Bifidobacterium adolescentis strains, methods and uses thereof for degrading starch and modulating intestinal flora in non-human mammals

Abstract

The invention relates to a novel host cell capable of degrading starch and / or using starch as a carbon source, wherein the host cell comprises one or more of the following sequences: (a) a polypeptide having at least 97% identity with a polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 2; (b) a polypeptide having at least 96% identity to the polypeptide set forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein the polypeptide is capable of binding to starch and / or has alpha-amylase enzymatic activity; (c) one or more polynucleotides encoding the one or more polypeptides as described in (a) to (b). In particular, the present invention relates to a novel probiotic strain of Bifidobacterium adolescentis selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" having a preservation number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" having a preservation number DSM 34065; as well as methods and uses of the novel probiotic strains for degrading starch and altering intestinal flora in non-human mammals.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0003] Deposited biological material

[0004] The present invention uses and / or relates to a deposited biological material which can only be made available by providing samples to an expert in accordance with Article 32(1) of the European Patent Convention (EPC) for those names sought in relation to a European patent until the date on which the grant of the European patent is published or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn: Bifidobacterium adolescentis designated strain "KIBAD2" having deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" having deposit number DSM 34065. Both strains were deposited on October 11, 2021 at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY. Technical Field

[0005] The present invention relates to a novel host cell capable of degrading starch and / or utilizing starch as a carbon source, wherein the host cell comprises one or more of the following sequences: (a) a polypeptide having at least 97% identity with the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 2; (b) a polypeptide having at least 96% identity with the polypeptide shown in SEQ ID NO: 3 or SEQ ID NO: 4, wherein the polypeptide is capable of binding to starch and / or has α-amylase enzymatic activity; (c) one or more polynucleotides encoding the one or more polypeptides as described in (a) to (b). Specifically, the present invention relates to a novel probiotic strain of Bifidobacterium adolescentis selected from the group consisting of: the designated strain "KI BAD2" of Bifidobacterium adolescentis having the deposit number DSM 34061, and the designated strain "KI BAD4" of Bifidobacterium adolescentis having the deposit number DSM 34065; and methods and uses of the novel probiotic strain for degrading starch and changing the intestinal flora in non-human mammals. Background Art

[0006] Carbohydrates constitute one of the basic ingredients of the diet of many livestock animals, including pigs. Typically, the main source for providing carbohydrates is starch, which is the main component of, for example, corn, cereals, yams, rice and potatoes. Starch consists of two types of polysaccharide molecules: amylose, which is a mostly straight-chain and flexible polymer composed of D-anhydroglucose units linked by α-1,4-D-glucosidic bonds, and amylopectin, which is a branched polymer of straight chains linked by α-1,6-D-glucosidic bonds. Starch is mainly digested in the small intestine of most mammals (e.g., pigs) by the enzyme α-amylase (e.g., WO2008087164). However, "resistant starch" can resist digestion and absorption in the small intestine of livestock mammals (e.g., pigs). Typically, the ingested resistant starch is passed to the large intestine, where it is fermented to a certain extent by the colonic microflora into short-chain fatty acids, especially butyrate and gas. In addition, due to the widespread and excessive use of antibiotics in animal husbandry, which has led to the destruction of the microbiome in healthy livestock animals (e.g., pigs), there is a need for novel probiotic strains that degrade resistant starch with improved properties in animal husbandry, as well as non-therapeutic methods based on the probiotic strains for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract (GIT) of livestock animals. Specifically, there is a need in the art for new methods to improve the intestinal flora in livestock species, especially to regulate the balance of beneficial and harmful (e.g., pathogenic) bacteria in the intestine of livestock species in favor of beneficial species.

[0007] Therefore, the basic technical problem of the present application is to comply with this requirement. The technical problem is solved by providing the embodiments reflected in the claims, described in the specification and illustrated in the following examples and drawings. Summary of the invention

[0008] The present invention relates to a deposited biological material which can only be made available by providing samples to an expert in accordance with Article 32(1) of the European Patent Convention (EPC) for those names for which a European patent is sought, until the date on which the grant of the European patent is published or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

[0009] In one aspect, the present invention relates to a host cell (e.g., an isolated and / or modified and / or recombinant host cell), characterized in that: (i) the host cell is capable of degrading starch, preferably the starch is resistant starch (RS), and further preferably the host cell has α-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and / or (ii) the host cell is capable of utilizing starch as a carbon source, preferably the starch is resistant starch (RS); wherein the host cell comprises one or more (e.g., 2, 3 or 4) of the following sequences: (a) with SEQ ID NO: 1 (e.g., BAD4NNHABGDB_01028 protein) or SEQ ID NO:2 (e.g., BAD2IGDMICFN_02026 protein) has at least 97% (e.g., at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO:2 (e.g., BAD2IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has α-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); (b) a polypeptide having at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO:3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO:4 (e.g., BAD2IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has α-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity). 3.2.1.1 enzymatic activity); (c) a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4; (d) a fragment of any one of (a) to (c) that is capable of binding (e.g., binds to) starch (e.g., the fragment is a starch-binding fragment) and / or has α-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); preferably, the fragment is at least 200 amino acids (e.g., at least 250, at least 300, at least 350, at least 400 amino acids) long; (e) one or more (e.g., 2, 3 or 4) polynucleotides (e.g., DNA or RNA, e.g., polycistronic or monocistronic) encoding the one or more polypeptides as in (a) to (d), preferably the one or more polynucleotides are capable of expressing (e.g., expressed) the one or more polypeptides.

[0010] In another aspect, the present invention relates to a host cell, which is selected from the group consisting of: Bifidobacterium adolescentis designated strain "KIBAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065 (for example, both strains were deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms on October 11, 2021, address: Inhofen Street 7B, D-38124, Brunswick, Germany), preferably wherein the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065 is at least 98.5% (for example, at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain "KIBAD2" with the deposit number DSM 34061.

[0011] In another aspect, the present invention relates to a polypeptide capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or having α-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), the polypeptide being selected from the group consisting of: (a) a polypeptide having at least 97% (e.g., at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein); (b) a polypeptide having at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein); or (c) a polypeptide having SEQ The polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0012] Still another aspect of the present invention relates to one or more polynucleotides encoding one or more polypeptides of the present invention.

[0013] Still another aspect of the present invention relates to one or more nucleic acid constructs or expression vectors capable of expressing one or more polynucleotides of the present invention.

[0014] In another aspect, the present invention relates to a composition or a kit comprising one or more of the following: (a) one or more host cells according to the host cells of the present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; (b) a combination of one or more host cells according to the present invention and one or more (e.g., 2) of the following probiotic strains: Enterococcus faecium strain and / or Pediococcus acidilactici strain; preferably Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065. A combination of "BAD4" and one or more (e.g., 2) of the following strains: Enterococcus faecium strain and Pediococcus acidilactici strain; (c) one or more (e.g., 2, 3 or 4) polypeptides according to the present invention; (d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing the one or more polypeptides; (e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); (f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing the one or more polynucleotides encoding the one or more polypeptides according to (c).

[0015] In another aspect, the invention relates to a composition or kit of the invention, wherein the composition or kit is a starch-degrading, nutritional (eg, nutritional supplement), prebiotic (eg, comprising resistant starch) and / or probiotic composition or kit.

[0016] In another aspect, the present invention relates to a food, an intermediate food; a forage, an intermediate forage; a feed, an intermediate feed; an additive (e.g., a food additive, a forage additive or a forage additive), an intermediate additive (e.g., a food intermediate additive, a forage intermediate additive or a feed intermediate additive); a nutritional supplement, an intermediate nutritional supplement; a prebiotic, an intermediate prebiotic, a probiotic, an intermediate probiotic and / or a mixture thereof, comprising one or more of the following: (a) one or more host cells in the host cells according to the present invention, preferably a strain selected from the group consisting of: the Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and the Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; (b) a combination of one or more host cells in the host cells according to the present invention and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain and a Pediococcus acidilactici strain; preferably, a Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065. A combination of "BAD4" and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain and a Pediococcus acidilactici strain; (c) one or more (e.g., 2, 3 or 4) polypeptides according to the present invention; (d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides; (e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); (f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c). (g) one or more (e.g., 2, 3 or 4) compositions according to the present invention.

[0017] In another aspect, the present invention relates to a method for degrading starch, preferably resistant starch (RS), comprising: (i) providing one or more of the following: (a) one or more host cells according to the present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with a deposit number of DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with a deposit number of DSM 34065; (b) one or more host cells of the host cells according to the present invention in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably, a Bifidobacterium adolescentis designated strain "KI BAD4" with a deposit number of DSM 34065. BAD4" and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); (c) one or more polypeptides according to any one of the preceding claims; (c) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., expressed) the one or more polypeptides; (d) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); (e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); (f) one or more (e.g., 2, 3 or 4) compositions according to the present invention; (g) one or more of the following: food, intermediate food; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and / or mixtures thereof; (ii) applying (i) to starch (e.g., resistant starch).

[0018] In another aspect, the present invention relates to a method for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract of a non-human mammal (e.g., a non-therapeutic method), the method comprising feeding the non-human mammal one or more of the following: (a) one or more host cells of the host cells according to the present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065; (b) one or more host cells of the host cells according to the present invention in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably, the Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065. BAD4" and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); (c) one or more (e.g., two, three or four) polypeptides according to the present invention; (d) one or more (e.g., two, three or four) polynucleotides encoding the one or more polypeptides as in (c), preferably the at least one polynucleotide is capable of expressing (e.g., has expressed) the one or more polypeptides; (e) one or more (e.g., two, three or four) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); (f) ) one or more (for example, 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); (g) one or more (for example, 2, 3 or 4) compositions according to the present invention; (h) one or more of the following: food, intermediate food; forage, intermediate forage; feed, intermediate feed; additive (for example, additive food, additive forage or feed additive), intermediate additive (for example, intermediate additive food, intermediate additive forage or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and / or mixtures thereof according to the present invention.

[0019] In another aspect, the invention relates to a method of the invention, wherein said method is an in vitro, ex vivo, in vivo and / or non-therapeutic method and / or said non-human mammal is healthy.

[0020] Overview of Sequence Listing

[0021] SEQ ID NO: 1 is the amino acid sequence of BAD4 NNHABGDB_01028 protein:

[0022]

[0023]

[0024] SEQ ID NO: 2 is the amino acid sequence of BAD2 IGDMICFN_02026 protein:

[0025]

[0026]

[0027] SEQ ID NO: 3 is the amino acid sequence of BAD4 NNHABGDB_01017 protein:

[0028]

[0029]

[0030] SEQ ID NO: 4 is the amino acid sequence of BAD2 IGDMICFN_02013 protein:

[0031]

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The inhibition of the harmful strain of E. coli O8K88 (F4) by means of supplementation with the BAD4 strain according to the invention, assessed by means of an agar spot assay (see Example 4), is shown. DETAILED DESCRIPTION

[0034] definition:

[0035] As referred to herein, "EC numbers" (Enzyme Commission Numbers) may be used to refer to enzymatic activities according to the Enzyme Nomenclature Database (available, for example, at https: / / enzyme.expasy.org / ) published on February 26, 2020. EC numbers refer to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, Calif., including Supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650, respectively.

[0036] The term "polypeptide" is used in this article as equivalent to the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, generally having less than 30 amino acids) contain one or more amino acids coupled to each other via covalent peptide bonds (thereby producing an amino acid chain). The term "polypeptide" as used herein describes a group of molecules, which, for example, are composed of more than 30 amino acids. Polypeptides can further form polymers, such as dimers, trimers and higher oligomers, i.e., are composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be identical or different. Therefore, the corresponding higher-order structures of such polymers are referred to as homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, the naturally occurring form of which is composed of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins, wherein the modifications are affected by, for example, post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well known in the art.

[0037] As used herein, the terms "polynucleotide" and "nucleic acid" or "nucleic acid molecule" should be interpreted synonymously. In general, nucleic acid molecules can include DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. In addition, the term "nucleic acid molecule" can refer to DNA or RNA or their hybrids or any modification thereof known in the art (see, for example, US 5525711, US 471 1955, US 5792608 or EP 302175 for modification examples). The polynucleotide sequence can be single-stranded or double-stranded, linear or circular, natural or synthetic, and without any size restrictions. For example, the polynucleotide sequence can be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribosomal RNA or DNA encoding such RNA or chimeroplast (Gamper et al., Nucleic Acids Res (2000), 28 (21): 4332-4339). The polynucleotide sequence can be in the form of a vector, plasmid or viral DNA or RNA. Also described herein are nucleic acid molecules complementary to the above nucleic acid molecules and nucleic acid molecules capable of hybridizing with the nucleic acid molecules described herein. The nucleic acid molecules described herein can also be fragments of nucleic acid molecules in the context of the present invention. Specifically, such fragments are functional fragments.

[0038] Sequence identity: the dependency between two amino acid sequences or between two nucleotide sequences is described by parameter " sequence identity ".For purposes of the present invention, use as the Needle program (EMBOSS:TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) of the EMBOSS bag, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) realized in preferably 5.0.0 or higher versions measures the sequence identity between two amino acid sequences.The parameter used can be 10 gap opening penalty points, 0.5 gap extension penalty points, and EBLOSUM62 (EMBOSS version of EBLOSUM62) substitution matrix.The Needle output labeled as " longest identity " (using no-brief option to obtain) is used as identity percentage, and is calculated as follows:

[0039] (Identical residues x 100) / (alignment length - total number of gaps in the alignment).

[0040] Alternatively, the parameters used can be a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "longest identity" (obtained using the no-brief option) is used as the identity percentage and is calculated as follows:

[0041] (identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment).

[0042] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] Expression vector: The term "expression vector" may refer to a linear or circular DNA molecule comprising a polynucleotide encoding a variant (polypeptide) and operably linked to control sequences that provide for the expression of the polynucleotide, in particular for the transcription of the polynucleotide.

[0044] Fragment: The term "fragment" may refer to a polypeptide having one or more (eg, several) amino acids not present at the amino terminus and / or carboxyl terminus of a mature polypeptide; wherein the fragment has an activity as described elsewhere herein.

[0045] Host cell: The term "host cell" may refer to any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The term "host cell" may also refer to all cells that contain a nucleotide sequence or expression vector to be expressed and are capable of producing or have produced an enzyme or polypeptide according to the present invention.

[0046] Nucleic acid construct: The term "nucleic acid construct" may refer to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in some manner to contain a nucleic acid segment that does not otherwise exist in nature or is synthetic, which includes one or more control sequences.

[0047] Operably linked: The term "operably linked" may refer to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0048] Control sequences: As used herein, the term "control sequences" may refer to nucleic acid sequences necessary for the expression of a polynucleotide encoding a polynucleotide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant, or may be native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. The control sequence comprises at least a promoter and transcription and translation termination signals. The control sequence may have a linker for the introduction of a specific restriction site, thereby facilitating the purpose of connecting the control sequence to the coding region of the polynucleotide of the present invention.

[0049] The term "food" may refer to a substance that has edible value.

[0050] The term "fodder" may refer to material fed to livestock.

[0051] The term "feed" may refer to substances used as food for livestock.

[0052] The term "additive" may refer to a compound or substance that is added to another product or substance, such as in small amounts, to affect desired properties and / or characteristics.

[0053] The term "prebiotic" may refer to a compound or substance that is capable of inducing the growth and / or activity of beneficial microorganisms.

[0054] The term "probiotic" may refer to microorganisms intended to have a health benefit when consumed or applied to the body.

[0055] The term "nutritional supplement" may refer to compounds or substances capable of supporting the nutritional content of a diet (eg, vitamins, minerals, and / or probiotics).

[0056] The term "intermediate" may refer to a compound or substance produced during a process to obtain a final product of the invention (e.g., during an intermediate stage of the process), such as a food, forage, feed; an additive (e.g., a food additive, a forage additive or a feed additive), a nutritional supplement, a probiotic or a prebiotic of the invention.

[0057] The term "starch" may refer to a glucose polymer in which the pyranose glucose units are bonded by α-bonds. It may consist of a mixture of amylose (15-20%) and amylopectin (80-85%). Amylose may consist of a linear chain of several hundred glucose molecules, while amylopectin is a branched molecule made of several thousand glucose units (each chain of 24-30 glucose units is an amylopectin unit). Thus, starch may consist of two types of polysaccharide molecules: amylose, which is a mostly linear and flexible polymer composed of D-anhydroglucose units linked by α-1,4-D-glucosidic bonds, and amylopectin, which is a branched polymer of linear chains linked by α-1,6-D-glucosidic bonds. Starch may have Formula I:

[0058]

[0059] The term "resistant starch" may refer to starch that resists digestion and absorption in the small intestine of, for example, mammals. Resistant starch (RS) can be divided into four categories based on the reason for resistance. RS1 is a starch that is physically inaccessible because the granules are enclosed in a protein matrix or plant cell wall. RS2 is a granular starch that resists digestion by pancreatic alpha-amylase. RS3 is a coagulated, non-granular starch or starch food. RS4 is a resistant starch that has bonds other than alpha-1,4-glycosidic bonds and alpha-1,6-D-glycosidic bonds. Resistant starch in the context of the present invention includes, but is not limited to, these four categories.

[0060] Alpha-amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) as used herein constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.

[0061] The term "starch binding" may refer to the functional property of a polypeptide having binding specificity to starch, particularly raw starch.

[0062] As used herein, "the balance of beneficial bacteria to harmful bacteria" is equivalent to the longer phrase "the balance of the levels of beneficial bacteria to harmful bacteria". As described below, a skilled person can determine such "levels", and as used herein, the "levels" are equivalent to "amounts". Therefore, the longer phrase as previously described can also be read as "the balance of the amounts of beneficial bacteria to harmful bacteria".

[0063] As used herein, the term "intestinal tract" is equivalent to the term "gastrointestinal tract." Thus, one term may be substituted for the other and vice versa.

[0064] The intestinal flora, i.e., the community of bacteria that normally inhabit the gastrointestinal tract, contains both beneficial and harmful types or species of bacteria. Whether a particular member of the intestinal flora is beneficial, harmful, or indifferent to the health of a non-human mammalian species in a particular situation can depend on many factors, but for the purposes of the present invention, certain types or species of bacteria may be considered beneficial, while other types or species of bacteria are considered harmful. Examples of beneficial members of the intestinal flora include bifidobacteria (species of the genus Bifidobacterium) and lactic acid bacteria, more specifically species of the genus Lactobacillus and Prevotella spp. bacteria.

[0065] Harmful bacteria include pathogenic bacteria or conditionally pathogenic bacteria. Examples of harmful members of the intestinal flora include pathogenic forms of Escherichia coli.

[0066] Gastrointestinal health generally depends on the maintenance of an appropriate balance between beneficial and harmful bacteria. An increase in the level of harmful bacteria and / or a decrease in the level of beneficial bacteria may be associated with a decrease in gastrointestinal health. Conversely, an increase in the level of beneficial bacteria and / or a decrease in the level of harmful bacteria may be associated with an improvement in gastrointestinal health, such as an improvement in health.

[0067] "Improvement" or "improvement" or other grammatical forms include "enhancement" or "improvement" of the balance between the levels of beneficial bacteria and harmful bacteria as described herein. "Improvement" herein means to tilt the balance in favor of the levels of beneficial bacteria, and thus may involve increasing the levels of beneficial bacteria and / or reducing the levels of harmful bacteria. In some embodiments of the invention, the enhancement of the balance results from a reduction in the levels of harmful bacteria (e.g., pathogenic bacteria) and an increase in the levels of beneficial bacteria.

[0068] As shown in Example 3 herein, the increase in the level of beneficial bacteria in the intestine of a non-human mammal species fed the products as described herein compared to a non-human mammal not fed the products as described herein encompasses an increase in the level of beneficial bacteria in the intestine of the non-human mammal by 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250% or more. Thus, in the context of the methods and uses of the present invention, the increase in the level of beneficial bacteria may advantageously be 100%, 125%, 150%, 175%, 200% or more.

[0069] During the course of the present invention, it was surprisingly discovered that two newly isolated strains of the species Bifidobacterium adolescentis, referred to herein as Bifidobacterium adolescentis BAD4 and Bifidobacterium adolescentis BAD2, can degrade starch, a property not found in other strains of this species.

[0070] It is further demonstrated that the B. adolescentis BAD4 and B. adolescentis BAD2 of the present invention carry a collection of genes associated with enzymatic starch degradation. According to the whole genome comparative analysis performed in the process of the present invention, these genes are not present in the genome of the reference strain of B. adolescentis that cannot utilize starch as a carbon source.

[0071] In a preferred embodiment of the present invention, feeding starch-degrading bacteria to animals (eg, livestock non-human mammals) as live probiotics can improve feed digestibility, nutrient intake and / or animal growth, especially for feeds containing starch.

[0072] Furthermore, it was surprisingly found that the combination of Bifidobacterium adolescentis BAD4 with two other strains (Enterococcus faecium E16 and Pediococcus acidilactici G2) had a beneficial effect on the intestinal microflora of pigs. The results showed that the relative abundance of several butyrate-producing bacteria inhabiting the pig intestine could be increased with the help of the strains of the invention (see the Examples section herein). Butyrate is known to help maintain the intestinal barrier function and is also known to have immunomodulatory and anti-inflammatory properties.

[0073] It was also surprisingly found that B. adolescentis BAD4 and B. adolescentis BAD2 had superior inhibitory activity against E. coli porcine pathogen strains compared to a reference strain of B. adolescentis, as shown by agar spot test (see the Examples section herein). Inhibition of naturally occurring zoonotic pathogens in the porcine intestine, such as E. coli, by applying B. adolescentis to animal feed is a promising measure for reducing the incidence of post-weaning diarrhea in pigs.

[0074] Livestock farmers (e.g., pig farmers) typically add several different feed additives (e.g., non-therapeutic additives) to the diet of their animals to maintain gut health and improve performance. In order to reduce the number of necessary products and also the cost, a product with a broad spectrum of functionality is needed, such as balancing a healthy gut microbiome, inhibiting potential pathogens and / or also enhancing digestibility and growth at the same time.

[0075] On the other hand, probiotics have been used for many years in human and animal nutrition and specific bacterial species have a long history of safe use. Most of the well-known probiotic products worldwide consist of a single strain. Commonly used species are Bacillus subtilis, Saccharomyces cerevisiae, Enterococcus faecium, Lactobacillus acidophilus. Compared to the previously named species, species of the genera Pediococcus spp. and Bifidobacterium spp. are rarely used in the development of feed additives.

[0076] Roselli et al., 2017, provided an overview of the main results of in vitro testing of porcine intestinal cell lines with probiotic strains and the effects of prebiotics and probiotics on the composition of the porcine microbiota and on pathogen adhesion and diarrhea in pigs.

[0077] However, in pigs, Lactobacillus acidophilus produces lactic acid and the enzyme amylase during the pig's growing phase. Lactobacillus casei and Enterococcus faecium complement Lactobacillus acidophilus, also producing these chemicals, but also enjoying a wide temperature and pH range. Both of these microorganisms create an acidic environment that is unfavorable to pathogenic bacteria. Bifidobacterium thermophilum is a delicate microorganism found in breast milk and in the gastrointestinal tract of humans and animals. Restoring this bacterium could be used for the health and growth of animals.

[0078] Despite this, there is no prior art describing the benefits of using Bifidobacterium adolescentis alone or in combination with other species as described herein as a probiotic feed additive for pigs. The present invention therefore meets this need by providing strains of the invention and related products as described herein that are able to reduce the number of ingredients to be added to a "standard" pig diet.

[0079] Thus, without being bound by theory, the present invention provides more sustainability for livestock farming because the levels of beneficial bacteria in the intestine can be increased - most likely they will colonize and remain in the intestine, which is more sustainable than simply adding beneficial bacteria through the feed (whereby such externally added beneficial bacteria may not colonize and remain in the intestine). Sustainability is therefore hypothesized to reside in the fact that in the long term, non-human mammalian species benefit from the increased levels of beneficial bacteria caused by - as shown in the examples - exogenous feeding of the probiotic strains of the present invention, rather than by constantly receiving other beneficial bacteria through the feed that may not colonize and reside in the intestine.

[0080] Based on the above, the products, methods and uses of the present invention as described herein have at least the following advantages over known products:

[0081] - Starch degradation (phenotypically demonstrated by API testing);

[0082] - α-amylase activity (genotype-specific by genome-wide comparative analysis);

[0083] - beneficial modulation of the swine intestinal flora by enhancing the relative abundance of Prevotella in the large intestine in combination with Enterococcus faecium and Pediococcus acidilactici; and / or

[0084] -Inhibits porcine pathogens (E. coli F4 and F18) that cause post-weaning diarrhea.

[0085] Items of the present invention

[0086] The present invention can also be summarized by the following items:

[0087] 1. A host cell (e.g., an isolated and / or modified and / or recombinant and / or transformed host cell), characterized in that:

[0088] i) the host cell is capable of degrading starch, preferably the starch is resistant starch (RS) (e.g., starch that resists digestion and absorption in the small intestine of, for example, a mammal), further preferably the host cell has α-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and / or

[0089] ii) the host cell is capable of utilizing starch as a carbon source, preferably the starch is resistant starch (RS);

[0090] wherein the host cell comprises one or more (e.g., 2, 3 or 4) of the following sequences:

[0091] a) a polypeptide having at least 88% (e.g., at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0092] b) a polypeptide having at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0093] c) a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4;

[0094] d) a polypeptide having at least 78% (e.g., at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 95%) identity to the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0095] e) a polypeptide having at least 82% (e.g., at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 94%) identity to the polypeptide shown in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0096] f) a polypeptide having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0097] g) a polypeptide having at least 93% (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0098] h) a polypeptide having at least 78% (e.g., at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 94%) identity to the polypeptide shown in SEQ ID NO:4 (e.g., BAD2 IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0099] i) A polypeptide according to (a) to (h), wherein the polypeptide is an α-amylase-like protein;

[0100] j) A fragment of any one of (a)-(i), which fragment is capable of binding (e.g., binds to) starch (e.g., the fragment is a starch-binding fragment) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); preferably, the fragment is at least 200 amino acids (e.g., at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100 or at least 1200 amino acids) long;

[0101] k) one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) polynucleotides (e.g., DNA or RNA, e.g., polycistronic or monocistronic) encoding the one or more polypeptides as in (a) to (j), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides.

[0102] 2. The host cell according to any of the preceding items, wherein the host cell is prokaryotic (e.g., Bifidobacterium spp., Bacteroides spp. (e.g., B. thetaiotaomicron), Ruminococcus spp. (e.g., R. bromii), Escherichia spp. (e.g., Escherichia coli), Bacillus spp. (e.g., Lactobacillus acidophilus), Lactobacillus spp. (e.g., Lactobacillus amylovora), amylovorus, Lactobacillus acidophilus, L. salivarius, L. plantarum, L. buchneri, L. fermentum, L. delbrueckii, L. brevis and / or L. bulgaricus) or a eukaryotic cell (e.g., a yeast cell, e.g., Saccharomyces cerevisiae).

[0103] 3. The host cell according to any one of the preceding items, wherein the host cell is a Bifidobacterium species strain (eg, a Bifidobacterium adolescentis strain).

[0104] 4. The strain according to any one of the preceding items, wherein the strain is selected from the group consisting of: the designated strain "KI BAD2" of Bifidobacterium adolescentis with the deposit number DSM 34061, and the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM 34065 (for example, both strains were deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms on October 11, 2021, address: Inhofenstrasse 7B, D-38124, Brunswick, Germany), preferably wherein the whole genome nucleotide sequence of the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM 34065 is at least 98.5% (for example, at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of the designated strain "KI BAD2" of Bifidobacterium adolescentis with the deposit number DSM 34061.

[0105] 5. The host cell according to any one of the preceding clauses, wherein the host cell has one or more of the following characteristics:

[0106] a) the host cell is a Gram-positive, non-motile and / or anaerobic bacterial cell / strain;

[0107] b) the host cell is a bacterial strain of Bifidobacterium adolescentis;

[0108] c) the host cell is capable of improving the balance of beneficial and harmful bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock ungulate mammal such as a horse, or a livestock ungulate mammal such as a cattle, sheep, goat or pig), preferably the non-human mammal is a Sus sp., such as a domestic pig (Sus domesticus);

[0109] d) A host cell according to (c), wherein the improvement comprises an increase in the level of beneficial bacteria and / or a decrease in the level of harmful bacteria, preferably the improvement is a modulation of the GIT microflora, for example by improving the relative abundance of bacteria of the genus Prevotella (e.g. in the large intestine of pigs);

[0110] e) A host cell according to (c) or (d), wherein said improvement comprises improving the digestibility of feed and / or the growth of said non-human mammal;

[0111] f) A host cell according to any one of (c) to (e), wherein the beneficial bacteria comprise one or more butyrate-producing bacterial strains (e.g., Eubacterium spp., such as E. rectale, and / or Prevotella strains, such as in the large intestine of non-human mammals, such as pigs); and / or the harmful bacteria comprise one or more Escherichia coli strains (e.g., pathogenic E. coli strains, such as pig pathogens causing post-weaning diarrhea (E. coli F4 and / or F18 strains); and / or

[0112] g) the host cell is capable of degrading and / or utilizing one or more of the following carbohydrates as a carbon source: L-arabinose, ribose, D-xylose, galactose, glucose, fructose, arbutin, esculin, salicin, maltose, lactose, melibiose, sucrose, raffinose, starch, glycogen, turanose, preferably the host cell is the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM34065.

[0113] 6. A polypeptide capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), the polypeptide being selected from the group consisting of:

[0114] a) a polypeptide having at least 97% (e.g., at least 98%, at least 99% or 100%) identity to the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein);

[0115] b) a polypeptide that is at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identical to the polypeptide set forth in SEQ ID NO:3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO:4 (e.g., BAD2IGDMICFN_02013 protein); or

[0116] c) a polypeptide having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;

[0117] d) a polypeptide having at least 88% (e.g., at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity);

[0118] e) a polypeptide having at least 82% (e.g., at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%, preferably at least 94%) identity to the polypeptide shown in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity).

[0119] 7. A polynucleotide encoding one or more polypeptides according to any one of the preceding items.

[0120] 8. A nucleic acid construct or expression vector capable of expressing one or more polynucleotides according to any one of the preceding items.

[0121] 9. A composition or kit comprising one or more of the following:

[0122] a) one or more host cells according to any of the preceding items, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065;

[0123] b) a combination of one or more host cells according to any of the preceding items with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain);

[0124] c) one or more (eg, 2, 3 or 4) polypeptides according to any one of the preceding items;

[0125] d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides;

[0126] e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d);

[0127] f) one or more (eg, 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c).

[0128] 10. The composition or kit according to any one of the preceding clauses, wherein the composition or kit is a starch degrading, nutritional (eg nutritional supplement), prebiotic and / or probiotic composition or kit.

[0129] 11. The composition or kit according to any one of the preceding clauses, further comprising starch (eg, resistant starch).

[0130] 12. A food, an intermediate food; a fodder, an intermediate fodder; a feed, an intermediate feed; an additive (e.g., a food additive, a fodder additive or a feed additive), an intermediate additive (e.g., a food intermediate additive, a fodder intermediate additive or a feed intermediate additive); a nutritional supplement, an intermediate nutritional supplement; a prebiotic, an intermediate prebiotic, a probiotic, an intermediate probiotic and / or a mixture thereof, comprising one or more of the following:

[0131] a) one or more host cells according to any of the preceding items, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065;

[0132] b) a combination of one or more host cells according to any of the preceding items with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain);

[0133] c) one or more (eg, 2, 3 or 4) polypeptides according to any one of the preceding items;

[0134] d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides;

[0135] e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d);

[0136] f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c);

[0137] g) one or more (e.g., 2, 3 or 4) compositions according to any one of the preceding clauses;

[0138] Preferably, the food, intermediate food; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and / or mixtures thereof are for non-human mammals (e.g., pigs).

[0139] 13. A method for degrading starch, preferably resistant starch (RS), the method comprising:

[0140] (i) provide one or more of the following:

[0141] a) one or more host cells according to any of the preceding items, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065;

[0142] b) a combination of one or more host cells according to any of the preceding items with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain);

[0143] c) one or more polypeptides according to any of the preceding items;

[0144] d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides;

[0145] e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d);

[0146] f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c);

[0147] g) one or more (e.g., 2, 3 or 4) compositions according to any one of the preceding clauses;

[0148] h) one or more of the following: food, intermediate food according to claim 10; forage, intermediate forage; feed, intermediate feed; additive (e.g. additive food, additive forage or feed additive), intermediate additive (e.g. intermediate additive food, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotics, intermediate prebiotics, probiotics, intermediate probiotics and / or mixtures thereof;

[0149] (ii) applying (i) to the starch.

[0150] 14. A method for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract of a non-human mammal, the method comprising feeding the non-human mammal one or more of:

[0151] a) one or more host cells according to any of the preceding items, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065;

[0152] b) a combination of one or more host cells according to any of the preceding items with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain);

[0153] c) one or more (eg, 2, 3 or 4) polypeptides according to any one of the preceding items;

[0154] d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably at least one of the polynucleotides is capable of expressing (e.g., has expressed) the one or more polypeptides;

[0155] e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d);

[0156] f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c);

[0157] g) one or more (e.g., 2, 3 or 4) compositions according to any one of the preceding clauses;

[0158] h) one or more of the following: food, intermediate food according to any of the preceding items; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotics, intermediate prebiotics, probiotics, intermediate probiotics and / or mixtures thereof.

[0159] 15. The method according to any of the preceding clauses, wherein the method is an in vitro, ex vivo, in vivo and / or non-therapeutic method, and / or the non-human mammal is healthy.

[0160] 16. One or more of the following:

[0161] a) one or more host cells according to any of the preceding items, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with deposit number DSM 34065;

[0162] b) a combination of one or more host cells according to any of the preceding items with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain);

[0163] c) one or more (eg, 2, 3 or 4) polypeptides according to any one of the preceding items;

[0164] d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably at least one of the polynucleotides is capable of expressing (e.g., has expressed) the one or more polypeptides;

[0165] e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d);

[0166] f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c);

[0167] g) one or more (e.g., 2, 3 or 4) compositions according to any one of the preceding clauses;

[0168] h) one or more of the following: food, intermediate food according to any of the preceding items; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and / or mixtures thereof;

[0169] For use in one or more of the following:

[0170] i) for degrading starch (e.g., resistant starch, RS);

[0171] ii) for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g. a livestock ungulate mammal), preferably the non-human mammal is a Sus species, e.g. a domestic pig; preferably, wherein the improvement comprises an increase in the level of beneficial bacteria and / or a decrease in the level of harmful bacteria, preferably the improvement is a modulation of the GIT microflora, e.g. by improving the relative abundance of bacteria of the genus Prevotella (e.g. in the large intestine); further preferably, wherein the improvement comprises improving the digestibility of feed and / or the growth of the non-human mammal; most preferably, wherein the beneficial bacteria comprise one or more butyrate-producing bacterial strains (e.g. a Eubacterium species, e.g. Eubacterium rectum, and / or a strain of the genus Prevotella, e.g. in the large intestine); and / or the harmful bacteria comprise one or more strains of Escherichia coli (e.g. a pathogenic E. coli strain, e.g. a pig pathogen causing post-weaning diarrhoea (E. coli F4 and / or F18 strains);

[0172] iii) The use according to (ii), wherein the non-human mammal is healthy;

[0173] iv) the use according to any one of (i) to (iii), wherein the use is a non-therapeutic use;

[0174] v) Any combination of (i) to (iv);

[0175] vi) The use according to (i) to (v), wherein the use is in vitro, ex vivo, in vivo and / or non-therapeutic.

[0176] 17. The method, host cell, composition, kit or use according to any one of the preceding clauses, wherein the non-human mammal is an ungulate mammal (e.g., a horse or livestock, such as cattle, sheep, goats or pigs), preferably the non-human mammal is a species of the genus Sus, such as a domestic pig.

[0177] ***

[0178] Unless otherwise stated, the following terms used in this document (including the specification and claims) have the definitions given below.

[0179] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. The present invention is intended to encompass such equivalents.

[0180] It should be noted that as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may be modified or substituted for the methods described herein.

[0181] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. The present invention is intended to encompass such equivalents.

[0182] The term "and / or" used anywhere herein includes the meanings of "and", "or" and "all or any other combinations of the elements connected by the term".

[0183] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5% of a given value or range. However, it also includes specific numbers, for example, about 20 includes 20.

[0184] Throughout the specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "include" and "comprising" should be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprises" may be replaced with the term "contains" or "including", or sometimes when used herein, may be replaced with the term "having".

[0185] When used in this article, "consisting of" excludes any elements, steps or ingredients not specified in the claim elements. When used in this article, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0186] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of may be replaced with any of the other two terms.

[0187] It should be understood that the present invention is not limited to the specific methods, protocols, materials, reagents and substances described herein, and therefore may vary. The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims.

[0188] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.) are hereby incorporated by reference in their entirety. Nothing herein shall be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. If material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material.

[0189] Example

[0190] The following examples illustrate the present invention. These examples should not be construed as limiting the scope of the present invention. The examples are included for illustrative purposes only, and the present invention is limited only by the claims.

[0191] Example 1: Starch degradation

[0192] Materials and methods

[0193] The carbohydrate metabolism of the Bifidobacterium adolescentis BAD4 strain of the invention was determined by the API 50CH test system (Biomerieux) and compared with the most advanced strains available.

[0194] Bacterial culture

[0195] Bacterial cultures were prepared from single colonies grown on LSM-Cys agar plates and incubated anaerobically for 48 h at 37° C. using AnaeroGen bags. After the incubation time, single colonies were inoculated into 10 mL of LSM-Cys broth and incubated anaerobically for 24 to 48 h at 37° C. using AnaeroGen bags. The purity of the ON cultures was checked via microscopy.

[0196] Inoculum and test strip preparation

[0197] Transfer the bacterial culture to a 15 mL test tube and centrifuge at 3000 rpm for 5 rpm. Place the API 50 test strip in the incubation tray just before use. After centrifugation, remove the supernatant and resuspend the pellet with 100 μl of 50 CHL medium. Fill each well of the test strip with 150 μL of the suspension and cover with 1-2 drops of paraffin oil and incubate in a box at 37° C. for 48 h. Evaluate the color reaction after 24 h and 48 h of incubation.

[0198] result

[0199] API 50 test

[0200] The carbohydrate utilization of the BAD4 strain of the present invention was compared with that of the BAD4 strain of Bifidobacterium adolescentis 126. Position 36 in Table 1 shows that BAD4 can utilize starch, whereas strain 126 cannot.

[0201] Table 1. Comparison of carbohydrate utilization of Bifidobacterium adolescentis strains:

[0202]

[0203]

[0204]

[0205] Example 2: Genotype exploration of genes related to starch degradation

[0206] Whole genome sequence analysis was performed on strains B. adolescentis BAD4 and BAD2 to assess the presence of genetic features associated with the degradation of resistant starch (RS). Two independent and different sequencing strategies were used to obtain the most complete genomes possible for these strains: conventional double-end short read sequencing using Illumina, and long read sequencing using a new generation sequencing platform from Oxford Nanopore according to the manufacturer's instructions. This assembly was used for protein annotation and comparative genomic analysis.

[0207] Materials and methods

[0208] Sequencing strategy

[0209] Long-read MinION sequencing

[0210] Long read sequencing was performed in-house on a Mk1C device from Oxford Nanopore. Purified DNA was used as input for sequencing library preparation using a ligation sequencing kit (SQK-LSK109) and then sequenced with a run time of 48 h. Base calling and demultiplexing of Oxford Nanopore sequencing data were performed using guppy_basecaller (version 3.4.3+f4fc735) and the fast algorithm (parameters: -configdna_r9.4.1_450bps_fast.cfg -barcode_kits'EXP-NBD104 EXP-NBD114' -min_qscore 7 -trim_strategy dna-recursive) (https: / / community.nanoporetech.com / protocols / Guppy-protocol / v / gpb_2003_v1_revo_14dec2018 / linux-guppy—last accessed on 2021-02-17). Trimming and filtering of long reads were included during base calling and demultiplexing.

[0211] Short-read Illumina sequencing

[0212] The Allegro Targeted Genotyping Kit (TECAN) was used by LGC Genomics GmbH according to the manufacturer's instructions and then the plexWell TM 384 Library Preparation Kit (seqWell TM ) performed Illumina sequencing. The library was amplified for 14 cycles in emulsion PCR using standard Illumina primers. Size selection of the Illumina library was performed on LMP-agarose gel electrophoresis, selecting a range between 300bp and 800bp. The final library purification step and library quality control were completed with BioAnalyzer and Qubit. On the NextSeq 500 / 550V2Illumina instrument, the prepared library was sequenced by double-end 2×150bp. Sequencing reads were trimmed based on quality and Illumina adapters were removed using Trimmomatic (version 0.39) (parameters: ILLUMINACLIP:NexteraPE-PE.fa:2:30:10LEADING:3TRAILING:3SLIDINGWINDOW:10:20MINLEN:30), and reads shorter than 30bp were discarded.

[0213] Assembly and protein annotation

[0214] Hybrid assembly was performed by combining short and long sequencing reads. For this purpose, the assembly pipeline Unicycler (version 0.4.8, parameters: default) (Wick et al., 2017) was used. This pipeline assembles Illumina short sequencing reads with SPAdes (Bankevich et al., 2012) and performs long read assembly with Miniasm and multiple rounds of polishing using Racon (Vaser et al., 2017), using Illumina contigs as anchors to improve the assembly.

[0215] De novo assembled contigs were annotated using the software Prokka (version 1.13.7 with parameters: -centre XXX) (https: / / github.com / tseemann / prokka) (Seemann 2014) to identify open reading frames (ORFs) and proteins.

[0216] Comparative genomic analysis

[0217] Comparative genomic analysis was performed with PATRIC and RASTk to identify proteins present in some genomes but not in others. A special focus was set on proteins belonging to key pathways of carbohydrate metabolism. After identifying these specific features, a global alignment of the protein sequences with the sequences of amylolytic enzymes from strain Bifidobacterium adolescentis P2 / P3 (Jung et al., 2020) was performed. The alignment was performed with the Needleman-Wunsch alignment algorithm implemented in the Needle software from EMBOSS (Rice et al., 2000).

[0218] The genomes included in the comparative genomic analysis were selected based on information on the ability to degrade resistant starch (Table 2).

[0219] Table 2. B. adolescentis strains included in the comparative genomic analysis:

[0220]

[0221] result

[0222] Identification of genes associated with amylase activity

[0223] Based on comparative genomic analysis, the genomes listed in Table 2 were screened for unique features that were present in only some genomes but not in others. In particular, genetic features classified as alpha-amylase (EC 3.2.1.1) were found in the genomes of BAD4 and BAD2, and in addition in the genomes of Eg1, L2-32, P2 / P3 and Ru424 (Table 3). These sequences were not detected in the genome of the model strain E194a of Bifidobacterium adolescentis, nor in the genome of strain E298b. The sequences of these genetic components were extracted for further comparison.

[0224] Table 3. Identification of unique genetic components that may perform α-amylase function:

[0225]

[0226]

[0227] Local alignment of amylase-related sequences

[0228] All coding sequences identified in the genomes of B. adolescentis BAD4 and BAD2 were compared with a set of 19 genes encoding amylolytic enzymes described for B. adolescentis P2 / P3 (Jung et al., 2020). Two sequences with similarity to the amylolytic enzymes present in P2 / P3 were identified in BAD4 (NNHABGDB_01028 and NNHABGDB_01017), and three sequences with similarity to the amylolytic enzymes were identified in BAD2 (IGDMICFN_02026, IGDMICFN_02014, and IGDMICFN_02013). These sequences are not present in the model strain B. adolescentis E194a (DSM20083 / ATCC 15703) (Table 4).

[0229] Based on similar blast comparisons, these sequences were also absent in strain Bifidobacterium adolescentis E298b (DSM20086 / ATCC15705). Other amylolytic enzymes present in P2 / P3 were also present in strains BAD4 and BAD2 with high percent identity and coverage (Table 4).

[0230] Based on the available protein function data for P2 / P3, the identified sequences have α-amylase activity and the presence of these enzymes confers the ability to utilize resistant starch. Since two of the starch-binding sequences from P2 / P3 were found in both BAD4 and BAD2, further characterization of these sequences was performed. The remaining 16 sequences of P2 / P3 associated with amylase activity described by Jung et al. (2020) were also identified in BAD4, BAD2, and a model strain of Bifidobacterium adolescentis that is unable to utilize RS, suggesting that these enzymes may be involved in the general degradation of other starches and other carbohydrates.

[0231] Table 4. Coverage and percent identity based on local alignment with protein sequences of B. adolescentis P2 / P3 (Jung et al., 2020). B. adolescentis BAD4 and B. adolescentis BA2 are able to degrade resistant starch, while B. adolescentis E298b is unable to degrade resistant starch. The relevant proteins for the degradation of resistant starch are marked in bold:

[0232]

[0233]

[0234]

[0235]

[0236] Global alignment of starch-binding proteins

[0237] There is high identity between the sequences of the starch-binding proteins identified in BAD4 and BA2, as shown below.

[0238] Proteins similar to CV760_07855

[0239] A global alignment of the amino acid sequences of starch-binding proteins similar to CV760_07855 in P2 / P3 identified in BAD4 (CDS NNHABGDB_01028) and BAD2 (CDSIGDMICFN_02026) showed a high percentage of identity (94.88%) between the two strains. The overall similarity between the two proteins and the CV760_07855 coding sequence was also estimated, showing lower similarity (77.3% for NNHABGDB_01028 and 77.5% for IGDMICFN_02026).

[0240] Proteins similar to CV760_07945

[0241] Similarly, a global alignment of the amino acid sequences of starch-binding proteins similar to CV760_07855 in P2 / P3 identified in BAD4 (labeled here as NNHABGDB_01017) and BA2 (labeled as IGDMICFN_02013) showed a high identity percentage of 93.47% between the two strains. The overall similarity between the two proteins and the CV760_07855 coding sequence was also estimated, showing lower similarity (81.20% for NNHABGDB_01017 and 77.5% for IGDMICFN_02013).

[0242] Whole-genome comparison

[0243] The whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 was determined to be 98.5% identical to that of Bifidobacterium adolescentis designated strain "KIBAD2" having the deposit number DSM 34061.

[0244] Example 3: Beneficial modulation of intestinal microflora

[0245] A feeding trial was performed on piglets to investigate the effect of a combination of Bifidobacterium adolescentis BAD4, Enterococcus faecium and Pediococcus acidilactici on the intestinal microbiota.

[0246] Materials and methods

[0247] Feeding trial

[0248] The piglet trial was conducted from June 18 to July 30, 2020 at Swine Services Unlimited, Inc. (Minnesota, US).

[0249] Four rooms were used, with a total of 64 pens, each accommodating 8 pigs. Treatments were distributed throughout the rooms to reduce room effects. The pigs were housed for a total of 42 days, during which time performance was measured for each of the 3 stages. Blood and feces were collected on the 21st day and also on the 42nd day, and a subset of the pigs were necropsyed on the 42nd day.

[0250] A prototype was tested at three different doses (low-medium-high). The prototype contained a combination of Bifidobacterium adolescentis BAD4, Pediococcus acidilactici, Enterococcus faecium. The low dose had a total microbial count of 2.5E+08 cfu / kg feed, the medium dose had a total microbial count of 5.0E+08 cfu / kg feed, and the high dose had a total microbial count of 7.5E+08 cfu / kg feed.

[0251] Table 5. Feeding trial results (NC - negative control).

[0252] # Dietary treatment Fences Animals / Fences 1 Basic diet (NC) 8 8 2 NC+ Prototype 1PS-1 Low Dose 8 8 3 NC+ Prototype 1PS-1 Medium Dose 8 8 4 NC+ Prototype 1PS_1 High Dose 8 8

[0253] Microbiome analysis

[0254] Intestinal digesta (ileum and colon) and fecal samples were sequenced at LGC Genomics (Illumina 16S amplicon sequencing). 16s rRNA sequences were provided to assess the effects of probiotic supplementation on the taxonomic profile of the piglet gut microbiota. Data were collected from pig small intestinal digesta from animals fed 3 different doses of probiotic supplementation (low, medium, and high) on day 42 of the feeding trial. Differences between controls and treatments were assessed using a Dada2-based pipeline followed by Calypso downstream analysis.

[0255] BioIT Pipeline

[0256] The reads were preprocessed according to the parameters provided in the data preprocessing report ( / ngsstorage1 / JGI / pigstar2 / Data_description.pdf). The input to Dada2 version 1.16 (1) was a collection of reads without primers and sequence adapters. These reads were processed according to the steps provided in the provided command line document ( / ngsstorage1 / JGI / PSEU / commandlines.Rhistory), and for taxonomic classification, RDP classification training data formatted for DADA2 version 18 was used (https: / / zenodo.org / record / 4310151). The Dada2 output was processed to generate the files needed to be loaded as input to Calypso (2) and normalized using the CSS+log method. Data analysis was performed on this platform as previously described (e.g., Callahan et al., Nature Methods, Vol. 13, 7 (2016): 581-3; Zakrzewski et al., Bioinformatics, Vol. 33, No. 5, March 1, 2017).

[0257] result

[0258] Table 6. Fold changes in relative abundance of bacterial genera in colon samples from animals fed a medium dose of prototype 1 (PS-1med) compared to negative controls without probiotic supplementation, obtained by differential abundance analysis (DeSeq2):

[0259] Taxonomic group P-value Mean value of PS-1-Med Control mean Fold change Prevotella 0.0048 12.93 10.92 1.18 Lactobacillus 0.0054 6.53 10.37 -1.6 Roseburia 0.014 9.86 5.56 1.77 Megasphaera 0.016 8.99 3.2 2.81 Faecalibacterium 0.022 9.46 7.78 1.22

[0260] Table 7. Fold changes in relative abundance of bacterial genera in colon samples from animals fed Prototype 1 at dose 2 (PS-1-high) compared to negative controls without probiotic supplementation, obtained by differential abundance analysis (DeSeq2):

[0261] Taxonomic group P-value PS-1-High mean Control mean Fold change Prevotella 0.024 12.67 10.92 1.16 Lactobacillus 0.057 8.65 10.37 -1.2 Roseburia 0.088 8.45 5.56 1.52 Megasphaera 0.086 7.79 3.2 2.43 Faecalibacterium 0.13 9.12 7.78 1.17

[0262] Prevotella-driven enterotypes were positively correlated with animal traits including feed intake, feed efficiency, weight gain, and diarrhea incidence. Prevotella, Megasphaera elsdenii, and Roseburia have previously been reported as SCFA producers, which are beneficial for strengthening the intestinal cell wall barrier and have beneficial immunomodulatory effects.

[0263] Example 4: Pathogen Inhibition

[0264] Materials and methods

[0265] To determine the ability of B. adolescentis BAD to inhibit the porcine pathogen E. coli, the following protocol was followed.

[0266] Day 1: Prepare an overnight culture of probiotics in LSM-Cys and incubate anaerobically at 37 °C overnight.

[0267] Day 2: Prepare LSM-Cys agar plates (20 mL agar / plate); prepare preculture of probiotics and incubate to desired OD690: Bifidobacterium: OD690 0.250 ± 0.02); spot probiotics (10E+5 cells / spot) onto LSM-Cys agar; incubate plates anaerobically at 37°C for 48 h using AnaeroGen bags for bacterial spot formation.

[0268] Day 3: Prepare an overnight culture of pathogenic bacteria (E. coli) in Nutrient Broth (NB) and incubate under the desired incubation conditions.

[0269] Day 4: Prepare 0.6% soft nutrient agar; prepare preculture of pathogen in NB; inoculate preculture of pathogen strain (final concentration 7 log cfu / mL) into soft agar (wait until cooled to 50°C) and pour the soft agar onto plates (10 mL / plate); incubate plates anaerobically at 37°C for 24 h (agar spot assay).

[0270] After the incubation period, the inhibition zone around the probiotic colonies was measured by using the HiAntibiotic ZoneScale (from the outer edge of the bacterial spot to the outer edge of the clearing zone).

[0271] The antibacterial activity of the probiotic strains after incubation at 37°C for 24 h was recorded by measuring the width of the clear zone (R).

[0272]

[0273] The growth inhibitory index (GII) was calculated according to the equation.

[0274] Strains tested: Bifidobacterium adolescentis (BAD 4 - strain of interest; BRC 126 - DSM 20083 and BRC1260 - DSM 28530). Pathogen: Escherichia coli O8 K88 (BRC Nr. 104) = F4.

[0275] result

[0276] Inhibition of Escherichia coli O8 K88(F4)

[0277] It was observed that B. adolescentis BAD4 could inhibit the growth of E. coli O8 K88(F4) strain ( Figure 1 ).

Claims

1. A host cell (e.g., an isolated and / or modified and / or recombinant host cell), characterized in that: jj) the host cell is capable of degrading starch, preferably the starch is resistant starch (RS), further preferably the host cell has α-amylase enzymatic activity (eg, has EC 3.2.1.1 enzymatic activity); and / or ii) the host cell is capable of utilizing starch as a carbon source, preferably the starch is resistant starch (RS); wherein the host cell comprises one or more (e.g., 2, 3 or 4) of the following sequences: a) a polypeptide having at least 97% (e.g., at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has α-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); b) a polypeptide having at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identity with the polypeptide shown in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2IGDMICFN_02013 protein), preferably the polypeptide is capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); c) a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4; d) a fragment of any one of (a) to (c) that is capable of binding (e.g., binding to) starch (e.g., the fragment is a starch-binding fragment) and / or has alpha-amylase enzymatic activity (e.g., has EC 3.2.1.1 enzymatic activity); preferably, the fragment is at least 200 amino acids (e.g., at least 250, at least 300, at least 350, at least 400 amino acids) long; e) one or more (e.g., 2, 3 or 4) polynucleotides (e.g., DNA or RNA, e.g., polycistronic or monocistronic) encoding the one or more polypeptides as in (a) to (d), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides.

2. The host cell according to claim 1, wherein the host cell is a strain of Bifidobacterium species.

3. The strain according to claim 2, wherein the strain is selected from the group consisting of: the designated strain "KI BAD2" of Bifidobacterium adolescentis with the deposit number DSM34061, and the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM 34065 (for example, both strains were deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms on October 11, 2021, address: 7B, Inhofen Street, Brunswick, Germany, D-38124), preferably wherein the whole genome nucleotide sequence of the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM34065 is at least 98.5% (for example, at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of the designated strain "KI BAD2" of Bifidobacterium adolescentis with the deposit number DSM 34061.

4. A host cell according to any one of the preceding claims, wherein the host cell has one or more of the following properties: a) the host cell is a Gram-positive, non-motile and / or anaerobic bacterial cell / strain; b) the host cell is a bacterial strain of Bifidobacterium adolescentis; c) the host cell is capable of improving the balance of beneficial and harmful bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock ungulate mammal), preferably a Sus spp., e.g., a domestic pig; d) A host cell according to (c), wherein the improvement comprises an increase in the level of beneficial bacteria and / or a decrease in the level of harmful bacteria, preferably the improvement is a modulation of the GIT microflora, for example by improving the relative abundance of bacteria of the genus Prevotella (e.g. in the large intestine of pigs); e) A host cell according to (c) or (d), wherein said improvement comprises improving the digestibility of feed and / or the growth of said non-human mammal; f) A host cell according to any one of (c) to (e), wherein the beneficial bacteria comprise one or more butyrate-producing bacterial strains (e.g., Eubacterium species, such as Eubacterium rectum, and / or Prevotella species strains, such as in the large intestine of pigs); and / or the harmful bacteria comprise one or more Escherichia coli strains (e.g., pathogenic E. coli strains, such as a pig pathogen causing post-weaning diarrhea (E. coli F4 and / or F18 strains); and / or g) the host cell is capable of degrading and / or utilizing one or more of the following carbohydrates as a carbon source: L-arabinose, ribose, D-xylose, galactose, glucose, fructose, arbutin, esculin, salicin, maltose, lactose, melibiose, sucrose, raffinose, starch, glycogen, turanose, preferably the host cell is the designated strain "KI BAD4" of Bifidobacterium adolescentis with the deposit number DSM34065.

5. A polypeptide capable of binding (e.g., binding to) starch (e.g., the polypeptide is a starch-binding protein) and / or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), the polypeptide being selected from the group consisting of: a) a polypeptide having at least 97% (e.g., at least 98%, at least 99% or 100%) identity to the polypeptide shown in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2IGDMICFN_02026 protein); b) a polypeptide that is at least 96% (e.g., at least 97%, at least 98%, at least 99% or 100%) identical to the polypeptide set forth in SEQ ID NO:3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO:4 (e.g., BAD2IGDMICFN_02013 protein); or c) a polypeptide having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

6. A polynucleotide encoding one or more polypeptides according to claim 5.

7. A nucleic acid construct or expression vector capable of expressing one or more polynucleotides according to claim 6.

8. A composition or kit comprising one or more of the following: a) one or more host cells according to any of the preceding claims, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; b) a combination of one or more host cells according to any one of the preceding claims with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); c) one or more (e.g., 2, 3 or 4) polypeptides according to any one of the preceding claims; d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides; e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); f) one or more (eg, 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c).

9. The composition or kit of claim 8, wherein the composition or kit is a starch degrading, nutritional (eg, supplement) and / or probiotic composition or kit.

10. A food, an intermediate food; a fodder, an intermediate fodder; a feed, an intermediate feed; an additive (e.g., a food additive, a fodder additive or a feed additive), an intermediate additive (e.g., a food intermediate additive, a fodder intermediate additive or a feed intermediate additive); a nutritional supplement, an intermediate nutritional supplement; a prebiotic, an intermediate prebiotic, a probiotic, an intermediate probiotic and / or a mixture thereof, comprising one or more of the following: a) one or more host cells according to any of the preceding claims, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; b) a combination of one or more host cells according to any one of the preceding claims with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); c) one or more (e.g., 2, 3 or 4) polypeptides according to any one of the preceding claims; d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides; e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); g) one or more (e.g., 2, 3 or 4) compositions according to claims 8-9; Preferably, the food, intermediate food; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and / or mixtures thereof are for non-human mammals (e.g., pigs).

11. A method for degrading starch, preferably resistant starch (RS), the method comprising: (i) provide one or more of the following: a) one or more host cells according to any of the preceding claims, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; b) a combination of one or more host cells according to any one of the preceding claims with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); c) one or more polypeptides according to any one of the preceding claims; d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably the one or more polynucleotides are capable of expressing (e.g., have expressed) the one or more polypeptides; e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); g) one or more (e.g., 2, 3 or 4) compositions according to claims 8-9; h) one or more of the following: food, intermediate food according to claim 10; forage, intermediate forage; feed, intermediate feed; additive (e.g. food additive, forage additive or feed additive), intermediate additive (e.g. food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotics, intermediate prebiotics, probiotics, intermediate probiotics and / or mixtures thereof; (ii) Apply (i) to starch.

12. A method for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract of a non-human mammal, the method comprising feeding the non-human mammal one or more of: a) one or more host cells according to any of the preceding claims, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; b) a combination of one or more host cells according to any one of the preceding claims with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); c) one or more (e.g., 2, 3 or 4) polypeptides according to any one of the preceding claims; d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably at least one of the polynucleotides is capable of expressing (e.g., has expressed) the one or more polypeptides; e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); g) one or more (e.g., 2, 3 or 4) compositions according to claims 8-9; h) one or more of the following: food, intermediate food according to claim 10; forage, intermediate forage; feed, intermediate feed; additive (e.g., food additive, forage additive or feed additive), intermediate additive (e.g., food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotics, intermediate prebiotics, probiotics, intermediate probiotics and / or mixtures thereof.

13. The method according to any one of the preceding claims, wherein the method is an in vitro, ex vivo, in vivo and / or non-therapeutic method, and / or the non-human mammal is healthy.

14. One or more of the following: a) one or more host cells according to any of the preceding claims, preferably selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" with the deposit number DSM 34061, and Bifidobacterium adolescentis designated strain "KI BAD4" with the deposit number DSM 34065; b) a combination of one or more host cells according to any one of the preceding claims with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); preferably a combination of Bifidobacterium adolescentis designated strain "KI BAD4" having the deposit number DSM 34065 and one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., an Enterococcus faecium strain E16) and a Pediococcus acidilactici strain (e.g., a Pediococcus acidilactici G2 strain); c) one or more (e.g., 2, 3 or 4) polypeptides according to any one of the preceding claims; d) one or more (e.g., 2, 3 or 4) polynucleotides encoding the one or more polypeptides as in (c), preferably at least one of the polynucleotides is capable of expressing (e.g., has expressed) the one or more polypeptides; e) one or more (e.g., 2, 3 or 4) nucleic acid constructs capable of expressing the one or more polynucleotides according to (d); f) one or more (e.g., 2, 3 or 4) expression vectors capable of expressing one or more polynucleotides encoding the one or more polypeptides according to (c); g) one or more (e.g., 2, 3 or 4) compositions according to claims 8-9; h) one or more of the following: food, intermediate food according to claim 10; forage, intermediate forage; feed, intermediate feed; additive (e.g. food additive, forage additive or feed additive), intermediate additive (e.g. food intermediate additive, forage intermediate additive or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotics, intermediate prebiotics, probiotics, intermediate probiotics and / or mixtures thereof; For use in one or more of the following: i) for degrading starch (e.g., resistant starch, RS); ii) for improving the balance of beneficial and harmful bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g. a livestock ungulate mammal), preferably the non-human mammal is a Sus species, e.g. a domestic pig; preferably, wherein the improvement comprises an increase in the level of beneficial bacteria and / or a decrease in the level of harmful bacteria, preferably the improvement is a modulation of the GIT microflora, e.g. by improving the relative abundance of bacteria of the genus Prevotella (e.g. in the large intestine); further preferably, wherein the improvement comprises improving the digestibility of feed and / or the growth of the non-human mammal; most preferably, wherein the beneficial bacteria comprise one or more butyrate-producing bacterial strains (e.g. a Eubacterium species, e.g. Eubacterium rectum, and / or a strain of the genus Prevotella, e.g. in the large intestine); and / or the harmful bacteria comprise one or more strains of Escherichia coli (e.g. a pathogenic E. coli strain, e.g. a pig pathogen causing post-weaning diarrhoea (E. coli F4 and / or F18 strains); iii) The use according to (ii), wherein the non-human mammal is healthy; iv) the use according to any one of (i) to (iii), wherein the use is a non-therapeutic use; v) Any combination of (i) to (iv); vi) The use according to (i) to (v), wherein the use is in vitro, ex vivo, in vivo and / or non-therapeutic.

15. The method or use according to any one of the preceding claims, wherein the non-human mammal is a livestock ungulate mammal, preferably the non-human mammal is a species of the genus Sus, such as the domestic pig.

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