WO2024047155A1 - Bifidobacterium adolescentis strains, methods and uses thereof for starch degradation and modifying gut flora in non-human mammals - Google Patents
Bifidobacterium adolescentis strains, methods and uses thereof for starch degradation and modifying gut flora in non-human mammals Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the invention uses and/or relates to a deposited biological material, which is to be made available only by the issue of a sample to an expert pursuant to Rule 32(1) EPC in respect to those designations in which a European Patent is sought until the publication of the mention of grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. Both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY on 11 October 2021.
- the present invention relates to novel host cells capable of degrading starch and/or utilizing starch as a carbon source wherein said host cell comprising one or more of the following sequences: (a) a polypeptide having at least 97% identity to the polypeptide set forth 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 said polypeptide is capable of binding starch and/or having alpha-amylase enzymatic activity; (c) one or more polynucleotides encoding said one or more polypeptides as in (a)-(b).
- the present invention relates to novel probiotic strains of Bifidobacterium adolescentis selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 as well as methods and used thereof for starch degradation and modifying gut flora in non-human mammals.
- Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061
- Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 as well as methods and used thereof for starch degradation and modifying gut flora in non-human mammals.
- Carbohydrates constitute one of the basic ingredients in the diet of many livestock animals including pigs. Frequently the primary source for providing carbohydrate is starch, which is the main constituent of e.g. corn, cereals, yams, rice and potatoes. Starch is composed of two types of polysaccharide molecules, amylose, a mostly linear and flexible polymer of D-anhydroglucose units linked by alpha-1 , 4-D-glucosidic bonds, and amylopectin, a branched polymer of linear chains linked by alpha-1 , 6-D-glucosidic bonds.
- starch is composed of two types of polysaccharide molecules, amylose, a mostly linear and flexible polymer of D-anhydroglucose units linked by alpha-1 , 4-D-glucosidic bonds, and amylopectin, a branched polymer of linear chains linked by alpha-1 , 6-D-glucosidic bonds.
- Starch is digested predominantly in the small intestine of most mammals (e.g., pigs) by the enzyme alpha-amylase (e.g., W02008087164).
- alpha-amylase e.g., W02008087164
- resistant starch can resist digestion and absorption in the small intestine of livestock mammals (e.g., pigs).
- ingested resistant starch passes into the large intestine where it is fermented to some extent by colonic microflora to short chain fatty acids, particularly butyrate and gases.
- the invention relates to a deposited biological material, which is to be made available only by the issue of a sample to an expert pursuant to Rule 32(1) EPC in respect to those designations in which a European Patent is sought until the publication of the mention of grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn.
- the present invention relates to a host cell (e.g., an isolated and/or modified and/or recombinant host cell) characterized in that: (i) said host cell is capable of degrading starch, preferably said starch is a resistant starch (RS), further preferably said host cell having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and/or (ii) said host cell is capable of utilizing starch as a carbon source, preferably said starch is a resistant starch (RS); wherein said host cell comprising one or more (e.g., 2, 3, or 4) of the following sequences: (a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_ 01028 protein) or SEQ ID NO: 2 (e.g., BAD4 NNHABG
- the present invention relates to a host cell selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 (e.g., both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr.
- the present invention relates to a polypeptide capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), said polypeptide selected from the group consisting of: (a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth 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% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NN
- the present invention relates to one or more polynucleotides encoding one or more polypeptides of the present invention.
- 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.
- the present invention relates to a composition or kit comprising one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells according to present invention in combination with one or more (e.g., 2) of the probiotic following strains: an Enterococcus faecium strain and/or a Pediococcus acidilactici strain; preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faec
- nucleic acid constructs e.g., 2, 3 or 4 capable of expressing said one or more polynucleotides according to (d);
- expression vectors e.g., 2, 3 or 4 capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c).
- the present invention relates to the composition or kit of the present invention, wherein said composition or kit is a starch-degrading, nutritional (e.g., nutritional supplement), prebiotic (e.g., comprising resistant starch) and/or probiotic composition or kit.
- nutritional e.g., nutritional supplement
- prebiotic e.g., comprising resistant starch
- the present invention relates to a foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s t thereof, comprising one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells according to present invention in combination with one or more (e.g., 2) of the following strains: an Enterococc
- the present invention relates to a method for degrading starch, preferably a resistant starch (RS), comprising: (i) providing one or more of the following: (a) one or more of the host cells according to the present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more 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., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the B
- the present invention relates to a method (e.g., non-therapeutic method) for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of a non-human mammal, comprising feeding said non-human mammal with one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells according to present invention in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g.,
- the present invention relates to a method of the present 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.
- SEQ ID NO: 1 is the amino acid sequence of BAD4 NNHABGDB_01028 protein:
- SEQ ID NO: 2 is the amino acid sequence of BAD2 IGDMICFN_02026 protein:
- SEQ ID NO: 3 is the amino acid sequence of BAD4 NNHABGDB_01017 protein:
- SEQ ID NO: 4 is the amino acid sequence of BAD2 IGDMICFN_02013 protein:
- HFTSDWS AIDSDWQNTDYFHKRTGCDGPNGEIKDYSNRYKVTQCHLLGLWDLNTQSQTVADRMQSFLK
- Figure 1 shows inhibition of E. coli deleterious strain 08 K88 (F4) by the means of supplementation of BAD4 strain of the present invention assessed by agar spot assay (see Example 4).
- EC numbers Enzyme Commission numbers
- the EC number refers 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.
- polypeptide is equally used herein with the term “protein”. Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids).
- polypeptide(s) as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical.
- heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains.
- polypeptide and protein also refer to naturally modified polypeptides/proteins wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
- nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules.
- nucleic acid molecule may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711, US 471 1955, US 5792608 or EP 302175 for examples of modifications).
- the polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation.
- the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper et al., Nucleic Acids Res (2000), 28 (21): 4332-4339).
- Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA.
- nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein.
- a nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment.
- Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
- sequence identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
- the parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
- the output of Needle labeled “longest identity” is used as the percent identity and is calculated as follows:
- the parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
- the output of Needle labeled “longest identity” (obtained using the no-brief option) is used as the percent identity and is calculated as follows:
- Expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
- Expression vector may refer to a linear or circular DNA molecule that comprises a polynucleotide encoding a variant (polypeptide) and is operably linked to control sequences that provide for its expression, in particular for its transcription.
- fragment may refer to a polypeptide having one or more (e.g. several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has an activity as described elsewhere herein.
- Host cell may refer to any cell type that is susceptible to transformation, transfection, transduction, or the like 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 containing either a nucleotide sequence to be expressed, or an expression vector, and which is able to produce or already producing an enzyme or a polypeptide according to the invention.
- nucleic acid construct may refer to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
- 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 expression of the coding sequence.
- control sequences may refer to nucleic acid sequences necessary for 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 native or foreign to each other.
- control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter, signal peptide sequence, and transcription terminator.
- the control sequences include a promoter, and transcriptional and translational stop signals.
- the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide of the present invention.
- the term “foodstuff’ may refer to a substance having a food value.
- binder may refer to a substance fed to domestic animals.
- feed may refer to a substance used as food for livestock.
- additive may refer to a compound or substance added to another product or substance, e.g., in a small amount, to affect a desired property and/or characteristics.
- prebiotic may refer to a compound or substance capable of inducing the growth and/or activity of beneficial microorganisms.
- probiotic may refer to microorganisms that are intended to have health benefits when consumed or applied to a body.
- the term “nutritional supplement” may refer to a compound or substance capable to support the nutritional content of the diet, e.g., vitamins, minerals and/or probiotic bacteria.
- intermediate may refer to a compound or substance produced during the process (e.g., during an intermediate stage of the process) of obtaining an end-product of the present invention, e.g., foodstuff, fodder, fodder; feed, additive (e.g., foodstuff-, fodder- or feed additive), nutritional supplement, probiotic or prebiotic of the present invention.
- an end-product of the present invention e.g., foodstuff, fodder, fodder; feed, additive (e.g., foodstuff-, fodder- or feed additive), nutritional supplement, probiotic or prebiotic of the present invention.
- starch may refers to a glucose polymer in which glucopyranose units are bonded by a/pha-linkages. It may be made up of a mixture of amylose (15-20%) and amylopectin (80-85%). Amylose may consist of a linear chain of several hundred glucose molecules, and Amylopectin is a branched molecule made of several thousand glucose units (every chain of 24-30 glucose units is one unit of Amylopectin).
- starch may be composed of two types of polysaccharide molecules, amylose, a mostly linear and flexible polymer of D-anhydroglucose units linked by alpha-1 ,4-D-glucosidic bonds, and amylopectin, a branched polymer of linear chains linked by alpha-1 ,6-D-glucosidic bonds, Starch may have Formula I:
- resistant starch may refer to starch that resists digestion and absorption in the small intestine of e.g. mammals.
- Resistant starch may be classified into four categories depending on the causes of resistance.
- RS1 is a physically inaccessible starch due to entrapment of granules within a protein matrix or within a plant cell wall.
- RS2 is a granular starch that resists digestion by pancreatic alpha-amylase.
- RS3 is a retrograded, non-granular starch or starch food.
- RS4 is a resistant starch that has linkages other than alpha-1 ,4- and alpha-1 ,6-D-glucosidic bonds. Resistant starch in the context of the present invention includes, but is not limited to these four categories.
- Alpha-amylases (alpha-1 , 4-glucan-4-glucanohydrolases, EC 3.2.1.1) as used herein constitute a group of enzymes, which catalyzes hydrolysis of starch and other linear and branched 1 ,4-gluosidic oligo- and polysaccharides.
- binding starch may refer to a functional characteristic of a polypeptide having binding specificity for starch, in particular raw starch.
- the gut flora i.e., the community of bacteria resident in the gastrointestinal tract, comprises both beneficial and deleterious bacterial types or species. Whether a particular member of the gut flora is beneficial, deleterious or inconsequential to the health of the non- human mammalian species in particular circumstances can depend on a number of factors, but for the purposes of the present invention certain types or species of bacteria can be considered beneficial and others deleterious.
- beneficial members of the gut flora include bifidobacteria (species of the genus Bifidobacterium) and lactic acid bacteria, more particularly species of the genus Lactobacillus as well as Prevotella spp. bacteria.
- Deleterious bacteria include pathogenic bacteria or opportunistic pathogenic bacteria. Examples of deleterious members of the gut flora include pathogenic forms of Escherichia coli.
- Gastrointestinal health typically depends on maintenance of an appropriate balance of beneficial and deleterious bacteria. An increase in the level of deleterious bacteria and/or a decrease in the level of beneficial bacteria can be associated with a decline in gastrointestinal health. Conversely, an increase in the level of beneficial bacteria and/or a decrease in the level of deleterious bacteria can be associated with an improvement in gastrointestinal health, for example improvement of health.
- “Improvement” or “improving” or other grammatical forms include “enhancing” or “enhancement” of the balance between the level of beneficial and deleterious bacteria as described herein.
- An “improvement” herein means shifting the balance in favor of the level of beneficial bacteria, and thus can involve an increase in the level beneficial bacteria and/or a decrease in the level deleterious bacteria.
- enhancement of the balance arises from both a reduction in the level of deleterious, e.g. pathogenic, bacteria and an increase in the level of beneficial bacteria.
- An increase in the level beneficial bacteria in the gut of non-human mammalian species fed with products as described herein encompasses an increase of 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250% or more in the level of beneficial bacteria in the gut of non-human mammals in comparison to non-human mammals not fed with products as described herein as is shown in Example 3 herein. Accordingly, an increase in the level of beneficial bacteria in the context of the methods and uses of the present invention may advantageously be 100%, 125%, 150%, 175%, 200% or more.
- B. adolescentis BAD4 and B. adolescentis BAD2 can degrade starch, a property which was not found in other strains of this species.
- B. adolescentis BAD4 and B. adolescentis BAD2 of the present invention carry a set of gene which are correlated with enzymatic starch degradation. According to whole genome comparison analysis carried out in the course of the present invention, these genes are not present in genomes of B. adolescentis reference strains, which cannot utilize starch as carbon source.
- starch-degrading bacteria fed as live probiotics to animals (e.g., livestock non-human mammals) are capable of improving feed digestibility, nutrient uptake and/or animal growth, especially with starch comprising feeds.
- B. adolescentis BAD4 beneficial effects of B. adolescentis BAD4 in combination with two other strains (Enterococcus faecium E16 and Pediococcus acidilactici G2) on pig gut microbiota were surprisingly found. It was shown that the relative abundance of several genera of butyrate-producing bacteria, inhabiting the pig intestine, could be increased by the means of the strains of the present invention (see examples section herein). Butyrate is known to contribute to maintenance of the gut barrier function and is also known for its immunomodulatory and anti-inflammatory properties. [0058] It was also surprisingly found that B. adolescentis BAD4 and B.
- adolescentis BAD2 had superior inhibiting activity against Escherichia coli pig pathogen stains, compared to B. adolescentis reference strain, as shown by agar spot tests (see examples section herein).
- the inhibition of zoonotic pathogens naturally occurring in the gut of pigs, such as E. coli, by applying B. adolescentis to the animal feed is a promising measure for reducing the incidences of post weaning diarrhea in pigs.
- Livestock farmers e.g., pig producers usually include several different feed additives (e.g., non-therapeutic additives) to animal’s diet to maintain gut health and improve performance.
- feed additives e.g., non-therapeutic additives
- probiotics are used in human and animal nutrition for many years and there is a long history of safe use for specific bacterial species.
- Most global known probiotic products are comprised of a single strain.
- Commonly used species are Bacillus subtilis, Saccharomyces cerevisiae, Enterococcus faecium, Lactobacillus acidophilus.
- Species of Pediococcus spp. and Bifidobacterium spp. are rarely used for development of feed additives, compared to formerly named species.
- Roselli et al. 2017 provides an overview of main results of in vitro test with probiotic strains on pig intestinal cell lines and influence of prebiotics and probiotics on porcine microbiota composition and on pathogen adhesion and diarrhea in pigs.
- Lactobacillus acidophilus produces lactic acid and the enzyme amylase during its growth phase.
- Lactobacillus casei and Enterococcus faecium complement it, also producing those chemicals, but also enjoys a wide temperature and pH range. Both of these microorganisms create an acidic environment hostile to pathogenic bacteria.
- Bifidobacterium thermophilum is a delicate microorganism found in mother’s milk, as well as human and animal gastrointestinal tracts. Restoring this bacterium is useful for animal’s well being and growth.
- the present invention satisfies such need by providing strains and related products of the present invention as described herein, which are capable of reducing number of ingredients to be added to a “standard” pig diet.
- the present invention provides for more sustainability in livestock farming, since the level of beneficial bacteria can be increased in the gut - most likely they settle and can remain in the gut, which is more sustainable than just being added beneficial bacteria through feed, whereby such externally added beneficial bacteria may be not able to settle and remain in the gut.
- the sustainability is assumed to reside in the fact that non-human mammal species have in the long run a benefit from the increase of the level of beneficial bacteria caused - as is shown in the Examples - by an exogenously fed probiotic strains of the present invention, rather than by constantly receiving other beneficial bacteria through feed which may no be able to setle and reside in the gut.
- the products, methods and uses of the present invention as described herein have at least the following advantages over known products: - starch degradation (phenotypically shown by API test); alpha amylase activity (shown genotypically by whole genome comparison analysis); in combination with Enterococcus faecium and Pediococcus acidilactici, beneficial modulation of the pig gut microbiota, by enhancing relative abundance of Prevotella in the large intestine; and/or inhibition of pig pathogens causing post weaning diarrhea (E. coli F4 and F18).
- a host cell e.g., isolated and/or modified and/or recombinant and/or transformed host cell/s
- said host cell is capable of degrading starch, preferably said starch is a resistant starch (RS) (e.g., a starch that resists digestion and absorption in the small intestine of e.g.
- RS resistant starch
- said host cell having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and/or ii) said host cell is capable of utilizing starch as a carbon source, preferably said starch is a resistant starch (RS); wherein said host cell comprising one or more (e.g., 2, 3, or 4) of the following sequences: a) a polypeptide having at least 88% identity (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
- polypeptide set forth in SEQ ID NO: 1 e.g., BAD4 NNHABGDB_01028 protein
- SEQ ID NO: 2 e.g., BAD2 IGDMICFN_02026 protein
- said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch- binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity);
- a polypeptide having at least 96% identity e.g., at least 97%, at least 98%, at least 99% or 100%
- SEQ ID NO: 3 e.g., BAD4 NNHABGDB_01017 protein
- SEQ ID NO: 4 e.g., BAD2 IGDMICFNJ32013 protein
- polypeptide set forth in SEQ ID NO: 4 e.g., BAD2 IGDMICFN_02013 protein
- said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity);
- a polypeptide according to (a)-(h) wherein said polypeptide is an alpha- amylase like protein;
- the host cell according to any one of preceding items, wherein said host cell is a prokaryotic (e.g., Bifidobacterium spp.,, Bacteroides spp., (e.g., B. thetaiotaomicron), Ruminococcus spp., e.g., R. bromii, Escherichia spp., (e.g., E. coli), Bacillus spp., (e.g., B. subtilis), Lactobacillus spp., (e.g., Lactobacillus amylovorus, Lactobacillus acidophilus, L. salivarius, L. plantarum, L. buchneri, L.
- a prokaryotic e.g., Bifidobacterium spp.,, Bacteroides spp., (e.g., B. thetaiotaomicron), Ruminococcus spp.
- the host cell according to any one of preceding items, wherein said host cell is a Bifidobacterium sp. strain (e.g., a Bifidobacterium adolescentis strain).
- a Bifidobacterium sp. strain e.g., a Bifidobacterium adolescentis strain.
- strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 (e.g., both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr.
- the host cell of any one of the preceding items wherein said host cell has one or more of the following characteristics: a) said host cell is a gram-positive, non-motile and/or anaerobic bacterial cell/strain; b) said host cell is a Bifidobacterium adolescentis bacterial strain; c) said host cell is capable of improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock hoofed mammal, e.g., horse or livestock hoofed mammal, e.g., as catle, sheep, goats or pigs), preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig); d) the host cell according to (c), wherein said improvement comprises an increase in level of beneficial bacteria and/or a decrease in level of deleterious bacteria, preferably said improvement is a modul
- the host cell (e.g., in the large intestine of a pig); e) the host cell according to (c) or (d), wherein said improvement comprises an improvement of digestibility of feed and/or growth of said non-human mammal; f) the host cell according to any one of (c) - (e), wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale, and/or Prevotella spp.
- butyrate-producing bacterial strains e.g., Eubacterium spp., e.g., E. rectale, and/or Prevotella spp.
- strains e.g., in the large intestine of a non-human mammal, e.g., a pig
- the deleterious bacteria comprise one or more of Escherichia coli strains (e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea (E.
- said 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 said host cell is Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065.
- a polypeptide capable of binding e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), said polypeptide selected from the group consisting of: a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth 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% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO:
- a composition or kit comprising one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2
- composition or kit according to any one of the preceding items wherein said composition or kit is a starch-degrading, nutritional (e.g., nutritional supplement), prebiotic and/or probiotic composition or kit.
- composition or kit according to any one of the preceding items further comprising starch (e.g., resistant starch).
- Pediococcus acidilactici strain e.g., Pediococcus acidilactici G2 strain
- a method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of a non-human mammal comprising feeding said non-human mammal with one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a
- Pediococcus acidilactici strain e.g., Pediococcus acidilactici G2 strain
- a) one or more of the host cells according to any one of the preceding items preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065.
- Enterococcus faecium strain e.g., Enterococcus faecium E16 strain
- a Pediococcus acidilactici strain e.g., Pediococcus acidilactici G2 strain
- said improvement comprises an improvement of digestibility of feed and/or growth of said non- human mammal; most preferably wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale and/or Prevotella spp. strains, e.g., in the large intestine); and/or the deleterious bacteria comprise one or more of Escherichia coli strains (e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea (E.
- Escherichia coli strains e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea
- non-human mammal is a hoofed mammal (e.g., horse or livestock, e.g., catle, sheep, goat or pig), preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig).
- a hoofed mammal e.g., horse or livestock, e.g., catle, sheep, goat or pig
- Sus sp. e.g., Sus domesticus (domestic pig).
- Carbohydrate metabolism of B. adolescentis BAD4 strain of the present invention was determined by API 50 CH test system (Biomerieux) and compared to state-of-the art strains.
- Bacterial culture was prepared form a single colony grown on LSM-Cys agar plate, incubate anaerobically, using AnaeroGen bags at 37°C for 48h. After incubation time, take a single colony was inoculated in 10 mL of LSM-Cys broth and incubate anaerobically, using AnaeroGen bags at 37°C for 24 to 48h. Purity of ON cultures was examined via microscopy.
- Bacterial culture was transferred in 15-mL tubes and centrifuged at 3000 rpm for 5 min.
- API 50 test strips were placed just before use in the incubation tray. After centrifugation the supernatant was removed and pellet resuspended with 100pl of 50 CHL media.
- Each well of the test strip was filled with 150 pL of suspension and covered with 1-2 drops of paraffin oil and incubated in a box at 37°C for 48 h. Color reaction was evaluated after 24 and 48 h of incubation.
- Example 2 Genotypic discovery of genes related to starch degradation
- Illumina sequencing was performed by LGC Genomics GmbH using first the Allegro Targeted Genotyping kit (TECAN), followed by the plexWellTM384 Library Preparation Kit (seqWellTM) according to the manufacturer’s instructions. Libraries were amplified in an emulsion PCR for 14 cycles using standard Illumina primers. Size selection for Illumina libraries was done on LMP-agarose gel electrophoresis selecting a range between 300 and 800bp. Final library purification step and quality control of libraries was done with the BioAnalyzer and Qubit. The prepared libraries were sequenced on a NextSeq 500/550 V2 Illumina instrument, paired end 2x150bp.
- TECAN Allegro Targeted Genotyping kit
- seqWellTM Library Preparation Kit
- the sequencing reads were trimmed based on quality and Illumina adaptors were removed using Trimmomatic (version 0.39) (parameters: ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 LEADINGS TRAILING:3 SLIDINGWINDOW:10:20 MINLEN:30) and reads shorter than 30bp were discarded.
- a hybrid assembly was performed by combining the short and long sequencing reads.
- the assembly pipeline Unicycler version 0.4.8, parameters: default
- This pipeline assembles the Illumina short sequencing reads with SPAdes (Bankevich et al. 2012), and performs long read assembly with Miniasm and multiple rounds of polishing with Racon (Vaser et al. 2017), using the Illumina contigs as anchor points to improve the assembly.
- Example 3 Beneficial modulation of gut microbiota
- a feeding trial in piglets was performed to investigate effects of a combination of B. adolescentis BAD4, E. faecium and P. acidilactici on intestinal gut microbiota.
- Treatments were distributed throughout the rooms to reduce any effect of room. Pigs were housed for a total of 42 days during which time performance was measured at each of the 3 phases. Blood and feces were collected at day 21 and also at day 42 at which time a necropsy on a subset of pigs occurred.
- Intestinal digests ileum and colon
- fecal samples were sequenced at LGC Genomics (Illumina 16S amplicon sequencing). 16s rRNA sequences were provided to assess the effect of probiotic additive on the taxonomic profile of piglet gut microbiota. The data is collected from digests of pig colon at day 42 of the feeding trial from animals fed with 3 different dosages of the probiotic additive (low, medium, high). A Dada2 based pipeline followed by Calypso downstream analysis was used for the assessment of differences between control and the treatments.
- Prevotella-driven enterotype has shown positive associations with animal traits including feed intake, feed efficiency, weight gain and incidence of diarrhea.
- Prevotella Megasphaera elsdenii and Roseburia have been previously reported as SCFAs producer, which is beneficial for strengthening the intestinal cell wall barrier and for immunomodulatory effects.
- Day 1 prepare overnight culture of probiotic bacteria in LSM-Cys and incubate anaerobically at 37°C, overnight.
- Day 2 prepare LSM-Cys agar plates (20 mL agar / plate); prepare pre-culture of probiotic bacteria and incubate till desired OD690: Bifidobacterium: OD690 0.250 ⁇ 0.02); spot probiotic bacteria (10E+5 cell/spot) on LSM-Cys agar; to develop bacterial spots, incubate plates anaerobically, using AnaeroGen bags, at 37°C for 48 h.
- Day 3 prepare overnight culture of pathogen bacteria (E. coli) in Nutrient Broth (NB) and incubate under desired incubation conditions.
- pathogen bacteria E. coli
- NB Nutrient Broth
- Day 4 prepare 0.6 % soft Nutrient Agar; prepare pre-culture of pathogen bacteria in NB; inoculate soft agar (wait until cooled to 50°C) with pre-culture of the pathogen strains (with final concentration 7 log cfu/mL) and pour it over a plate (10 mL / plate); incubate plates for 24 h at 37°C, anaerobically (agar spot assay). [00146] After incubation period, measure inhibition zones around the probiotic colonies (from the outer edge of the bacteria spot to the outer edge of the clear zones) by using HiAntibiotic ZoneScale.
Abstract
The present invention relates to novel host cells capable of degrading starch and/or utilizing starch as a carbon source wherein said host cell comprising one or more of the following sequences: (a) a polypeptide having at least 97% identity to the polypeptide set forth 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 said polypeptide is capable of binding starch and/or having alpha-amylase enzymatic activity; (c) one or more polynucleotides encoding said one or more polypeptides as in (a)-(b). Particularly, the present invention relates to novel probiotic strains of Bifidobacterium adolescentis selected from the group consisting of: Bifidobacterium adolescentis designated strain "KI BAD2" having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain "KI BAD4" having Accession No.: DSM 34065 as well as methods and used thereof for starch degradation and modifying gut flora in non-human mammals.
Description
Bifidobacterium adolescentis strains, methods and uses thereof for starch degradation and modifying gut flora in non-human mammals
SEQUENCE LISTING
[001] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
DEPOSITED BIOLOGICAL MATERIAL
[002] The invention uses and/or relates to a deposited biological material, which is to be made available only by the issue of a sample to an expert pursuant to Rule 32(1) EPC in respect to those designations in which a European Patent is sought until the publication of the mention of grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. Both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY on 11 October 2021.
FIELD OF THE INVENTION
[003] The present invention relates to novel host cells capable of degrading starch and/or utilizing starch as a carbon source wherein said host cell comprising one or more of the following sequences: (a) a polypeptide having at least 97% identity to the polypeptide set forth 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 said polypeptide is capable of binding starch and/or having alpha-amylase enzymatic activity; (c) one or more polynucleotides encoding said one or more polypeptides as in (a)-(b). Particularly, the present invention relates to novel probiotic strains of Bifidobacterium adolescentis selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 as well as methods and used thereof for starch degradation and modifying gut flora in non-human mammals.
BACKGROUND OF THE INVENTION
[004] Carbohydrates constitute one of the basic ingredients in the diet of many livestock animals including pigs. Frequently the primary source for providing carbohydrate is starch, which is the main constituent of e.g. corn, cereals, yams, rice and potatoes. Starch is composed of two types of polysaccharide molecules, amylose, a mostly linear and flexible polymer of D-anhydroglucose units linked by alpha-1 , 4-D-glucosidic bonds, and amylopectin, a branched polymer of linear chains linked by alpha-1 , 6-D-glucosidic bonds. Starch is digested predominantly in the small intestine of most mammals (e.g., pigs) by the enzyme alpha-amylase (e.g., W02008087164). However, “resistant starch” can resist digestion and absorption in the small intestine of livestock mammals (e.g., pigs). Typically, ingested resistant starch passes into the large intestine where it is fermented to some extent by colonic microflora to short chain fatty acids, particularly butyrate and gases. Furthermore, due to a widespread and excessive antibiotic use in livestock farming resulting in disrupted microbiomes in healthy livestock animals (e.g., pigs) there is a need for novel probiotic resistant starch degrading bacterial strains with improved properties in livestock farming as well as non-therapeutic methods based thereon for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) in livestock animals. In particular, there is a need in the art for new methods of modifying gut flora, especially adjusting the balance of beneficial and deleterious (e.g., pathogenic) bacteria in the gut in favor of beneficial species, in livestock species.
[005] The technical problem underlying the present application is thus to comply with this need. The technical problem is solved by providing the embodiments reflected in the claims, described in the description and illustrated in the examples and figures that follow.
SUMMARY OF THE INVENTION
[006] The invention relates to a deposited biological material, which is to be made available only by the issue of a sample to an expert pursuant to Rule 32(1) EPC in respect to those designations in which a European Patent is sought until the publication of the mention of grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn. [007] 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) said host cell is capable of degrading starch, preferably said starch is a resistant starch (RS), further preferably said host cell having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and/or (ii) said host cell is capable of utilizing starch as a carbon source, preferably said starch is a resistant starch (RS); wherein said host cell comprising one or more (e.g., 2, 3, or 4) of the following sequences: (a) a polypeptide having at least 97% identity (e.g., at
least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_ 01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_ 02026 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); (b) a polypeptide having at least 96% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); (c) a polypeptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-4; (d) a fragment of any one of (a)-(c) capable of binding (e.g., bound to) starch (e.g., said fragment is a starch-binding fragment) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); preferably said fragment is at least 200 amino acids long (e.g., at least 250, at least 300, at least 350, at least 400 amino acids); (e) one or more (e.g., 2, 3, or 4) polynucleotides (e.g., DNA or RNA, e.g., polycistronic or monocistronic) encoding said one or more polypeptides as in (a)-(d), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides.
[008] In another aspect the present invention relates to a host cell selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 (e.g., both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY on 11 October 2021), preferably wherein the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 is at least 98.5% (e.g., at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061.
[009] In a further aspect the present invention relates to a polypeptide capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), said polypeptide selected from the group consisting of: (a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth 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% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth 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 ID NO: 1 , 2, 3, or 4.
[0010] In a yet further aspect the present invention relates to one or more polynucleotides encoding one or more polypeptides of the present invention.
[0011] In a yet further aspect 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.
[0012] In another aspect the present invention relates to a composition or kit comprising one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells according to present invention in combination with one or more (e.g., 2) of the probiotic following strains: an Enterococcus faecium strain and/or a Pediococcus acidilactici strain; preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain and a Pediococcus acidilactici strain; (c) one or more polypeptides (e.g., 2, 3 or 4) according to the present invention; (d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing said one or more polypeptides;
(e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); (f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c).
[0013] In another aspect the present invention relates to the composition or kit of the present invention, wherein said composition or kit is a starch-degrading, nutritional (e.g., nutritional supplement), prebiotic (e.g., comprising resistant starch) and/or probiotic composition or kit.
[0014] In a further aspect the present invention relates to a foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s t thereof, comprising one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells
according to present invention in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain and a Pediococcus acidilactici strain; preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain and a Pediococcus acidilactici strain; (c) one or more polypeptides (e.g., 2, 3 or 4) according to the present invention; (d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; (e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); (f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c); (g) one or more compositions (e.g., 2, 3 or 4) according to the present invention.
[0015] In a further aspect the present invention relates to a method for degrading starch, preferably a resistant starch (RS), comprising: (i) providing one or more of the following: (a) one or more of the host cells according to the present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more 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., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4" having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); (c) one or more polypeptides according to any one of the preceding claims; (c) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; (d) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); (e) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c); (f) one or more compositions (e.g., 2, 3 or 4) according to the present invention; (g) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or
mixture/s thereof according to the present invention; (ii) applying (i) to starch (e.g., resistant starch).
[0016] In another aspect the present invention relates to a method (e.g., non-therapeutic method) for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of a non-human mammal, comprising feeding said non-human mammal with one or more of the following: (a) one or more of the host cells according to present invention, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; (b) one or more of the host cells according to present invention in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); (c) one or more polypeptides (e.g., 2, 3 or 4) according to the present invention; (d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said at least one polynucleotide is capable of expressing (e.g., expressed) said one or more polypeptides; (e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); (f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c); (g) one or more compositions (e.g., 2, 3 or 4) according to the present invention; (h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s according to the present invention.
[0017] In another aspect the present invention relates to a method of the present 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.
OVERVIEW OF THE SEQUENCE LISTING
[0018] SEQ ID NO: 1 is the amino acid sequence of BAD4 NNHABGDB_01028 protein:
MKHRKPAPAWHRLGLKISKKVWGITAAATAFGGLAIASTAAQASTDRDSYADTVENAAFEQARKRYG LAQQMSEGATLHAWEWSFKTIEENIPAIAEAGYTSVQTEPISAIHNGGKGKIFTENWYYVYQPTDTTI GNWVMGTEDDLKSLCDTAHKYGVRI I I DWANHMTATWNAI ADRWKKSEYYHHDCNDGNVQDGNNRYQ VTHCKLLGLYDINTENTETANMMHDYLVQAVNDGVDGIRFDAAKHIELPGEYNNSQYWDIVLNNGAQF QYGEVLQDSISRDADYAALFSKYSRYGGGVTASSYGLKLRNALKSYSLDAGSLSNWSNPASAGNLTSW VESHDNYSNNPNADDASTKMSEWQMTMGWGVIGSRSQTMPLYFDRPVGSGGDQPQFAEKSQLGDAGAD SWKDPQWAVNHFRNTMNNSKASEYLRNCGANSCLMVERYIKDGNFKNDGVTITNMGDTQELSGTATN LDDGTYKDQVSGGTITVSGGKITSGSAPGGKISVFFTDNSGSVSATPGDSSFKTDTTTVTLNANNVTD
ATYTTSEGKSGSYQDGDTITIGASTAIGDTITVKLQGKDADGQTVSATYKYTKKDPAATSTAYAKKPS AWSNLYAYVYVDDSSATTLKENAKWPGEPMTQVASGDTCGKDGEYKYEI PDDLEGSDTRI I FNDGNAT NTKKYPADTTEGEDAAGLKIDGNYAWDGNTSSGTWEARNCWTPTKSVKIDQSDYTTDLSGGVATKQL TATTDPKGASVSWSSSDKDVATVSPKGWTPKKAGKATITAKSGTKTSSITVTVTGQLPPDPVKDNTI YAAKPSGWGKIYAYVYTGDGATAANNAAWPGVEMTAPSATDGCQQTELYKYWPDSLAKGAKVIFNDG GSQQYPGSRQPGLDYNGGIVKWDGSSAALGALECETTIPVTSVSVSGDGVSGGKLSLKSGASVQLTAT VKPDNATDRKVTWTSSDSSVANVMGTGWTAGSKAGKATVTATAGGVSASVEVTVEAVGKQPMTVWYK PDSSWKTVKANYRVYSTPQSAGHGVELKATCGGWYKLTVPDTKGAEVSIAFTNGSKWDSNGGKNYIGS GAVMAVSDGRVSFKTPDCPAAKPMTVWYKPDSSWTTVKANYRVYSTVQKTGYGAVLEKSCGGWYKLTV PDTEGAKVS I AFTNGSKWDSNGGKNY I AAGETMAVSGGQS I PDVTPNCAI TTKQ
[0019] SEQ ID NO: 2 is the amino acid sequence of BAD2 IGDMICFN_02026 protein:
MKHRKPAPAWHRLGLKISKKVWGITAAATAFGGLAIASTAAQASTDRDSYADTVENTTFEQARKHYG LAQQMSEGATLHAWEWS FKT I EENI PAI AEAGYTS VQTEP I SAI HNGGKGKI FTENWYYVYQPTDTT I GNWVMGTEDDLKSLCDTAHKYGVRI I I DWANHMTATWNAIADRWKKSEYYHHDCNDGKVQDWNNRYQ VTHCKLLGLYDINTENTETANMMHDYLVQAVNDGVDGIRFDAAKHIELPGEYNNSQYWDIVLNNGAQF QYGEVLQDSISRDADYAALFSKYSRYGGGVTASSYGLKLRNALKSYSLDAGSLSNWSNPASAGNLTSW VESHDNYSNDPNDDDASTKMSEWQMTMGWGVIGSRSQTMPLYFDRPVGSGGSQPQFAEKSKLGDAGAD SWKDAQWAVNHFRNTMNNNKASEYLRNCGANSCLMVERYIKDGNFKNDGVTITNMGDTQELSGTATN LDDGTYKDQVSGGTITVSGGKITSGSAPGGKISVFFTDNSGSVSATPGDSSFKTDTTTVTLNANNVTD ATYTTSEGKSGSYQDGDTITIGASTAIGDTITVKLQGKDADGQTVSATYKYTKKDPAATSTAYAKKPS
AWSNLYAYVYVDDSSATTLKENAKWPGEPMTQVASGDTCGKDGEYKYEIPDDLEGSNTRIIFNDGNAT NTQKYPADTTEGVDAAGLKIDGNYAWDGNTSSGTWEARNCWTPTKSVKIDQSDYSVDLSNGVTTKQL TATTDPKGASVSWSSSDKDVATVSPKGWTPRKAGKATITAKSGTKTSSITVTVTGQLPPDPVKDNTI YAAKPSGWGKIYAYVYTGDGATAVNNAAWPGVKMTAPSATDGCQQTELYKYWPDNLAKGAKVIFNDG GSQQHPGSREPGLDYNGGIVKWDGSSAALAAVECETTIPVTSVSVSGDGVSGGKLSLKSGASAQLTAT VKPDNAT DRKVTWT S S DS S VANVMGTG WTAGNKAGT AT I KAT AGGKS AS VQVT VTDVPKQPMTVWYK
PDSSWKTVKANYRVHSTPQSAVHGAGLKAACGGWYKLTVPDTKGAEVSIAFTNGSKWDSNGGKNYIGS
GTVMAVSDGRISFKAPDCPWKPMTVWYKPDSSWKTVKANYRVYSAVQKTGNGWLEKSCGGWYSLTV
PDTEGARVSIAFTNGSKWDSNGGKNYIGAGDSMAVSGGQWSDVTPNCTINAK
[0020] SEQ ID NO: 3 is the amino acid sequence of BAD4 NNHABGDB_01017 protein:
MRLSTHTFAGMANALTRKEPPHDDQPELMTRAERRRAARKARAAKIWKKVAAGTVAVATLFGGMGVAS TALAADRDSYQDTVGNSSFEAARNQYGLTKHMKNGAILHAWMWSFKNITANMEAIAKAGYTSVQTEPM SKI KYVPANGKKFDENWYYVYQ P ANT S VGN FWGTEDDLKEMTATAHK YGVR 11 VDVVANH FTS DWS A IDSDWQNTDYFHKRTGCDGPNGEIKDYSSRYKVTQCHLLGLWDLNTQSQTVADRMQSFLKEAVADGVD GFRYDAAKHVELPSEVFDNKQSNYWNTILKNGAQFQYGEVLQGDSGLDYKAYANMFRDNSSDGGGNTA SAYGGTIRAAISSGNLSTKMVQSISTGGANEDQLVTWVESHDNYANKEQTGSDGVKKGVSTELTDYEL
MMGWAIVGSRKAGAPLYFNRPKESGGKDQNGNLRPQFAEKSQLGDTGDDMWKNTSWAVNHFRNAMDG
KSEYLQNCGDKSCLMIERFTKDKQANDGVTIANMGGEQNLAGMSTNLDDGTYPDEVNGGKLVVKNGKI
ESGTAKANSVSVFYVKGKALPYVSAEPASIEFSTDNVKVTLHAVYASNLKYTTSEGKTGTFEDGKVIS
VGDSLSVGQSATITVTGTASKTVENVQQGTTLSNKVTVKKVEVPKQNLASQYGTNKVGTGVKKTINFT
AGKDASIADWDSSMLIAQGAANDDPRVYRPNSMYEVPIDLYALYGAYDDDNLYLMWEMTNVQDWDTG
DDYPLSQGHLWQTQNLPFHIAIDTKDDSTRIGNNGGLQTGGSLWASNITWGGEQKLNNWTISTNGSN
GPWIYKGDETGLNANAAYGPAANAKTNTKKSGIKFGYGNGILSKDVIGIDGGWGESNGRWGDMKADN
ENNAKWVNFNDKGHNSDRMDDHYEIAIPLEELGTTAERIATSGIGIELAATFGLSAMDSLPYDLAMND
NADLPDTNTQVNNSFEKSDEDMFTVKMANIGGSGPLVETKSVKINQGNYTVDLSKGVTTKQLTATTDP KGAS VSWS SSDKDVATVS PKGWT PKKAGKAT I TAKSGTKTS S I TVTVTGQLPPDPVKDNTI YAAKPS
GWGKIYAYVYTGDGATAANNAAWPGVEMTAPSATDGCQQTELYKYWPDSLAKGAKVIFNDGGSQQYP
GSRQPGLDYNGGIVKWDGSSAALGALECETTIPVTSVSVSGDGVSGGKLSLKSGASVQLTATVKPDNA
TDRKVTWTSSDSSVANVMGTGWTAGSKAGKATVTATAGGVSASVEVTVEAVGKQPMTVWYKPDSSWK
TVKANYRVYSTPQSAGHGVELKATCGGWYKLTVPDTKGAEVSIAFTNGSKWDSNGGKNYIGSGTVMAV
SDGRVSFKTPDCPAVKPMTVWYKPDSSWTTVKANYRVYSAVQKTGNGWLEKSCGGWYSLTVPDTEGA RVSIAFTNGSRWDSNGGKNYIAAGDTMAVSGGQWPDVTPNCTINAK
[0021] SEQ ID NO: 4 is the amino acid sequence of BAD2 IGDMICFN_02013 protein:
MQGSREGKVRLSTHTFAGMANALTRKEPPHENQPELMTRAERRRAARKARA'VKTWKKVAAGTVAVATL
FGGMGVASTALAADRDS YQDT I GNS S FEAARQQYGLTKHMKNGAI LHAWMWSFKNI TANMEAI AKAGY TSVQTEPMSKIKFVPSNGKKFDENWYYVYQPANTSIGNFVVGSEDDLKEMTATAHKYGVRVIVDWAN
HFTSDWSAIDSDWQNTDYFHKRTGCDGPNGEIKDYSNRYKVTQCHLLGLWDLNTQSQTVADRMQSFLK
TAVADGVDGFRYDAAKHVELPSEVFDNKKSNYWNTILKNGSQFQYGEVLQGDSGLDYKAYANMFSDNS
SDGGGNTASAYGGTVRAAISSGNLSTKMVQSISTGGASEDQLVTWVESHDNYANKEQTGSDGVKKGVS
TELTDYELMMGWAIVGSRKAGAPLYFNRPKESGGKDANGNIRPQFAEKSQLGDTGDDMWKNTSWAVN
HFRNAMDGKSEYLQNCGDKSCLMIERFTKDKQANDGVTIANMGGEQNLAGMSTNLDDGTYPDEVNGGK
LWKNGKIESGTAKANSVSVFYVKGKVLPYVSAEPASIDFSTDSVKVTLHAVYASNLKYTTSEGKTGT
FQDGDVISVGSSLSVGQSATITVTGTASKTVESVQQGTALSNKVTVNKVDVPPQNLAVQYGTNKVGNG VKKTINFTAGKDASIADWDSSMLIAQGAANDDPRVYRPNSMYEVPIDLYALYGAYDDDNLYLMWEMTN VQDWDTGDDYPLSQGHLWQTQNLPFHIAIDTKDDSTRIGNNGGLQTGGSLWASNITWGGEQKLNNW TISTNGSNGPWIYKGDESGLNANAVYGPAANAATNTKKSNIKFGYGNGILSKDVIGIDGGWGESNGRV VGDMKADNENNAKWVNFNEKGHNSDRMDDHYEIAIPLEEIGTTADRIASSGIGIELAATFGLSAMDSL PYDLAMNDNADLPDTGSQVNNSFEKSDDDMFSVKMANIGGEEPPVETKSVKIDQSDYSVDLSNGVTTK QLTATTDPKGASVSWSSSDKDVATVSPKGWTPRKAGKATITAKSGTKTSSITVTVTGQLPPDPVKDN TIYAAKPSGWGKIYAYVYTGDGATAVNNAAWPGVKMTAPSATDGCQQTELYKYWPDNLAKGAKVIFN DGGSQQHPGSREPGLDYNGGIVKWDGSSAALAAVECETTIPVTSVSVSGDGVSGGKLSLKSGASAQLT AT VKP DNAT DRKVT WT S S D S S VAN VMGT G WT AGNKAGT AT I KAT AGGKS AS VQVT VT DVPKQ PMT VW
YKPDSSWKTVKANYRVHSTPQSAVHGAGLKAACGGWYKLTVPDTKGAEVSIAFTNGSKWDSNGGKNYI GSGTVMAVSDGRISFKAPDCPWKPMTVWYKPDSSWKTVKANYRVYSAVQKTGNGWLEKSCGGWYSL TVPDTEGARVSIAFTNGSKWDSNGGKNYIGAGDSMAVSGGQWSDVTPNCAVTNKQ
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 shows inhibition of E. coli deleterious strain 08 K88 (F4) by the means of supplementation of BAD4 strain of the present invention assessed by agar spot assay (see Example 4).
DETAILED DESCRIPTION OF THE INVENTION [0023] Definitions:
[0024] As referred herein “EC numbers” (Enzyme Commission numbers) may be used to refer to enzymatic activity according to the Enzyme nomenclature database, Release of February 26, 2020 (e.g., available at https://enzyme.expasy.org/). The EC number refers 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.
[0025] The term “polypeptide" is equally used herein with the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide(s)" as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo-
or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
[0026] As used herein, the terms , .polynucleotide” and ..nucleic acid" or ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711, US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper et al., Nucleic Acids Res (2000), 28 (21): 4332-4339). Said polynucleotide sequence may be in the form of a vector, plasmid or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment.
[0027] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity". For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the no-brief option) is used as the percent identity and is calculated as follows:
(Identical Residues* 100)/(Length of Alignment-Total Number of Gaps in Alignment).
Alternatively, the parameters used may be gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output
of Needle labeled “longest identity” (obtained using the no-brief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotidesx100)/(Length of Alignment-Total Number of Gaps in Alignment).
[0028] Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
[0029] Expression vector: The term "expression vector" may refer to a linear or circular DNA molecule that comprises a polynucleotide encoding a variant (polypeptide) and is operably linked to control sequences that provide for its expression, in particular for its transcription.
[0030] Fragment: The term "fragment" may refer to a polypeptide having one or more (e.g. several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has an activity as described elsewhere herein.
[0031] Host cell: The term "host cell" may refer to any cell type that is susceptible to transformation, transfection, transduction, or the like 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 containing either a nucleotide sequence to be expressed, or an expression vector, and which is able to produce or already producing an enzyme or a polypeptide according to the invention.
[0032] Nucleic acid construct: The term "nucleic acid construct” may refer to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
[0033] 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 expression of the coding sequence.
[0034] Control sequences: The term "control sequences" as used herein may refer to nucleic acid sequences necessary for 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 native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with
linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide of the present invention.
[0035] The term “foodstuff’ may refer to a substance having a food value.
[0036] The term “fodder” may refer to a substance fed to domestic animals.
[0037] The term “feed” may refer to a substance used as food for livestock.
[0038] The term “additive” may refer to a compound or substance added to another product or substance, e.g., in a small amount, to affect a desired property and/or characteristics.
[0039] The term “prebiotic” may refer to a compound or substance capable of inducing the growth and/or activity of beneficial microorganisms. [0040] The term “probiotic” may refer to microorganisms that are intended to have health benefits when consumed or applied to a body.
[0041] The term “nutritional supplement” may refer to a compound or substance capable to support the nutritional content of the diet, e.g., vitamins, minerals and/or probiotic bacteria.
[0042] The term “intermediate" may refer to a compound or substance produced during the process (e.g., during an intermediate stage of the process) of obtaining an end-product of the present invention, e.g., foodstuff, fodder, fodder; feed, additive (e.g., foodstuff-, fodder- or feed additive), nutritional supplement, probiotic or prebiotic of the present invention.
[0043] The term “starch” may refers to a glucose polymer in which glucopyranose units are bonded by a/pha-linkages. It may be made up of a mixture of amylose (15-20%) and amylopectin (80-85%). Amylose may consist of a linear chain of several hundred glucose molecules, and Amylopectin is a branched molecule made of several thousand glucose units (every chain of 24-30 glucose units is one unit of Amylopectin). Accordingly, starch may be composed of two types of polysaccharide molecules, amylose, a mostly linear and flexible polymer of D-anhydroglucose units linked by alpha-1 ,4-D-glucosidic bonds, and amylopectin, a branched polymer of linear chains linked by alpha-1 ,6-D-glucosidic bonds, Starch may have Formula I:
[0044] The term “resistant starch” may refer to starch that resists digestion and absorption in the small intestine of e.g. mammals. Resistant starch (RS) may be classified into four
categories depending on the causes of resistance. RS1 is a physically inaccessible starch due to entrapment of granules within a protein matrix or within a plant cell wall. RS2 is a granular starch that resists digestion by pancreatic alpha-amylase. RS3 is a retrograded, non-granular starch or starch food. RS4 is a resistant starch that has linkages other than alpha-1 ,4- and alpha-1 ,6-D-glucosidic bonds. Resistant starch in the context of the present invention includes, but is not limited to these four categories.
[0045] Alpha-amylases (alpha-1 , 4-glucan-4-glucanohydrolases, EC 3.2.1.1) as used herein constitute a group of enzymes, which catalyzes hydrolysis of starch and other linear and branched 1 ,4-gluosidic oligo- and polysaccharides. [0046] The term "binding starch" may refer to a functional characteristic of a polypeptide having binding specificity for starch, in particular raw starch.
[0047] The “balance of beneficial and deleterious bacteria” as used herein is equivalent to the longer phrase “balance of the level of beneficial and deleterious bacteria". As described herein below, the skilled person can determine such “level” which, as used herein, is equivalent to an “amount”. Thus, the longer phrase as described before may also read “balance of the amount of beneficial and deleterious bacteria”.
[0048] When used herein, the term “gut” is equal to the term “gastrointestinal tract". Thus, one term can replace the other and, vice versa.
[0049] The gut flora, i.e., the community of bacteria resident in the gastrointestinal tract, comprises both beneficial and deleterious bacterial types or species. Whether a particular member of the gut flora is beneficial, deleterious or inconsequential to the health of the non- human mammalian species in particular circumstances can depend on a number of factors, but for the purposes of the present invention certain types or species of bacteria can be considered beneficial and others deleterious. Examples of beneficial members of the gut flora include bifidobacteria (species of the genus Bifidobacterium) and lactic acid bacteria, more particularly species of the genus Lactobacillus as well as Prevotella spp. bacteria.
[0050] Deleterious bacteria include pathogenic bacteria or opportunistic pathogenic bacteria. Examples of deleterious members of the gut flora include pathogenic forms of Escherichia coli. [0051] Gastrointestinal health typically depends on maintenance of an appropriate balance of beneficial and deleterious bacteria. An increase in the level of deleterious bacteria and/or a decrease in the level of beneficial bacteria can be associated with a decline in gastrointestinal health. Conversely, an increase in the level of beneficial bacteria and/or a decrease in the level of deleterious bacteria can be associated with an improvement in gastrointestinal health, for example improvement of health.
[0052] “Improvement” or “improving” or other grammatical forms include "enhancing" or "enhancement" of the balance between the level of beneficial and deleterious bacteria as described herein. An “improvement” herein means shifting the balance in favor of the level of beneficial bacteria, and thus can involve an increase in the level beneficial bacteria and/or a decrease in the level deleterious bacteria. In some embodiments of the invention, enhancement of the balance arises from both a reduction in the level of deleterious, e.g. pathogenic, bacteria and an increase in the level of beneficial bacteria.
[0053] An increase in the level beneficial bacteria in the gut of non-human mammalian species fed with products as described herein encompasses an increase of 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250% or more in the level of beneficial bacteria in the gut of non-human mammals in comparison to non-human mammals not fed with products as described herein as is shown in Example 3 herein. Accordingly, an increase in the level of beneficial bacteria in the context of the methods and uses of the present invention may advantageously be 100%, 125%, 150%, 175%, 200% or more.
[0054] In the course of the present invention it was surprisingly found that two newly isolated strains of the species of Bifidobacterium adolescentis, referred to herein as B. adolescentis BAD4 and B. adolescentis BAD2, can degrade starch, a property which was not found in other strains of this species. [0055] It was further demonstrated that B. adolescentis BAD4 and B. adolescentis BAD2 of the present invention carry a set of gene which are correlated with enzymatic starch degradation. According to whole genome comparison analysis carried out in the course of the present invention, these genes are not present in genomes of B. adolescentis reference strains, which cannot utilize starch as carbon source.
[0056] In preferred embodiments of the present invention starch-degrading bacteria, fed as live probiotics to animals (e.g., livestock non-human mammals) are capable of improving feed digestibility, nutrient uptake and/or animal growth, especially with starch comprising feeds.
[0057] Furthermore, beneficial effects of B. adolescentis BAD4 in combination with two other strains (Enterococcus faecium E16 and Pediococcus acidilactici G2) on pig gut microbiota were surprisingly found. It was shown that the relative abundance of several genera of butyrate-producing bacteria, inhabiting the pig intestine, could be increased by the means of the strains of the present invention (see examples section herein). Butyrate is known to contribute to maintenance of the gut barrier function and is also known for its immunomodulatory and anti-inflammatory properties.
[0058] It was also surprisingly found that B. adolescentis BAD4 and B. adolescentis BAD2 had superior inhibiting activity against Escherichia coli pig pathogen stains, compared to B. adolescentis reference strain, as shown by agar spot tests (see examples section herein). The inhibition of zoonotic pathogens naturally occurring in the gut of pigs, such as E. coli, by applying B. adolescentis to the animal feed is a promising measure for reducing the incidences of post weaning diarrhea in pigs.
[0059] Livestock farmers (e.g., pig producers) usually include several different feed additives (e.g., non-therapeutic additives) to animal’s diet to maintain gut health and improve performance. To reduce the number of necessary products and also costs, there is a need for a product with a broad spectrum of functionalities, such as balancing a healthy gut microbiota, inhibiting potential pathogenic bacteria and/or at the same time also enhancing digestibility and growth.
[0060] On the other hand, probiotics are used in human and animal nutrition for many years and there is a long history of safe use for specific bacterial species. Most global known probiotic products are comprised of a single strain. Commonly used species are Bacillus subtilis, Saccharomyces cerevisiae, Enterococcus faecium, Lactobacillus acidophilus. Species of Pediococcus spp. and Bifidobacterium spp. are rarely used for development of feed additives, compared to formerly named species.
[0061] Roselli et al. 2017 provides an overview of main results of in vitro test with probiotic strains on pig intestinal cell lines and influence of prebiotics and probiotics on porcine microbiota composition and on pathogen adhesion and diarrhea in pigs.
[0062] However, in swine, Lactobacillus acidophilus produces lactic acid and the enzyme amylase during its growth phase. Lactobacillus casei and Enterococcus faecium complement it, also producing those chemicals, but also enjoys a wide temperature and pH range. Both of these microorganisms create an acidic environment hostile to pathogenic bacteria. Bifidobacterium thermophilum is a delicate microorganism found in mother’s milk, as well as human and animal gastrointestinal tracts. Restoring this bacterium is useful for animal’s well being and growth.
[0063] Nonetheless there is no prior art describing a benefit of using Bifidobacterium adolescentis alone or in combination of other species as described herein as a probiotic feed additive for swine. Accordingly, the present invention satisfies such need by providing strains and related products of the present invention as described herein, which are capable of reducing number of ingredients to be added to a “standard” pig diet.
[0064] Therefore, without being bound by theory, the present invention provides for more sustainability in livestock farming, since the level of beneficial bacteria can be increased in
the gut - most likely they settle and can remain in the gut, which is more sustainable than just being added beneficial bacteria through feed, whereby such externally added beneficial bacteria may be not able to settle and remain in the gut. Thus, the sustainability is assumed to reside in the fact that non-human mammal species have in the long run a benefit from the increase of the level of beneficial bacteria caused - as is shown in the Examples - by an exogenously fed probiotic strains of the present invention, rather than by constantly receiving other beneficial bacteria through feed which may no be able to setle and reside in the gut.
[0065] 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: - starch degradation (phenotypically shown by API test); alpha amylase activity (shown genotypically by whole genome comparison analysis); in combination with Enterococcus faecium and Pediococcus acidilactici, beneficial modulation of the pig gut microbiota, by enhancing relative abundance of Prevotella in the large intestine; and/or inhibition of pig pathogens causing post weaning diarrhea (E. coli F4 and F18).
[0066]
ITEMS OF THE INVENTION
[0067] The present invention may also be summarized by the following items:
1. A host cell (e.g., isolated and/or modified and/or recombinant and/or transformed host cell/s) characterized in that: i) said host cell is capable of degrading starch, preferably said starch is a resistant starch (RS) (e.g., a starch that resists digestion and absorption in the small intestine of e.g. mammals), further preferably said host cell having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and/or ii) said host cell is capable of utilizing starch as a carbon source, preferably said starch is a resistant starch (RS); wherein said host cell comprising one or more (e.g., 2, 3, or 4) of the following sequences: a) a polypeptide having at least 88% identity (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%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein), preferably said polypeptide is
capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch- binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); b) a polypeptide having at least 96% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFNJ32013 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); c) a polypeptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-4; d) a polypeptide having at least 78% identity (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%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN _02026 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); e) a polypeptide having at least 82% identity (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%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB-01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); f) a polypeptide having at least 90% identity (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%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity);
g) a polypeptide having at least 93% identity (e.g., at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); h) a polypeptide having at least 78% identity (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%) to the polypeptide set forth in SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); i) a polypeptide according to (a)-(h), wherein said polypeptide is an alpha- amylase like protein; j) a fragment of any one of (a)-(i) capable of binding (e.g., bound to) starch (e.g., said fragment is a starch-binding fragment) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); preferably said fragment is at least 200 amino acids long (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 lest 1200 amino acids); 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 said one or more polypeptides as in (a)-(j), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides. The host cell according to any one of preceding items, wherein said host cell is a prokaryotic (e.g., Bifidobacterium spp.,, Bacteroides spp., (e.g., B. thetaiotaomicron), Ruminococcus spp., e.g., R. bromii, Escherichia spp., (e.g., E. coli), Bacillus spp., (e.g., B. subtilis), Lactobacillus spp., (e.g., Lactobacillus amylovorus, Lactobacillus acidophilus, L. salivarius, L. plantarum, L. buchneri, L. fermentum, L. delbrueckii, L. brevis and/or L. bulgaricus) or eukaryotic cell (e.g., an yeast cell, e.g., Saccharomyces cerevisiae).
The host cell according to any one of preceding items, wherein said host cell is a Bifidobacterium sp. strain (e.g., a Bifidobacterium adolescentis strain). The strain according to any one of preceding items, wherein said strain is selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 (e.g., both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY on 11 October 2021), preferably wherein the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 is at least 98.5% (e.g., at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061.
The host cell of any one of the preceding items, wherein said host cell has one or more of the following characteristics: a) said host cell is a gram-positive, non-motile and/or anaerobic bacterial cell/strain; b) said host cell is a Bifidobacterium adolescentis bacterial strain; c) said host cell is capable of improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock hoofed mammal, e.g., horse or livestock hoofed mammal, e.g., as catle, sheep, goats or pigs), preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig); d) the host cell according to (c), wherein said improvement comprises an increase in level of beneficial bacteria and/or a decrease in level of deleterious bacteria, preferably said improvement is a modulation of GIT microbiota, e.g., by improving a relative abundance of bacteria of Prevotella spp. (e.g., in the large intestine of a pig); e) the host cell according to (c) or (d), wherein said improvement comprises an improvement of digestibility of feed and/or growth of said non-human mammal; f) the host cell according to any one of (c) - (e), wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale, and/or Prevotella spp. strains, e.g., in the large intestine of a non-human mammal, e.g., a pig); and/or the deleterious
bacteria comprise one or more of Escherichia coli strains (e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea (E. coli F4 and/or F18 strains); and/or g) said 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 said host cell is Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. A polypeptide capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), said polypeptide selected from the group consisting of: a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth 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% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth 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 ID NO: 1 , 2, 3, or 4; d) a polypeptide having at least 88% identity (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%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB 01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch- binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); e) a polypeptide having at least 82% identity (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%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein), preferably said polypeptide is capable of binding (e.g., is bound to)
starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity).
7. A polynucleotide encoding one or more polypeptides according to any one of preceding items.
8. A nucleic acid construct or expression vector capable of expressing one or more polynucleotides according to any one of the preceding items. 9. A composition or kit comprising one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding items; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c).
The composition or kit according to any one of the preceding items, wherein said composition or kit is a starch-degrading, nutritional (e.g., nutritional supplement), prebiotic and/or probiotic composition or kit. The composition or kit according to any one of the preceding items, further comprising starch (e.g., resistant starch). A foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff- , fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof, comprising one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a
Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain) c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding items; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c);
g) one or more compositions (e.g., 2, 3 or 4) according to any one of the preceding items; preferably said foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof are for a non-human mammal (e.g., pig). 13. A method for degrading starch, preferably a resistant starch (RS), comprising:
(i) providing one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain)", preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain) c) one or more polypeptides according to any one of the preceding items; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c); g) one or more compositions (e.g., 2, 3 or 4) according to any one of the preceding items;
h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof of claim 10;
(ii) applying (i) to said starch; 14. A method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of a non-human mammal, comprising feeding said non-human mammal with one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a
Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding items; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said at least one polynucleotide is capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c);
g) one or more compositions (e.g., 2, 3 or 4) according to any one of the preceding items; h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof according to any one of the preceding items. 15. The method of any one of the preceding items, wherein said method is an in vitro, ex vivo, in vivo and/or non-therapeutic method and/or said non-human mammal is healthy.
16. Use of one or more of the following: a) one or more of the host cells according to any one of the preceding items, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding items in combination with one or more (e.g., two) of the following strains: an
Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain) c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding items; d) one or more polynucleotide/s (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said at least one polynucleotide is capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid construct/s (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vector/s (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c);
g) one or more compositions (e.g., 2, 3 or 4) according to any one of the preceding items; h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof according to any one of the preceding items; for/in one or more of the following: i) for degrading starch (e.g., a resistant starch, RS); ii) for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock hoofed mammal), preferably said non-human mammal is a Sus spp., e.g., Sus domesticus (domestic pig); preferably, wherein said improvement comprises an increase in level of beneficial bacteria and/or a decrease in level of deleterious bacteria, preferably said improvement is a modulation of GIT microbiota, e.g., by improving a relative abundance of bacteria of Prevotella spp. (e.g., in the large intestine); further preferably, wherein said improvement comprises an improvement of digestibility of feed and/or growth of said non- human mammal; most preferably wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale and/or Prevotella spp. strains, e.g., in the large intestine); and/or the deleterious bacteria comprise one or more of Escherichia coli strains (e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea (E. coli F4 and/or F18 strains); iii) the use according to (ii), wherein said non-human mammal is healthy; iv) the use according to any one of (i)-(iii), wherein said use is a non-therapeutic use; v) any combination of (i)-(iv); vi) the use according to (i)-(v), wherein said use is an in vitro, ex vivo, in vivo and/or non-therapeutic use. The method, host cell, composition, kit or use according to any one of the preceding items, wherein said non-human mammal is a hoofed mammal (e.g., horse or livestock, e.g., catle, sheep, goat or pig), preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig).
[0068] Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.
[0069] Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
[0070] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
[0071] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. 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. Such equivalents are intended to be encompassed by the present invention.
[0072] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".
[0073] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.
[0074] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will 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 integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.
[0075] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
[0076] In each instance herein any of the terms "comprising", "consisting essentially of’ and "consisting of may be replaced with either of the other two terms.
[0077] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
[0078] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
EXAMPLES
[0079] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.
[0080] Example 1: Starch degradation
[0081] Materials and methods
[0082] Carbohydrate metabolism of B. adolescentis BAD4 strain of the present invention was determined by API 50 CH test system (Biomerieux) and compared to state-of-the art strains.
[0083] Bacterial culture
[0084] Bacterial culture was prepared form a single colony grown on LSM-Cys agar plate, incubate anaerobically, using AnaeroGen bags at 37°C for 48h. After incubation time, take a single colony was inoculated in 10 mL of LSM-Cys broth and incubate anaerobically, using AnaeroGen bags at 37°C for 24 to 48h. Purity of ON cultures was examined via microscopy.
[0085] Inoculum and strips preparation
[0086] Bacterial culture was transferred in 15-mL tubes and centrifuged at 3000 rpm for 5 min. API 50 test strips were placed just before use in the incubation tray. After centrifugation the supernatant was removed and pellet resuspended with 100pl of 50 CHL media. Each well of the test strip was filled with 150 pL of suspension and covered with 1-2 drops of paraffin oil and incubated in a box at 37°C for 48 h. Color reaction was evaluated after 24 and 48 h of incubation.
[0087] Results
[0088] API 50 test
[0089] Carbohydrate utilization of B. adolescentis BAD4 of the present invention was compared to B. adolescentis 126 strain. Position 36 in Table 1 shows that BAD4 can utilize starch whereas strain 126 cannot.
[0090] Table 1. Carbohydrate utilization comparison of the B. adolescentis strains:
[0091] Example 2: Genotypic discovery of genes related to starch degradation
[0092] Whole genome sequence analysis of the strains Bifidobacterium adolescentis BAD4 and BAD2 were performed to evaluate the presence of genetic features related to the degradation of resistant starch (RS). Two separate and different sequencing strategies were employed to obtain the most complete genome possible for these strains: the traditional paired-end short-read sequencing with Illumina, and long-read sequencing using a new generation of sequencing platform from Oxford Nanopore according to the manufacturer’s instructions. This assembly was used for protein annotation and comparative genome analysis.
[0093] Materials and methods
[0094] Sequencing strategies
[0095] Long-read MinlON sequencing
[0096] Long-read sequencing was performed in-house on Mk1C devices from Oxford Nanopore. The purified DNA was used as input for sequencing library preparation using the Ligation sequencing kit (SQK-LSK109), followed by sequencing with a runtime of 48h. Basecalling and demultiplexing of the Oxford Nanopore sequencing data was performed using guppy_basecaller (version 3.4.3+f4fc735) with the Fast algorithm (parameters: -config dna_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 date: 2021-02-17). Trimming and filtering of long reads was included during the basecalling and demultiplexing.
[0097] Short-read Illumina sequencing
[0098] Illumina sequencing was performed by LGC Genomics GmbH using first the Allegro Targeted Genotyping kit (TECAN), followed by the plexWell™384 Library Preparation Kit (seqWell™) according to the manufacturer’s instructions. Libraries were amplified in an emulsion PCR for 14 cycles using standard Illumina primers. Size selection for Illumina libraries was done on LMP-agarose gel electrophoresis selecting a range between 300 and 800bp. Final library purification step and quality control of libraries was done with the
BioAnalyzer and Qubit. The prepared libraries were sequenced on a NextSeq 500/550 V2 Illumina instrument, paired end 2x150bp. The sequencing reads were trimmed based on quality and Illumina adaptors were removed using Trimmomatic (version 0.39) (parameters: ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 LEADINGS TRAILING:3 SLIDINGWINDOW:10:20 MINLEN:30) and reads shorter than 30bp were discarded.
[0099] Assembly and protein annotation
[00100] A hybrid assembly was performed by combining the short and long sequencing reads. For this, the assembly pipeline Unicycler (version 0.4.8, parameters: default) was used (Wick et al. 2017). This pipeline assembles the Illumina short sequencing reads with SPAdes (Bankevich et al. 2012), and performs long read assembly with Miniasm and multiple rounds of polishing with Racon (Vaser et al. 2017), using the Illumina contigs as anchor points to improve the assembly.
[00101] The de-novo assembled contigs were annotated to identify open reading frames (ORF) and proteins with the software Prokka (version 1.13.7 and with parameters: - centre XXX) (https://github.com/tseemann/prokka) (Seemann 2014).
[00102] Comparative genome analysis
[00103] Comparative genome analyses were performed with PATRIC and RASTk to identify proteins present in some genomes but absent in others. Particular focus was set on proteins belonging to key pathways for carbohydrate metabolism. After identification of these specific features, global alignment of the protein sequences was performed against the sequences of amylolytic enzymes from the strain Bifidobacterium adolescentis P2/P3 (Jung et al. 2020). The alignments were performed with the Needleman-Wunsch alignment algorithm implemented in the software Needle from EMBOSS (Rice et al. 2000).
[00104] The genomes included in the comparative genome analysis were selected based on information on the ability to degrade resistant starch (Table 2).
[00105] Table 2. B. adolescentis strains included in the comparative genome analyses:
[00106] Results
[00107] Identification of genes related to amylase activity
[00108] Based on the comparative genome analysis, the genomes listed in Table 2 were screened for unique features, present only in some genomes, but absent in the others. Genetic features classified as alpha-amylase (EC 3.2.1.1) were identified particularly in the genomes of BAD4 and BAD2, and additionally in the genomes of Eg1, L2_32, P2/P3 and Ru424 (Table 3). These sequences were not detected in the genomes of the B. adolescentis type strain E194a, nor in the strain E298b. The sequences of these genetic components were extracted for further comparisons.
[00109] Table 3. Identification of unique genetic components potentially performing an alpha-amylase function:
[00110] Local alignments of amylase-related sequences
[00111] All coding sequences identified in the genomes of B. adolescentis BAD4 and BAD2 were used for comparisons to a set of 19 genes encoding for amylolytic enzymes described for B. adolescentis P2/P3 (Jung et al. 2020). Two sequences with similarity to amylolytic enzymes present in P2/P3 were identified in BAD4 (NNHABGDB_01028 and NNHABGDB-01017), and three sequences in BAD2 (IGDMICFN_02026, IGDMICFN_02014 and IGDMICFN_02013). These sequences were absent in the type strain B. adolescentis E194a (DSM 20083 / ATCC 15703) (Table 4). [00112] These sequences are also absent in the strain B. adolescentis E298b (DSM
20086 / ATCC 15705) based on similar blast comparisons. The other amylolytic enzymes present in P2/P3 were also present in the strains BAD4 and BAD2, with high percentage of identity and coverage (Table 4).
[00113] Based on the protein function data available for P2/P3, the identified sequences have alpha-amylase activity, and the presence of these enzymes confers the ability to utilize resistant starch. Since two of the starch-biding sequences from P2/P3 were found in both BAD4 and BAD2, further characterization of these sequences was performed. The remaining 16 sequences described by Jung et al. (2020) for P2/P3 related to amylase activity were also identified in the BAD4, BAD2 and the B. adolescentis type strain, which is not able to utilize RS, indicating that these enzymes are probably involved in the general degradation on other starches and other carbohydrates.
[00114] Table 4. Percent of coverage and Identity based on local alignments to the protein sequences of B. adolescentis P2P3 (Jung et al.2020). B. adolescentis BAD4 and B. adolescentis BA2 can degrade resistance starch while B. adolescentis E298b cannot degrade resistance starch. Relevant proteins for the degradation of resistant starch are marked in bold:
[00115] Global alignments of starch-binding proteins
[00116] A high identity between the sequences of the starch-binding proteins identified in BAD4 and BA2, as it is shown below.
[00117] Proteins similar to CV760_07855
[00118] Global alignment of the amino acid sequences of the starch-binding proteins identified in BAD4 (CDS NNHABGDB_01028) and BAD2 (CDS IGDMICFN .02026) that were similar to CV760_07855 in P2/P3 showed a high percent of identity (94.88%) between the two strains. The global similarity was also estimated between these two proteins and the CV760_07855 coding sequence, showing a lower similarity (77.3% for NNHABGDB_01028 and 77.5% for IGDMICFN_02026).
[00119] Proteins similar to CV760 JJ7945
[00120] Similarly, global alignment of the amino acid sequences of the starch-binding proteins identified in BAD4 (labeled here as NNHABGDB_01017) and BA2 (labeled as IGDMICFN_02013) that were similar to CV760_07855 in P2/P3 showed a high percent of identity, of 93.47% between the two strains. The global similarity was also estimated between these two proteins and the CV760_07855 coding sequence, showing a lower similarity (81.20% for NNHABGDB_01017 and 77.5% for IGDMICFN .02013).
[00121] Whole genome comparison
[00122] It was determined that the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 is 98.5% identical to the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061.
[00123] Example 3: Beneficial modulation of gut microbiota
[00124] A feeding trial in piglets was performed to investigate effects of a combination of B. adolescentis BAD4, E. faecium and P. acidilactici on intestinal gut microbiota.
[00125] Materials and methods
[00126] Feeding trial
[00127] The piglet trial was conducted at Swine Services Unlimited, Inc. (Minnesota, US) from 18th June - 30th July 2020. [00128] 4 rooms were utilized for a total of 64 pens containing 8 pigs per pen.
Treatments were distributed throughout the rooms to reduce any effect of room. Pigs were housed for a total of 42 days during which time performance was measured at each of the 3 phases. Blood and feces were collected at day 21 and also at day 42 at which time a necropsy on a subset of pigs occurred.
[00129] One prototype in three different dosages was tested (low-medium-high). The prototype contained a combination of B. adolescentis BAD4, P. acidilactici, E. faecium. Total microbial counts for low dosage were 2.5E+08cfu/kg feed, for medium dosage 5.0E+08cfu/kg feed and for high dosage 7.5E+08 cfu/kg feed.
[00130] Table 5. Feeding trial results (NC-negatlve control).
[00131] Microbiome analysis
[00132] Intestinal digests (ileum and colon) and fecal samples were sequenced at LGC Genomics (Illumina 16S amplicon sequencing). 16s rRNA sequences were provided to assess the effect of probiotic additive on the taxonomic profile of piglet gut microbiota. The data is collected from digests of pig colon at day 42 of the feeding trial from animals fed with 3 different dosages of the probiotic additive (low, medium, high). A Dada2 based pipeline followed by Calypso downstream analysis was used for the assessment of differences between control and the treatments.
[00133] BiolT pipeline
[00134] Reads were pre-processed according to the parameters provided in the data preprocessing report (/ngsstorage1/JGI/pigstar2/Data_description.pdf). The input for Dada2 version 1.16(1) was a set of reads without primers and sequence adapters. These reads were processed following the steps provided in the provided command line document (/ngsstorage1/JGI/PSEU/commandlines.Rhistory), for taxonomic classification the RDP taxonomic training data formated for DADA2 version 18 was used (https://zenodo.org/record/4310151). The Dada2 output was processed to generate the files necessary to be loaded as input for Calypso (2) and normalized with CSS+log method. Analysis of the data was done on this platform as previously described (e.g., Callahan et al.
Nature methods vol. 13,7 (2016): 581-3; Zakrzewski et al., Bioinformatics, Volume 33, Issue 5, 1 March 2017).
[00135] Results
[00136] Table 6. Fold change of relative abundance of bacterial genera in colon samples, from animals fed with Prototype 1 with medium dosage (PS-1 med) compared to negative control group without probiotic additives, obtained by differential abundance analysis (DeSeq2):
[00137] Table 7. Fold change of relative abundance of bacterial genera in colon samples, from animals fed with Prototype 1 with dosage 2 (PS-1 -High) compared to negative control group without probiotic additives, obtained by differential abundance analysis (DeSeq2):
[00138] Prevotella-driven enterotype has shown positive associations with animal traits including feed intake, feed efficiency, weight gain and incidence of diarrhea. Prevotella Megasphaera elsdenii and Roseburia have been previously reported as SCFAs producer, which is beneficial for strengthening the intestinal cell wall barrier and for immunomodulatory effects.
[00139] Example 4: Pathogen Inhibition
[00140] Materials and methods
[00141] For determination of the ability of B. adolescentis BAD to inhibit E. coli swine pathogens, the following protocol was followed.
[00142] Day 1 : prepare overnight culture of probiotic bacteria in LSM-Cys and incubate anaerobically at 37°C, overnight.
[00143] Day 2: prepare LSM-Cys agar plates (20 mL agar / plate); prepare pre-culture of probiotic bacteria and incubate till desired OD690: Bifidobacterium: OD690 0.250 ± 0.02); spot probiotic bacteria (10E+5 cell/spot) on LSM-Cys agar; to develop bacterial spots, incubate plates anaerobically, using AnaeroGen bags, at 37°C for 48 h.
[00144] Day 3: prepare overnight culture of pathogen bacteria (E. coli) in Nutrient Broth (NB) and incubate under desired incubation conditions.
[00145] Day 4: prepare 0.6 % soft Nutrient Agar; prepare pre-culture of pathogen bacteria in NB; inoculate soft agar (wait until cooled to 50°C) with pre-culture of the pathogen strains (with final concentration 7 log cfu/mL) and pour it over a plate (10 mL / plate); incubate plates for 24 h at 37°C, anaerobically (agar spot assay).
[00146] After incubation period, measure inhibition zones around the probiotic colonies (from the outer edge of the bacteria spot to the outer edge of the clear zones) by using HiAntibiotic ZoneScale.
[00147] The antibacterial activity pf probiotic strain after 24 h incubation at 37°C, was recorded by measuring the width of clear zone (R) Equation:
Total inhibition zone 2r [mm] ratioactMty - colony 2r [mm]
(Total inhibition zone 2r [mm] - colony 2r[mm])
Inhibition zone r [mm] = 2
[00148] The growth inhibitory indices (Gils) were calculated according to equation.
[00149] Strains tested: B. adolescentis (BAD 4 - strain of interest; BRC 126 - DSM
20083 and BRC 1260 - DSM 28530). Pathogen: E.coli 08 K88 (BRC Nr. 104) = F4.
[00150] Results
[00151] Inhibition of E. coll 08 K88 (F4)
[00152] It was observed that B. adolescentis BAD4 was able to inhibit E. coli 08 K88 (F4) strain (Figure 1 ).
Claims
CLAIMS A host cell (e.g., an isolated and/or modified and/or recombinant host cell) characterized in that: jj) said host cell is capable of degrading starch, preferably said starch is a resistant starch (RS), further preferably said host cell having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); and/or ii) said host cell is capable of utilizing starch as a carbon source, preferably said starch is a resistant starch (RS); wherein said host cell comprising one or more (e.g., 2, 3, or 4) of the following sequences: a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 1 (e.g., BAD4 NNHABGDB_01028 protein) or SEQ ID NO: 2 (e.g., BAD2 IGDMICFN_02026 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); b) a polypeptide having at least 96% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth in SEQ ID NO: 3 (e.g., BAD4 NNHABGDB_01017 protein) or SEQ ID NO: 4 (e.g., BAD2 IGDMICFN_02013 protein), preferably said polypeptide is capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); c) a polypeptide having a sequence selected from the group consisting of: SEQ ID NOs: 1-4; d) a fragment of any one of (a)-(c) capable of binding (e.g., bound to) starch (e.g., said fragment is a starch-binding fragment) and/or having alpha- amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity); preferably said fragment is at least 200 amino acids long (e.g., at least 250, at least 300, at least 350, at least 400 amino acids); e) one or more (e.g., 2, 3, or 4) polynucleotides (e.g., DNA or RNA, e.g., polycistronic or monocistronic) encoding said one or more polypeptides as in (a)-(d), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides. The host cell of claim 1 , wherein said host cell is a Bifidobacterium sp. strain.
The strain of claim 2, wherein said strain is selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 (e.g., both strains deposited with Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Address: Inhoffenstr. 7B, D-38124 Braunschweig, GERMANY on 11 October 2021), preferably wherein the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 is at least 98.5% (e.g., at least 99%, at least 99.5% or 100%) identical to the whole genome nucleotide sequence of Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061. The host cell of any one of the preceding claims, wherein said host cell has one or more of the following characteristics: a) said host cell is a gram-positive, non-motile and/or anaerobic bacterial cell/strain; b) said host cell is a Bifidobacterium adolescentis bacterial strain; c) said host cell is capable of improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock hoofed mammal), preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig); d) the host cell according to (c), wherein said improvement comprises an increase in level of beneficial bacteria and/or a decrease in level of deleterious bacteria, preferably said improvement is a modulation of GIT microbiota, e.g., by improving a relative abundance of bacteria of Prevotella spp. (e.g., in the large intestine of a pig); e) the host cell according to (c) or (d), wherein said improvement comprises an improvement of digestibility of feed and/or growth of said non-human mammal; f) the host cell according to any one of (c) - (e), wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale and/or Prevotella spp. strains, e.g., in the large intestine of a pig); and/or the deleterious bacteria comprise one or more of Escherichia coli strains (e.g,, a pathogenic Escherichia coli strains, e.g,, pig pathogens causing post-weaning diarrhea (E. coli F4 and/or F18 strains); and/or
g) said 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 said host cell is Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. A polypeptide capable of binding (e.g., is bound to) starch (e.g., said polypeptide is a starch-binding protein) and/or having alpha-amylase enzymatic activity (e.g., having EC 3.2.1.1 enzymatic activity), said polypeptide selected from the group consisting of: a) a polypeptide having at least 97% identity (e.g., at least 98%, at least 99% or 100%) to the polypeptide set forth 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% identity (e.g., at least 97%, at least 98%, at least 99% or 100%) to the polypeptide set forth 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 ID NO: 1, 2, 3, or 4. A polynucleotide encoding one or more polypeptides of claim 5. A nucleic acid construct or expression vector capable of expressing one or more polynucleotides of claim 6. A composition or kit comprising one or more of the following: a) one or more of the host cells according to any one of the preceding claims, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding claims in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2)
of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding claims; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c). The composition or kit of claim 8, wherein said composition or kit is a starch-degrading, nutritional (e.g., supplement) and/or probiotic composition or kit. A foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff- , fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof, comprising one or more of the following: a) one or more of the host cells according to any one of the preceding claims, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding claims in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., 2) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain)
c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding claims; d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c); g) one or more compositions (e.g., 2, 3 or 4) according to claims 8-9; preferably said foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof are for a non-human mammal (e.g., pig). A method for degrading starch, preferably a resistant starch (RS), comprising:
(i) providing one or more of the following: a) one or more of the host cells according to any one of the preceding claims, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding claims in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain)", preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain) c) one or more polypeptides according to any one of the preceding claims;
d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said one or more polynucleotides are capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptides according to (c); g) one or more compositions (e.g., 2, 3 or 4) according to claims 8-9; h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof of claim 10;
(ii) applying (i) to starch;
A method for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract of a non-human mammal, comprising feeding said non-human mammal with one or more of the following: a) one or more of the host cells according to any one of the preceding claims, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065; b) one or more of the host cells according to any one of the preceding claims in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain); c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding claims;
d) one or more polynucleotides (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said at least one polynucleotide is capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid constructs (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vectors (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c); g) one or more compositions (e.g., 2, 3 or 4) according to claims 8-9; h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof of claim 10. The method of any one of the preceding claims, wherein said method is an in vitro, ex vivo, in vivo and/or non-therapeutic method and/or said non-human mammal is healthy. Use of one or more of the following: a) one or more of the host cells according to any one of the preceding claims, preferably a strain selected from the group consisting of: Bifidobacterium adolescentis designated strain “KI BAD2” having Accession No.: DSM 34061 and Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065. b) one or more of the host cells according to any one of the preceding claims in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain)', preferably the Bifidobacterium adolescentis designated strain “KI BAD4” having Accession No.: DSM 34065 in combination with one or more (e.g., two) of the following strains: an Enterococcus faecium strain (e.g., Enterococcus faecium E16 strain) and a Pediococcus acidilactici strain (e.g., Pediococcus acidilactici G2 strain) c) one or more polypeptides (e.g., 2, 3 or 4) according to any one of the preceding claims;
d) one or more polynucleotide/s (e.g., 2, 3 or 4) encoding said one or more polypeptides as in (c), preferably said at least one polynucleotide is capable of expressing (e.g., expressed) said one or more polypeptides; e) one or more nucleic acid construct/s (e.g., 2, 3 or 4) capable of expressing said one or more polynucleotides according to (d); f) one or more expression vector/s (e.g., 2, 3 or 4) capable of expressing one or more polynucleotides encoding said one or more polypeptide/s according to (c); g) one or more compositions (e.g., 2, 3 or 4) according to claims 8-9; h) one or more of the following: foodstuff, intermediate foodstuff; fodder, intermediate fodder; feed, intermediate feed; additive (e.g., foodstuff-, fodder- or feed additive), intermediate additive (e.g., foodstuff-, fodder- or feed intermediate additive); nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, probiotic, intermediate probiotic and/or mixture/s thereof of claim 10; for/in one or more of the following: i) for degrading starch (e.g., a resistant starch, RS); ii) for improving the balance of beneficial and deleterious bacteria in the gastrointestinal tract (GIT) of a non-human mammal (e.g., a livestock hoofed mammal), preferably said non-human mammal is a Sus spp., e.g., Sus domesticus (domestic pig); preferably, wherein said improvement comprises an increase in level of beneficial bacteria and/or a decrease in level of deleterious bacteria, preferably said improvement is a modulation of GIT microbiota, e.g., by improving a relative abundance of bacteria of Prevotella spp. (e.g., in the large intestine); further preferably, wherein said improvement comprises an improvement of digestibility of feed and/or growth of said non- human mammal; most preferably wherein the beneficial bacteria comprise one or more of butyrate-producing bacterial strains (e.g., Eubacterium spp., e.g., E. rectale and/or Prevotella spp. strains, e.g., in the large intestine); and/or the deleterious bacteria comprise one or more of Escherichia coll strains (e.g., a pathogenic Escherichia coli strains, e.g., pig pathogens causing post-weaning diarrhea (E. coli F4 and/or F18 strains); iii) the use according to (ii), wherein said non-human mammal is healthy; iv) the use according to any one of (i)-(iii), wherein said use is a non-therapeutic use;
v) any combination of (i)-(iv); vi) the use according to (i)-(v), wherein said use is an in vitro, ex vivo, in vivo and/or non-therapeutic use. The method or use according to any one of the preceding claims, wherein said non- human mammal is a livestock hoofed mammal, preferably said non-human mammal is a Sus sp., e.g., Sus domesticus (domestic pig).
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