically, ground feed ingredients are mixed, and sufficient amounts of essential vitamins and minerals are added according to specifications for the species in question. Bacterial cultures and optionally enzymes can be added as solid or liquid formulations. For example, for paste feed, solid or liquid culture formulations can be added before or during the ingredient mixing step. For pellet feed, (liquid or solid) Bacillus compositions can also be added before or during the feed ingredient step. Typically, the liquid Bacillus compositions of the present invention comprise bacterial strains and optionally polyols, such as glycerol, ethylene glycol, or propylene glycol, and are added after the pelleting or extrusion step, for example, by spraying the liquid formulation onto pellets or coarse pellets. The bacteria can also be incorporated into animal feed additives or premixes.

[0081] Another aspect of this disclosure relates to a method of feeding an animal, including administering the composition of this disclosure to the animal, particularly a companion animal or pet, such as a dog or a cat.

[0082] On the other hand, this disclosure relates to the use of animal feed, animal feed additives or premixes containing at least two Bacillus strains of this disclosure or at least two Bacillus strains of this disclosure to improve the gastrointestinal health and fecal scores of animals, particularly companion animals or pets, such as dogs or cats.

[0083] On the other hand, this disclosure relates to the application of compositions of this disclosure that increase the release of reducing sugars and the solubility of proteins in animal feed by adding at least two Bacillus strains of this disclosure to animal feed.

[0084] Preservation and expert solutions

[0085] The applicant requests that, prior to the date of this patent grant, in accordance with the effective regulations of the Industrial Property Offices of the Contracting Parties to the Budapest Treaty, samples of the following deposited microorganisms may only be provided to experts.

[0086] Table 1: In accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for use in patent proceedings, the applicant has made the following deposits at a depositary institution (Leibniz Institute DSMZ – German Center for the Preservation of Microorganisms and Cell Cultures, 7B38124 Inhofenstrasse, Braunschweig, Germany) that has obtained international depositary status.

[0087]

[0088] Implementation Plan

[0089] The invention is further defined by the following numbered paragraphs:

[0090] 1. Use of an animal feed, animal feed additive, or animal feed premix composition for improving gastrointestinal health and / or fecal scores in animals receiving said animal feed, animal feed additive, or animal feed premix, said composition comprising:

[0091] a.10 5 Up to 10 12 At least two Bacillus strains with a colony forming unit / gram (CFU / g), said strains being selected from:

[0092] i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324.

[0093] ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and

[0094] iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840; and

[0095] b. One or more animal feed, animal feed additives or animal feed premix components.

[0096] 2. As described in paragraph 1, the animal is a pet, such as a dog or cat, preferably a dog.

[0097] 3. For the purposes described in paragraph 2, to improve the fecal score of dogs.

[0098] 4. As described in any of the preceding paragraphs, the at least two Bacillus strains comprise Bacillus subtilis strains and Bacillus amyloliquefaciens strains.

[0099] 5. According to any of the uses described in the preceding paragraphs, the at least two Bacillus strains include the Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324 and the Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840.

[0100] 6. According to any of the uses described in the preceding paragraphs, the at least two Bacillus strains comprise, or consist of, the Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324, the Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840, and the Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325.

[0101] 7. As described in any of the preceding paragraphs, wherein the at least two Bacillus strains are present as spores.

[0102] 8. As described in any of the preceding paragraphs, the composition is an animal feed additive.

[0103] 9. As described in any of the preceding paragraphs, the composition is in the form of a dry powder, capsule, or oral paste.

[0104] 10. According to any of the uses described in the preceding paragraphs, the composition comprises one or more animal feed, animal feed additive or animal feed premix ingredients selected from vitamins, minerals, enzymes, amino acids, flavoring agents, yeast extracts, activated carbon, mineral clay, silica or combinations thereof.

[0105] 11. A method of feeding a pet animal, such as a dog or cat, comprising administering to the animal an animal feed, animal feed additive, or animal feed premix composition comprising at least two strains of Bacillus spp., said strains being selected from:

[0106] i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324.

[0107] ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and

[0108] iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

[0109] 12. A method for improving gastrointestinal health and fecal scores in pet animals, such as dogs or cats, the method comprising administering to the animal an effective amount of an animal feed, animal feed additive, or animal feed premix composition comprising at least two strains of Bacillus spp., said strains being selected from:

[0110] i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324.

[0111] ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and

[0112] iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

[0113] 13. A method for improving energy and protein intake in an animal, wherein the animal is a pet animal, such as a dog or cat, the method comprising feeding the animal with an animal feed, animal feed additive, or animal feed premix composition comprising at least two strains of Bacillus spp., wherein the Bacillus spp. strains are selected from:

[0114] (a) A strain of Bacillus subtilis deposited at the German Center for Microbial and Cell Culture Collection (DSMZ) with accession number DSM 32324, and

[0115] (b) Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and

[0116] (c) A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

[0117] 14. The method of any one of paragraphs 11-13, wherein the animal feed, animal feed additive, or animal feed premix composition comprises 10 [units of something] in the composition. 5 Up to 10 12 Each Bacillus strain with colony-forming units per gram (CFU / g).

[0118] 15. The method of any of paragraphs 11-14, wherein the animal is a dog.

[0119] 16. A composition of animal feed, animal feed additive, or animal feed premix, comprising:

[0120] a.10 5 Up to 10 12 At least two Bacillus strains with a colony forming unit / gram (CFU / g), said strains being selected from:

[0121] i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324.

[0122] ii. A strain of Bacillus subtilis deposited in DSMZ with accession number DSM 32325,

[0123] iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840; and

[0124] b. One or more animal feed, animal feed additives, or animal feed premix ingredients.

[0125] The at least two Bacillus strains thereon effectively improve gastrointestinal health and fecal scores in animals that receive the animal feed, animal feed additives or animal feed premixes.

[0126] 17. The composition of paragraph 16, wherein the at least two Bacillus strains comprise Bacillus subtilis strains and Bacillus amyloliquefaciens strains.

[0127] 18. The composition according to paragraph 16 or 17, wherein the at least two Bacillus strains comprise Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324 and Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840.

[0128] 19. The composition according to any one of paragraphs 16-18, wherein the at least two Bacillus strains consist of a Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324, a Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840, and a Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325.

[0129] 20. The composition according to any one of paragraphs 16-19, wherein the at least two Bacillus strains are present as spores.

[0130] 21. The composition according to any one of paragraphs 16-20, wherein the composition is an animal feed additive.

[0131] 22. The composition according to any one of paragraphs 16-21, wherein the composition is in the form of a dry powder, capsule, or oral paste.

[0132] 23. The composition according to any one of paragraphs 16-22, wherein the animal feed, animal feed additive or animal feed premix ingredient is selected from the list including: vitamins, minerals, enzymes, amino acids, flavorings, yeast extracts, activated carbon, mineral clay, silica or combinations thereof.

[0133] 24. A method of feeding animals, comprising administering an animal feed, animal feed additive, or animal feed premix composition comprising at least two strains of Bacillus spp., said strains being selected from:

[0134] i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324.

[0135] ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and

[0136] iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

[0137] 25. The method of paragraph 24, wherein the at least two Bacillus strains are administered in amounts that effectively improve gastrointestinal health and fecal scores in animals.

[0138] 26. The method described in paragraph 24 or 25, wherein the animal is a pet animal, such as a dog or a cat.

[0139] 27. A method for improving gastrointestinal health and fecal scores in animals, the method comprising administering to the animal in need an effective amount of the composition according to any one of paragraphs 16-23.

[0140] 28. A method for increasing sugar release and protein solubility in animal feed, comprising adding a composition containing at least two strains of Bacillus spp., wherein the Bacillus spp. strains are selected from:

[0141] (a) The strain deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM 32324, and

[0142] (b) The strain deposited in DSMZ with accession number DSM 32325, and

[0143] (c) The strain deposited in DSMZ with accession number DSM 25840.

[0144] Example

[0145] Example 1

[0146] In vitro studies were conducted to investigate the ability of compositions of this disclosure containing at least two Bacillus strains to release sugars from commercially available dried dog food roughage, thereby obtaining a measure of the degradation of the carbohydrate portion in the dried dog food roughage.

[0147] Samples of commercially available dog food roughage diets were tested with and without the addition of compositions containing at least two strains of the Bacillus genus as described in this disclosure.

[0148] The method involves mixing animal feed samples in a liquid medium containing the composition of this disclosure (probiotic sample, referred to as "PRO") and in a liquid medium without the composition of this invention (control sample, referred to as "Control"), and collecting the supernatant after growth under standardized conditions. The supernatant is then mixed with the yellow compound 3,5-dinitrosalicylic acid (DNS), which, in the presence of simple sugars (also known as reducing sugars because they act as reducing agents), transforms into the darker red / brown compound 3-amino-5-nitrosalicylic acid. The intensity of the dark color is measured using a spectrophotometer to determine the amount of reducing sugar released and the ability of the composition of this disclosure to break down carbohydrates into simple sugars.

[0149] Compositions containing probiotic strains belonging to the genus Bacillus, based on live spores of Bacillus amyloliquefaciens and Bacillus subtilis (PRO sample), resulting in increased release of reducing sugars from commercially available dog food roughage compared to untreated controls (control sample), regardless of the amount of protein and fiber in the dog food roughage. Figure 1 (Labeled as "glucose equivalent"). An additional 620 or 501 kJ of glucose / kg was released from the two different roughage diets. Unbound by theory, the increased glucose release is considered beneficial because it is a measure of the amount of energy released to the dog after ingestion of dog food roughage.

[0150] Example 2

[0151] Proteins are complex molecules present in all living organisms and provide many essential functions within the body. These molecules are formed by “building blocks” of 20 amino acids, which can be considered essential or non-essential—essential amino acids cannot be synthesized in the body and must be obtained through diet, while non-essential amino acids can be synthesized by the body through the metabolism of other amino acids. The number of essential amino acids depends on the species in question. Therefore, the dissolution of proteins is necessary to provide essential amino acids for building hair, skin, nails / toenails, muscles, tendons, ligaments, and cartilage. Proteins also play a major role in hormone production and promote immune system function.

[0152] In vitro assays were performed to study the effect of the compositions disclosed herein on protein solubility. The physical structure of a protein can affect its digestibility. By exposing proteins to proteases and other protein-degrading enzymes, their structure can be altered and their digestibility improved.

[0153] Samples of commercially available dog food roughage diets were tested with and without the addition of compositions containing at least two strains of the Bacillus genus as described in this disclosure.

[0154] The method involves mixing animal feed samples in a liquid medium containing the composition of this disclosure (probiotic sample, referred to as "PRO") or in a liquid medium without the composition of this invention (control sample, referred to as "Control"), and collecting the supernatant after growth under standardized conditions. The supernatant is then mixed with copper sulfate and dioctoctanic acid (BCA). The dissolved proteins reduce Cu2+ from copper sulfate to Cu1+, which then reacts with dioctoctanic acid (BCA) to produce a strong purple reaction product, which exhibits a strong linear absorbance at 562 nm with increasing protein concentration. Therefore, the more dissolved protein produced from the feed sample by the probiotics, the stronger the color reaction.

[0155] A composition containing probiotic strains belonging to the genus Bacillus, based on live spores of Bacillus amyloliquefaciens and Bacillus subtilis (PRO sample), resulting in increased protein solubility (in) compared to the untreated control (control sample), regardless of the amount of protein and fiber in the dog food roughage. Figure 2 (Label labeled as "grams of protein"). An additional 14 or 13 g of protein / kg of the corresponding coarse grain diet can be released from each of the two coarse grain diets, respectively. Unbound by theory, the increase in protein solubility is considered beneficial because it is a measure of the digestibility of the coarse grains after ingestion and the amino acids released to the dog as a result.

[0156] Example 3

[0157] In vivo studies were conducted in dogs in Spain. The aim of this study was to evaluate the effects of a composition containing probiotic strains belonging to the genus Bacillus, based on live spores of Bacillus amyloliquefaciens and Bacillus subtilis. Without being bound by theory, it was assumed that the probiotics would positively influence the dry matter content and stool score in feces.

[0158] A total of 40 healthy dogs were randomly assigned to two groups of 20 dogs each, maintaining a balance in terms of age, sex, and weight. Before the trial began, the dogs received only a control diet (complete diet) for a 21-day acclimatization period. After the acclimatization period, both groups of dogs received either the control diet (T1) or a control diet (T2) containing a combination of probiotic strains for 35 days.

[0159] The dogs were kept in captivity as a group, but fed individually. They were fed a completely dry, extruded diet, and given probiotics or a placebo once daily as a top dressing, corresponding to 1.0 x 10. 9 The target dose is cfu / kg of food.

[0160] Weigh the dogs weekly, and adjust the amount of food provided to them according to whether their weight loss or gain exceeds 5% of their initial weight. Obtain fecal samples on days 0, 7, 28, and 35. Perform daily feed intake and fecal scoring. Determine daily fecal consistency using a fecal score for each dog. Based on consistency, grade the feces on a scale ranging from 1 (hard, dry feces) to 5 (liquid feces), with a fecal score of 2.5 considered optimal (firm, well-formed feces). See Table 2 for a detailed scoring scale.

[0161] Table 3 reports the main results of this study. It can be concluded that a significant reduction in the number of days with unacceptable scores (>3) was observed in the probiotic-fed dogs (P = 0.041). Furthermore, it can be concluded that the fecal dry matter content increased at day 35 at the end of the study in the probiotic-fed dogs (P = 0.054). Further noteworthy is the finding that the significant improvement in fecal scores was not solely due to the increased dry matter content. For several measurements, the dry matter content was lower in the probiotic group than in the control group; however, this did not affect the overall improvement in fecal scores. This indicates an improvement in the overall gastrointestinal health of the animals.

[0162]

[0163]

[0164] The present invention has been described with reference to various embodiments, aspects, examples, etc. These elements are not intended to be read in isolation. Therefore, this disclosure provides for combinations of two or more embodiments, aspects, examples, etc.

[0165] All embodiments described herein are intended to be within the scope of the disclosed invention. These and other embodiments of the invention will become apparent to those skilled in the art upon reference to the complete specification and the following detailed description of preferred embodiments. The invention is not limited to any specific preferred embodiment disclosed. PCT / RO / 134 form

Claims

1. Use of an animal feed, animal feed additive, or animal feed premix composition for improving gastrointestinal health and / or fecal scores in animals receiving said animal feed, animal feed additive, or animal feed premix composition, said animal feed, animal feed additive, or animal feed premix composition comprising: a.10 5 Up to 10 12 At least two Bacillus strains with a colony forming unit / gram (CFU / g), said strains being selected from: i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324. ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and iii. The strain of *Bacillus amyloliquefaciens* deposited in DSMZ with accession number DSM 25840; and b. One or more animal feed, animal feed additives or animal feed premix ingredients.

2. The use according to claim 1, wherein the animal is a pet, such as a dog or cat, preferably a dog.

3. The use according to claim 2, for improving fecal scores in dogs.

4. The use according to any one of the preceding claims, wherein the at least two Bacillus strains comprise Bacillus subtilis strains and Bacillus amyloliquefaciens strains.

5. The use according to any one of the preceding claims, wherein the at least two Bacillus strains comprise Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324 and Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840.

6. The use according to any one of the preceding claims, wherein the at least two Bacillus strains comprise, or consist of, the Bacillus subtilis strain deposited in DSMZ with accession number DSM 32324, the Bacillus amyloliquefaciens strain deposited in DSMZ with accession number DSM 25840, and the Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325.

7. The use according to any one of the preceding claims, wherein the at least two Bacillus strains are present as spores.

8. The use according to any one of the preceding claims, wherein the composition is an animal feed additive.

9. The use according to any one of the preceding claims, wherein the composition is in the form of a dry powder, capsules or oral paste.

10. The use according to any one of the preceding claims, wherein the composition comprises one or more animal feed, animal feed additive or animal feed premix ingredients, said ingredients being selected from vitamins, minerals, enzymes, amino acids, flavoring agents, yeast extracts, activated carbon, mineral clay, silica or combinations thereof.

11. A method of feeding a pet animal, such as a dog or cat, the method comprising administering to the animal an animal feed, an animal feed additive, or an animal feed premix composition, said animal feed, animal feed additive, or animal feed premix composition comprising at least two Bacillus strains selected from: i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324. ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

12. A method for improving gastrointestinal health and fecal scores in pet animals, such as dogs or cats, the method comprising administering to the animal an effective amount of an animal feed, animal feed additive, or animal feed premix composition, said animal feed, animal feed additive, or animal feed premix composition comprising at least two Bacillus strains selected from: i. A strain of Bacillus subtilis deposited at the German Center for Microbiology and Cell Culture (DSMZ) with accession number DSM 32324. ii. The Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and iii. A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

13. A method for improving energy and protein intake in an animal, wherein the animal is a pet animal, such as a dog or cat, the method comprising feeding the animal with an animal feed, an animal feed additive, or an animal feed premix composition comprising at least two Bacillus strains, wherein the Bacillus strains are selected from: (a) A strain of Bacillus subtilis deposited at the German Center for Microbial and Cell Culture Collection (DSMZ) with accession number DSM 32324, and (b) Bacillus subtilis strain deposited in DSMZ with accession number DSM 32325, and (c) A strain of Bacillus amyloliquefaciens deposited in DSMZ with accession number DSM 25840.

14. The method according to any one of claims 11-13, wherein the animal feed, animal feed additive, or animal feed premix composition comprises 10% of the composition. 5 Up to 10 12 Each Bacillus strain with colony-forming units per gram (CFU / g).

15. The method according to any one of claims 11-14, wherein the animal is a dog.

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