Bifidobacterium longum transitional microorganisms

Abstract

The present invention relates to a strain of a bifidobacterium longum transition-type microorganism preserved in CNCM (China CNCM I-5942), or to a strain of a bifidobacterium longum transition-type microorganism having identification characteristics of a strain of a bifidobacterium longum transition-type microorganism preserved in CNCM I-5942, and to a method for identifying a strain of a bifidobacterium longum transition-type microorganism preserved in CNCM I-5942.

Description

Technical Field

[0001] This invention relates to a transitional strain of Bifidobacterium longum. Background Technology

[0002] Nutrition plays a crucial role in the development of infants and young children across all areas, including cognitive, motor, sensory, dental, musculoskeletal, immune, and social development. Furthermore, the gastrointestinal or “gut” microbiome during infancy plays a vital role in infant health and development throughout life (see, for example, Tanaka and Nakayama. 2017. Allergol. Int. 66(4): 515-522). Various factors, including diet, can significantly influence microbiome structure and thus affect infant health and development throughout infancy and later life.

[0003] During infancy, mammals (including humans) transition from a diet consisting entirely or primarily of breast milk to a solid food. This is known as the “transition period,” “transfeeding period,” or “weaning.” When this occurs, the gut microbiome structure may undergo significant changes due to changes in diet and other stressors during this time (see, for example, Vatanen et al., 2019. Nature Microbiology. 4:470-479; Dizzell et al., 2021. PLOS ONE. https: / / doi.org / 10.1371 / journal.pone.0248924; Moore and Townsend. 2019. Open Biol. Sep; 9(9):190128; Magne et al. 2006. FEMS Microbiology Ecology, 58(3): 563-571; and Edwards C.A. Ann Nutr Metab 2017; 70:246-250). Changes in the microbiome structure, in turn, can influence the physiology, cognition, anatomy, health, or other states or traits of mammals. Although several studies have been and are investigating the gut microbiome in infants and young children, its impact on the immediate and lifelong health and well-being of infants is far from well characterized. Similar to the lack of characterization and understanding of the gut microbiome in infants and young children, there is also a lack of compositions and formulations that can facilitate the development of a healthy gut microbiome suitable for use in infants or young children. Therefore, there is a need for improved characterization and understanding of the gut microbiome, as well as compositions and methods, to support and / or establish a healthy gut microbiome, particularly in infants and young children.

[0004] The transition from a milk-based diet (breastfed or formula-fed) to a protein- and fiber-rich solid diet leads to an increase in the number of bacteria in the gut, resulting in the evolution of the microbiome composition associated with adult individuals. Weaning is considered a stressful and complex process, and disruption of the gut microbiota can lead to dysbiosis, which is linked to the pathogenesis of intestinal diseases such as diarrhea, IBD, IBS, and celiac disease, as well as extraintestinal disorders such as allergies, asthma, metabolic syndrome, cardiovascular disease, and obesity. The goal is to reduce the stress caused by weaning in order to develop and maintain a healthy gut microbiota.

[0005] Vatanen et al. have described a subspecies of *Bifidobacterium longum* present in the gut microbiome of mammals, particularly humans, during the transitional feeding period, and demonstrated that this unique *Bifidobacterium longum* clade expands with the introduction of solid foods and carries enzymes for utilizing breast milk and solid food substrates (Cell; 10 Nov 2022; 185(23):4280-4297.e12). WO2023 / 278441 describes that the relative abundance of this *Bifidobacterium longum* clade during the transitional feeding period (e.g., weaning) is greater than that of *Bifidobacterium longum* subsp. *infantis* (*Bifidobacterium infantis*) or *Bifidobacterium longum* subsp. *longum*. Indeed, the relative abundance of *Bifidobacterium longum* subsp. *infantis* decreases at the beginning of the transitional feeding period and continues to increase until the end of the transitional feeding period, coinciding with the increase in the abundance of *Bifidobacterium longum* subsp. *longum*.

[0006] However, there is a need for compositions and methods that are particularly suitable for and conducive to supporting the transition of infants and young children between milk-based diets and solids. Summary of the Invention

[0007] This invention is based, at least in part, on providing a novel transitional strain of *Bifidobacterium longum*. This transitional strain is referred to herein as NCC 5025; and was deposited by Nestlé, Inc., on March 29, 2023, at the National Center for the Collection of Microbial Cultures (CNCM), Pasteur Institute, under the Budapest Treaty, with accession number CNCM I-5942.

[0008] Therefore, in a first aspect, the present invention provides a transitional strain of Bifidobacterium longum deposited at the National Center for Microbial Culture Collection (CNCM) with accession number CNCM I-5942, or a transitional strain of Bifidobacterium longum having the identification characteristics of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

[0009] In another aspect, the present invention provides a Bifidobacterium longum transitional microbial strain having at least 99% average nucleotide identity (ANI) compared to a Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942. Suitably, as described herein, the Bifidobacterium longum transitional microbial strain possesses at least one identifying characteristic of the Bifidobacterium longum transitional strain deposited with accession number CNCM I-5942.

[0010] On the other hand, the present invention provides a composition comprising a transitional strain of Bifidobacterium longum according to the present invention; suitably wherein the composition comprises at least one additional probiotic and / or prebiotic.

[0011] The present invention also provides the use of the Bifidobacterium longum transitional strain according to the invention for promoting and / or assisting the transition of infants and / or young children from milk-based diets to solid foods.

[0012] In another aspect, the present invention provides a method for promoting and / or assisting the transition of infants and / or young children from a milk-based diet to solid foods, the method comprising administering to the infants and / or young children a transitional strain of Bifidobacterium longum according to the present invention.

[0013] The present invention provides a transitional strain of Bifidobacterium longum that is believed to have several advantageous properties, making it particularly suitable for supporting the transition between milk-based and solid diets for infants and young children, for example, when used as a probiotic or as part of a synbiotic.

[0014] Unbound by theory, the present invention provides one or more of the following advantages of the Bifidobacterium longum transitional strain:

[0015] a) No antibiotic resistance was found in the antibiotic groups that EFSA considers relevant;

[0016] b) A unique spectrum of carbohydrate-active enzymes (CaZy), including the presence of GH43 subfamily 17 enzymes, which have not been characterized in Bifidobacterium longum species to date.

[0017] c) Favorable growth on 3-FL; not bound by theory, this ability is believed to make the present invention’s Bifidobacterium longum transitional strain competitive in the gut environment of weaned infants;

[0018] d) Favorable growth on a group of food-derived fibers (e.g., inulin and arabinogalactan).

[0019] Overall, the present invention's Bifidobacterium longum transitional strain is particularly well-adapted to the weaning period and may perform better than other Bifidobacterium longum transitional strains in this environment. Furthermore, the present invention's Bifidobacterium longum transitional strain may perform better than other Bifidobacterium longum transitional strains with diets containing food-derived fiber (e.g., in adulthood). Attached Figure Description

[0020] Figure 1 - UPGMA phylogenetic tree of Bifidobacterium longum genome

[0021] Figure 2 - Carbohydrate-active enzymes (CAZyme) carried by transitional strains of Bifidobacterium longum (including NCC 5025).

[0022] Figure 3 - The genetic region of NCC 5025 carrying the unique GH43_17 encoding gene.

[0023] Figure 4 - Growth curves of Bifidobacterium longum transitional strains, including NCC 5025 on 3-FL as the sole carbon source. The last figure shows the growth rate k obtained for each tested strain.

[0024] Figure 5 - Growth curves of different transitional strains of Bifidobacterium longum on substrates A) inulin and B) arabinogalactan Detailed Implementation

[0025] Preferred features and embodiments of the invention will now be described by way of non-limiting examples. Those skilled in the art will understand that they can combine all the features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0026] Any references to prior art literature in this specification should not be construed as an admission that such prior art is well-known or part of general common knowledge in the art. All publications mentioned in this specification are incorporated herein by reference.

[0027] As used in this specification, the words “including,” “comprising,” and similar terms should not be construed as having an exclusive or exhaustive meaning. In other words, they are intended to mean “including, but not limited to.” The terms “including / comprising” and similar terms also include the term “consisting of.”

[0028] Unless otherwise specified, the practice of this invention will employ conventional techniques applicable to those skilled in the art. Such techniques are described in the literature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0029] Numerical ranges include the numerical values ​​that define the range, and unless otherwise stated, all percentages disclosed herein are based on w / w. As used herein, the term “about” means approximately, nearby, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies the value or range by extending the boundaries to be above and below the stated numerical value. Generally, the terms “about” and “approximately” are used herein to modify numerical values ​​above and below the stated value by 10%.

[0030] Unless otherwise specified, all percentages are by weight.

[0031] As used herein, the terms “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, durations, etc., are intended to cover variations in a particular value and variations arising from that particular value, such as variations in a particular value and variations arising from that particular value being 1 / -10% or less, 1 / -5% or less, 1 / -1% or less, and + / 0.1% or less, provided that such variations are suitable for making in the disclosed invention. It should be understood that the values ​​referred to by the modifiers “about” or “approximately” are themselves specifically and preferably disclosed.

[0032] A reference to a sequence having a percentage identity with any of the SEQ ID NOs detailed herein may refer to a sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0033] Identity comparisons can be performed visually, or more typically, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0034] Percentage identity can be calculated on consecutive sequences, that is, by aligning one sequence to another, directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is called "vacancy-free" alignment. Typically, this type of vacancy-free alignment is only performed over a relatively short range of residues.

[0035] While this is a very simple and consistent approach, it fails to consider that, for example, in an otherwise identical sequence pair, an insertion or deletion in the nucleotide sequence can cause subsequent codons to become misaligned, potentially leading to a significant reduction in percentage identity during global alignment. Therefore, most sequence alignment methods are designed to produce optimal alignments that account for possible insertions and deletions without excessively penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0036] However, these more sophisticated methods assign a "vacancy penalty" to each empty space in the alignment, so that for the same number of identical amino acids, the alignment with the fewest possible vacancies (reflecting a higher correlation between the two compared sequences) will receive a higher score than the alignment with many vacancies. The "affine gap cost" is commonly used, which charges a relatively high cost for the presence of a vacancy and a smaller penalty for each subsequent residue within that vacancy. This is the most commonly used vacancy scoring system. A high vacancy penalty will naturally produce the best alignment with fewer vacancies. Most alignment programs allow modification of the vacancy penalty. However, when using such software for sequence alignment, the default values ​​are preferred. For example, when using the GCG Wisconsin Bestfit package, the default vacancy penalties for amino acid sequences are: vacancy: -12, each extension: -4.

[0037] Therefore, calculating the maximum percentage identity first requires generating the best alignment while taking into account gap penalties. Suitable computer programs for performing such alignments are the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), minimap, or Burrows-Wheeler Aligner (BWA). Examples of other software for sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid. – Chapter 18), FASTA (Atschul et al. (1990) J.Mol.Biol. 403-410), and the GNEWORKS suite of comparison tools. BLAST and FASTA can be used for both offline and online retrieval (see Ausubel et al. (1999) ibid., pp. 7-58–7-60). However, for some applications, the GCG Bestfit program is preferred. Another tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0038] Appropriately, percentage identity can be calculated using at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% coverage, based on the minimum coverage shared between the two sequences to be aligned.

[0039] The alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A commonly used example of such a matrix is ​​the BLOSUM62 matrix (the default matrix in the BLAST program suite). The GCG Wisconsin program typically uses either public defaults or a custom symbolic comparison table (if provided) (see the user manual for further details). For some applications, it is preferred to use the public defaults of the GCG package, or, for other software, a default matrix such as BLOSUM62.

[0040] Once the software produces the optimal alignment, the percentage of homology can be calculated, preferably the percentage of sequence identity. As part of sequence comparison, the software typically performs these calculations and produces numerical results.

[0041] The terms “subject,” “individual,” and “patient” are used interchangeably to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets.

[0042] The term “infant” refers to human subjects under 12 months of age or non-human animals of equivalent age.

[0043] As used herein, the term “toddler” or “walking infant” may refer to human subjects aged between 12 months and 5 years. Appropriately, “toddler” may refer to non-human animals of equivalent age.

[0044] The terms "supplementary feeding period," "supplementary period," "transition period," "transitional feeding period," and "weaning period" are used interchangeably and refer to the period during which milk (breast milk or formula) is replaced by other foods in an infant's or toddler's diet. This typically involves a gradual transition from exclusive breastfeeding (breast milk or formula) to a mixed diet that includes milk and / or solid foods. The transition period varies depending on the infant or toddler, but is generally from about 4 months to about 18 months of age, such as from about 6 months to about 18 months, but in some cases it can extend to about 24 months or longer. For humans, weaning typically begins between 4 and 6 months of age and is considered complete once the infant and / or toddler is no longer breastfed or formula-fed, usually around 24 months of age. In some implementations, the weaning period is from 4 to 24 months.

[0045] The term "composition" or "nutritional composition" refers to any kind of composition or formulation that provides nutritional benefits to an individual and is safe for consumption by humans or animals. A nutritional composition may be in solid (e.g., powder), semi-solid, or liquid form and may contain one or more macronutrients, micronutrients, food additives, water, etc. For example, a nutritional composition may contain the following macronutrients: protein sources, lipid sources, carbohydrate sources, and any combination thereof. Furthermore, a nutritional composition may contain the following micronutrients: vitamins, minerals, fiber, phytochemicals, antioxidants, prebiotics, probiotics, bioactive agents, metabolites (e.g., butyrate, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA)), and any combination thereof. The composition may also contain food additives, such as stabilizers (when provided in liquid or solid form) or emulsifiers (when provided in liquid form). The amounts of various components (e.g., oligosaccharides) may be expressed in g / 100g of composition on a dry weight basis when the composition is in solid form (e.g., powder), or in g / L of composition concentration when the composition is in liquid form (the latter also covers liquid compositions that can be obtained by reconstituted powder in a liquid (such as milk, water), such as reconstituted infant formula or stage 2 infant formula or infant cereal products or any other formulation designed to provide nutrition for infants or young children). Typically, nutritional compositions are formulated for oral, enteral, parenteral, or intravenous administration, and generally include one or more nutrients selected from a variety of nutrients: lipid or fat sources, protein sources, and carbohydrate sources. Preferably, the nutritional composition is intended for oral administration.

[0046] In this specific implementation, the nutrient composition is a "synthetic nutrient composition." The expression "synthetic nutrient composition" means a mixture obtained by chemical and / or biological methods.

[0047] Where appropriate, a nutritional composition may contain fiber; for example, as defined herein.

[0048] As used herein, the term "infant formula" refers to a food intended for specific nutritional purposes in the first few months after birth and which meets the nutritional needs of such infants (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and follow-up formula). It also refers to nutritional compositions intended for use in infants, as defined in the Codex Alimentarius Commission (STAN 72-1981) and in provisions concerning infant specialties (including foods for specific medical purposes). The term "infant formula" encompasses both "Stage 1 infant formula," "Stage 2 infant formula," and "follow-up formula."

[0049] Stage 2 infant formula or follow-up formula is introduced starting from the 6th month. Infant formula constitutes the main liquid component of this group's gradually diversifying diet.

[0050] The term "infant food" refers to food designed for specific nutritional purposes for infants or young children during the first few years of their lives.

[0051] The term "infant cereal composition" refers to a food intended for specific nutritional purposes for infants or young children during the first few years of their lives.

[0052] The term "GUM" refers to a milk-based beverage that is typically fortified with vitamins and minerals and is intended for use by toddlers or children.

[0053] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying or mixing with human milk, infant formula, growing milk, or human breast milk fortified with other nutrients. Therefore, a fortifier may be applied after being dissolved in human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or it may be applied as a standalone composition.

[0054] The compositions of the present invention can be supplements.

[0055] Supplements may be in the form of tablets, capsules, lozenges, or liquids. Supplements may also contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulation agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, and gelling agents. Supplements may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including but not limited to: water, gelatin of any origin, plant gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, coloring agents, wetting agents, fillers, etc.

[0056] In addition, supplements may contain organic or inorganic carrier materials suitable for oral or parenteral administration, as well as vitamins, trace minerals, and other micronutrients recommended by government agencies such as the USRDA.

[0057] The term "metabolism" is used here to refer to the ability of a substrate to be broken down, adsorbed, and / or utilized by microorganisms. For example, a substrate may promote and / or contribute to the growth and / or survival of microorganisms.

[0058] Appropriately, the term "capable of metabolizing glycan substrates" may mean that a Bifidobacterium longum transitional strain encodes at least one CAZyme capable of utilizing glycan substrates. For example, the CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within the glycan substrate. Appropriately, the Bifidobacterium longum transitional strain may encode at least one, at least two, at least three, at least four, or at least five CAZymes capable of utilizing glycan substrates. Appropriately, the term "capable of metabolizing glycan substrates" may mean that the glycan substrate (or fibers or components containing the glycan substrate) can promote the growth and / or survival of the Bifidobacterium longum transitional strain (e.g., when added to an anaerobic culture of the Bifidobacterium longum transitional strain). The growth and / or survival of the Bifidobacterium longum transitional strain over time can be determined by measuring the abundance using strain-specific genes, such as using qPCR methods or by measuring growth via optical density measurements of cells at 580 nm. An exemplary assay for measuring the growth of the Bifidobacterium longum transitional strain in the presence of a glycan substrate (e.g., in fiber form) is provided in this example.

[0059] Compared to the number of Bifidobacterium longum transitional bacteria in control anaerobic cultures without HMOs, a polysaccharide substrate that promotes the growth and / or survival of Bifidobacterium longum transitional strains can increase the number of Bifidobacterium longum transitional bacteria in anaerobic cultures by at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 100%. Suitablely, a polysaccharide substrate that promotes the growth and / or survival of Bifidobacterium longum transitional strains can increase the number of Bifidobacterium longum transitional bacteria in anaerobic cultures by a statistically significant amount (e.g., p-value < 0.05 determined by one-way ANOVA) compared to the number of Bifidobacterium longum transitional bacteria in control anaerobic cultures without a polysaccharide substrate.

[0060] "Glycan substrate" refers to a glycan that can be metabolized by microorganisms. A glycan substrate can be, for example, a glycoconjugate, oligosaccharide, or polysaccharide. Glycan conjugates can include N-linked or O-linked glycans within glycoproteins and proteoglycans or glycolipids. For example, O-linked glycans can include proteins or peptides in which the oxygen atom of a serine or threonine residue is linked to a monosaccharide, oligosaccharide, or polysaccharide, as in the case of glycosaminoglycans (GAGs). Other examples of "glycan substrates" are cellulose, a glycan composed of β-1,4-linked D-glucose, and chitin, a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. As used herein, "glycan substrate" includes, for example, oligosaccharides and polysaccharides.

[0061] "Oligosaccharide" can refer to a carbohydrate having more than two but relatively few monosaccharide units (typically three, four, five, six, and up to ten). Exemplary oligosaccharides include, but are not limited to, fructooligosaccharides, galactooligosaccharides (raffinose, stachyose, verbascose), maltodextrin, gentiosaccharides, cellulose oligosaccharides, lactoosaccharides (e.g., those found in mammary gland secretions), isomaltooligosaccharides, lactulose oligosaccharides, mannan oligosaccharides, melibiose-derived oligosaccharides, pectin oligosaccharides, and xylooligosaccharides.

[0062] The term "polysaccharide" can refer to a carbohydrate having more than ten monosaccharide units. Exemplary polysaccharides include, but are not limited to, starch, arabinogalactan, arabinogalactan, β-glucan, laminarin, golden kelp polysaccharide, xylan, arabinoxylan, mannan, fucoidan, rhamnogalacturonic acid polysaccharide, and galactomannan. It should be understood that there is no precise boundary or distinction between the terms oligosaccharide and polysaccharide, and such distinction is not necessary for carrying out this invention.

[0063] The term "glycosaminoglycan" (GAG), or mucopolysaccharide, refers to a long, linear polysaccharide composed of repeating disaccharide units (i.e., two sugar units). The repeating disaccharide units consist of aldoses and amino sugars, with galactose present at the aldose position, except for keratin. GAGs are classified into four groups based on their core disaccharide structure.

[0064] As used in this article, "mucins" can refer to a family of high-molecular-weight, highly glycosylated proteins (glycoconjugates). A key characteristic of mucins is their ability to form gels; therefore, they are key components in most gel-like secretions, playing roles ranging from lubrication to cell signaling to the formation of mechanical or chemical barriers.

[0065] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, suggesting that their important functions may not be directly related to their calorific value. It has been specifically noted in the art that these carbohydrates play a crucial role in early infant and toddler development, such as the maturation of the immune system. Many different types of HMOs have been found in human milk. Each individual oligosaccharide is based on a variety of combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine with these molecules, resulting in a large number of diverse oligosaccharides in human milk; over 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). Based on the presence of fucose and sialic acid in the oligosaccharide structure, HMOs can be classified as unfucosylated (neutral) or fucosylated (neutral) and sialylated (acidic) and non-sialylated molecules, respectively.

[0066] The term "fucosylated oligosaccharide" refers to oligosaccharides containing fucose residues. These oligosaccharides are neutral. Some examples are 2'-fucosylvose (2-FL), 3-fucosylvose (3-FL), difucosylvose (DiFL), lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasaccharide, lactose-N-difucohexasaccharide I, fucosylvose-N-hexasaccharide, fucosylvose-N-neohexose, difucosylvose-N-hexasaccharide I, difucosylvose-N-neohexose II, and any combination thereof. Fucosylated oligosaccharides represent the largest portion of human milk, with 2'-FL comprising up to 30% of the total HMOs. Fucosylated oligosaccharides are believed to reduce the risk of infection and inflammation and promote the growth and metabolic activity of certain symbiotic microorganisms, thereby reducing inflammatory responses.

[0067] The term "N-acetylated oligosaccharide" encompasses both "N-acetyllactoside" and "oligosaccharides containing N-acetyllactoside." Such oligosaccharides are neutral oligosaccharides having N-acetyllactosamine residues. Suitable examples are LNT (lactose-N-tetrasaccharide), para-lactose-N-neohexose (para-LNnH), LNnT (lactose-N-neohexose), DSLNT (disialyllactose-N-tetrasaccharide), and any combination thereof. Other examples are lactose-N-hexose, lactose-N-neohexose, para-lactose-N-hexose, para-lactose-N-neohexose, lactose-N-octasose, lactose-N-neohexose, isol-lactose-N-octasose, para-lactose-N-octasose, and lactose-N-decanose.

[0068] The expressions “at least one fucoidylated oligosaccharide” and “at least one N-acetylated oligosaccharide” should be understood as “at least one type of fucoidylated oligosaccharide” and “at least one type of N-acetylated oligosaccharide”.

[0069] The term "sialylated oligosaccharide" refers to an oligosaccharide containing charged sialic acid residues. This oligosaccharide is acidic. Some examples are 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), and sialyllactose-N-tetrasaccharide (Lst, such as Lst-a, Lst-b, or Lst-c).

[0070] Appropriately, the term "capable of metabolizing HMOs" may mean that a Bifidobacterium longum transitional strain encodes at least one CAZyme capable of utilizing HMOs. For example, this CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within the HMO. Appropriately, the Bifidobacterium longum transitional strain may encode at least one, at least two, at least three, at least four, or at least five CAZymes capable of utilizing HMOs. Appropriately, the term "capable of metabolizing HMOs" may mean that HMOs can promote the growth and / or survival of the Bifidobacterium longum transitional strain (e.g., when added to an anaerobic culture of the Bifidobacterium longum transitional strain). The growth and / or survival of the Bifidobacterium longum transitional strain over time can be determined by measuring the abundance using strain-specific genes, for example, by qPCR or by measuring growth via optical density measurements of cells at 580 nm.

[0071] The term fiber is used herein to refer to carbohydrates that are indigestible in humans or animals. Such fibers in relation to carbohydrates are also discussed herein. Suitablely, fiber can be fermented by the present invention's Bifidobacterium longum transitional strain. In the context of this invention, as used herein, the expressions "fiber" or "multiple fibers" or "dietary fiber" or "various dietary fibers" refer to the indigestible portion of plant-derived food that reaches the large intestine from the small intestine, comprising two main components: soluble fiber, which dissolves in water; and insoluble fiber. Mixtures of fibers are included within the scope of the terms mentioned above. Soluble fiber readily ferments in the colon into gases and physiologically active byproducts and can be prebiotic and viscous. Insoluble fiber is insoluble in water, metabolically inert, and swells, or it can be prebiotic and fermented in the large intestine. Chemically, dietary fiber consists of carbohydrate polymers having three or more monomeric units that are not hydrolyzed by endogenous enzymes in the upper digestive tract of the small intestine or digestive system, such as arabinoxylan, cellulose, and many other plant components such as resistant starch, resistant dextrin, inulin, lignin, chitin, pectin, arabinogalactan, arabinogalactan, galactan, xylan, β-glucan, and oligosaccharides. Non-limiting examples of dietary fiber include: prebiotic fibers such as fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), fruit fiber, plant fiber, cereal fiber, and resistant starch such as high-amylose corn starch.

[0072] As used herein, “added fiber” or “added dietary fiber” means an ingredient that consists primarily or entirely of fiber added to a nutritional supplement composition, and the amount of fiber in which it constitutes the total fiber content of the composition. The total fiber content of a nutritional supplement composition is provided by the sum of the amount of fiber naturally present in the ingredients used in the formulation (e.g., from whole grain flour) and the amount of added fiber.

[0073] Suitable of the invention, the composition may be a probiotic composition.

[0074] The term "prebiotic" refers to non-digestible carbohydrates that exert a beneficial effect on the host by selectively stimulating the growth and / or activity of beneficial healthy bacteria (such as Bifidobacteria in the human colon) (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995; 125:1401-12).

[0075] The term "probiotics" refers to a microbial cell preparation or microbial cell component that has a beneficial effect on the health or well-being of the host (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999: 10 107-10). The microbial cells according to the present invention are typically bacteria.

[0076] The term "cfu" should be understood as colony-forming unit.

[0077] The gut microbiota is the composition of microorganisms (including bacteria, archaea, and fungi) that live in the digestive tract.

[0078] The term “gut microbiome” can include the “gut microbiota” and their “site of activity”, which can include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolites (signaling molecules, toxins, organic and inorganic molecules), and molecules produced by the coexisting host and structured by the surrounding environmental conditions (Berg, G. et al., 2020. Microbiome, 8(1), pp. 1-22).

[0079] Bifidobacterium longum transitional microorganism strain

[0080] In a first aspect, the present invention provides a transitional strain of Bifidobacterium longum deposited at the National Center for Microbial Culture Collection (CNCM) with accession number CNCM I-5942, or a transitional strain of Bifidobacterium longum having the identification characteristics of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

[0081] Appropriately, the transitional strain of Bifidobacterium longum may be referred to as the juvenile subspecies of Bifidobacterium longum in this article.

[0082] Appropriately, the identification characteristics of the Bifidobacterium longum transitional strain of the present invention may refer to one or more of the phenotypic or genotypic characteristics described herein.

[0083] On the other hand, the present invention provides a Bifidobacterium longum transitional microbial strain that has at least 99% average nucleotide identity (ANI) with the Bifidobacterium longum transitional strain deposited in CNCM with accession number CNCM I-5942.

[0084] In some implementations, compared with the Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942, the transitional strain of Bifidobacterium longum has an ANI of at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%.

[0085] Preferably, compared with the Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942, the transitional strain of Bifidobacterium longum has at least 99.9% ANI.

[0086] Appropriately, as described herein, compared with the Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942, the transitional strain of Bifidobacterium longum has an ANI of at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, and has at least one identifying characteristic of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

[0087] Appropriately, as described herein, compared with the Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942, the transitional strain of Bifidobacterium longum has an ANI of at least 98.4%, at least 98.5%, at least 98.6%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, and has at least one identifying characteristic of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

[0088] Methods for microbial genome sequencing are known in the art (see, for example, Segerman; Front. Cell. Infect. Microbiol.; 2020; 10; Article 527102 & Donkor; Genes; 2013;4(4); 556-572). Metagenomic methods can be used by way of example. For example, shotgun sequencing data can be used for suitable metagenomic methods. Suitable metagenomic methods are known in the art and include, for example, MetaPhlAn 3.0 (see Beghini et al.; eLife 2021; 10: e65088; https: / / huttenhower.sph.harvard.edu / metaphlan) for metagenomic sequencing.

[0089] "Average Nucleotide Identity (ANI)" is a term in this field referring to a distance-based method of characterizing species based on pairwise comparisons of genome sequences, and is a computerized alternative to the traditional DNA-DNA hybridization (DDH) technique already used for the phylogenetic definition of species (Goris et al., 2007, "DNA-DNA hybridization values ​​and their relationship to whole-genome sequence similarities", Int. J. Syst. Evol. Microbiol. 57: 81-91). Based on DDH, strains with a correlation greater than 70% are considered to belong to the same species (see, for example, Wayne et al., 1987, Report of the Ad-Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37: 463-464). ANI is similar to the aforementioned 70% DDH cutoff value and can be used for species characterization. ANI has been evaluated in multiple laboratories and has become the gold standard for species demarcation (see, for example, Kim et al., 2014, “Towards ataxonomic coherence between average nucleotide identity and 16S rRNA genesequence similarity for species demarcation of prokaryotes”, Int. J. Syst. Evol. Micr. 64: 346-351; Richter et al., 2009, “Shifting the genomic goldstandard for the prokaryotic species definition”, P Natl Acad Sci USA 106:19126-19131; and Chan et al., 2012, “Defining bacterial species in the genomic era: insights from the genus Acinetobacter”, Bmc. Microbiol. 12).

[0090] The ANI (Average Nucleus Indices) of shared genes between two strains is a powerful tool for comparing genetic relationships between strains, and approximately 95% of ANI values ​​correspond to the 70% DNA-DNA hybridization criterion used to define species. See, for example, Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102(7):2567-72 (2005); and Goris et al., Int Syst Evol Microbiol.57(Pt 1):81-91 (2007). The ANI between two bacterial genomes is calculated by pairwise comparisons of all sequences common to any two strains and can be determined, for example, using any of a number of publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al., Antonie van Leeuwenhoek 110:1281-1286 (2017)); ANI calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)); and JSpeciesWS (Richter et al., Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI between two genomes are known in the art. See, for example, Konstantinidis, KT and Tiedje, JM, Proc. Natl. Acad. Sci. USA, 102: 2567-2572 (2005); and Varghese et al., Nucleic Acids Research, 43(14):6761-6771 (2015). In one specific implementation, the ANI between two bacterial genomes can be determined, for example, by averaging the nucleotide identity of homologous genes identified as bidirectional best hits (BBHs). Protein-coding genes in the first genome (genome A) and the second genome (genome B) are compared at the nucleotide level using a similar search tool, such as NSimScan (Novichkov et al., Bioinformatics 32(15): 2380-23811 (2016)). The results are then filtered to show at least 70% sequence identity for BBHs that retain only those shorter than 70% of the shorter sequence in each BBH pair. The ANI between genome A and genome B is defined as the percentage of identity multiplied by the sum of the alignment lengths of all BBH genes, then divided by the sum of the lengths of the BBH genes. These and ANI determination techniques are known in the art.

[0091] According to the present invention, the reference genome for comparison with the test genome is represented by the transitional strain of Bifidobacterium longum deposited in CNCM with accession number CNCM I-5942.

[0092] In some embodiments, the Bifidobacterium longum transitional strain of the present invention is isolated from humans.

[0093] In some other implementations, the transitional strain of Bifidobacterium longum is neither the subspecies *B. longum* subsp. longum nor *B. longum* subsp. infantis*.

[0094] Suitable, the Bifidobacterium longum transitional strain is provided in the form of a probiotic. Suitable, the Bifidobacterium longum transitional strain is provided in the composition.

[0095] Antibiotic resistance

[0096] Suitablely, the present invention's Bifidobacterium longum transitional strain does not carry transferable antibiotic resistance to one or more antibiotics, preferably one or more antibiotics associated with the European Food Standards Agency (EFSA) (see European Food Safety Authority. 2012. Guidance on the assessment of bacterial susceptibility to antimicrobial alsof human and veterinary importance. EFSA J 10:2740).

[0097] Antibiotic resistance refers to the ability of microorganisms to tolerate antibiotic treatment. The overuse or abuse of antibiotics is associated with the emergence and spread of resistant microorganisms, rendering treatment ineffective and posing a serious risk to public health. Furthermore, the widespread use of antibiotics means that providing bacterial strains that do not exhibit transferable resistance to one or more EFSA-associated antibiotics is becoming increasingly challenging.

[0098] It is known that a single gene can instill antibiotic resistance to a specific antibiotic, and that bacteria can transfer genes via horizontal gene transfer through conjugation, transduction, or transformation. Therefore, it is known that antibiotic resistance can be transferred between bacteria via horizontal gene transfer, including within the gut microbiome.

[0099] Therefore, it is advantageous that the Bifidobacterium longum transition strain of the present invention does not carry transferable antibiotic resistance to one or more antibiotics, because when the Bifidobacterium longum transition strain of the present invention is used as a probiotic, this reduces the risk of antibiotic resistance being transferred to other components of the microbiome.

[0100] Antibiotic resistance has been well described and can be determined using any suitable assay known in the art. Phenotypic and / or genetic methods can be used, by way of example. Phenotypic methods typically involve measuring the growth of test bacteria in the presence of an appropriate concentration of the antibiotic of consideration. Furthermore, many genes mediating antibiotic resistance are known. Therefore, genetic methods for determining antibiotic resistance include determining the presence of one or more antibiotic resistance genes in the genome of the test bacteria (e.g., by PCR, DNA microarrays, whole-genome sequencing and metagenomics, and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry). Appropriately, phenotypic antibiotic tests can be performed according to EFSA recommendations (EFSA J 16,e05206, doi:10.2903 / j.efsa.2018.5206 (2018)); for example, following the official method ISO 10932. Exemplary methods for determining antibiotic resistance are described in detail in this embodiment.

[0101] Antibiotic resistance and potential genes present in Bifidobacteria are known in the art (see, for example, Duranti et al.; Appl Environ Microbiol. 2017 Feb 1; 83(3):e02894-16.). Therefore, those skilled in the art can determine whether a Bifidobacterium is resistant to one or more antibiotics.

[0102] Suitable, the present invention provides a transitional strain of Bifidobacterium longum that is not resistant to at least one, at least two, at least three, at least four, at least five, at least six, or at least seven EFSA-associated antibiotics.

[0103] Appropriately, transitional strains of Bifidobacterium longum are not resistant to either tetracycline or erythromycin.

[0104] Appropriately, transitional strains of Bifidobacterium longum are not resistant to any of tetracycline, erythromycin, clindamycin, and ampicillin.

[0105] Appropriately, transitional strains of Bifidobacterium longum are not resistant to any of tetracycline, erythromycin, clindamycin, ampicillin, gentamicin, streptomycin, chloramphenicol, and vancomycin.

[0106] Tetracycline resistance may be provided by the tet(W) or tet(Q) gene encoding a ribosomal protective protein. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the tet(W) gene. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the tet(W) gene encoding a polypeptide as shown in SEQ ID NO: 1 or a variant sharing at least 80% sequence identity with SEQ ID NO: 1. Suitablely, the variant may share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1.

[0107] SEQ ID NO: 1

[0108] MKIINIGILAHVDAGKTTTLTESLLYASGAISEPGSVEKGTTRTDTMLLERQRGITIQAAVTSFQWHRCKVNIVDTPGHMDFLAEVYRSLAVLDGAILVISAKDGVQAQTRILFHALRKMNIPTVIFINKIDQAGVDLQSVVQSVRDKLSADIIIKQTVS LSPEIVLEENTDIEAWDAVIENNDKLLEKYIAGEPISREKLVREEQRRVQDASLFPVYYGSAKKGLGIQPLMDAVTGLFQPIGEQGSAALCGSVFKVEYTDCGQRRVYLRLYSGTLRLRDTVALAGREKLKITEMRIPSKGEIVRTDTAYPGEIVILPSD SVRLNDVLGDPTRLPRKRWREDPLPMLRTSIAPKTAAQRERLLDALTQLADTDPLLRCEVDSITHEIILSFLGRVQLEVVSALLSEKYKLETVVKEPTVIYMERPLKAASHTIHIEVPPNPFWASIGLSVTPLPLGSGVQYKSRVSLGYLNQSFQNAVRD GIRYGLEQGLFGWNVTDCKICFEYGLYYSPVSTPADFRSLAPIVLEQALKESGTQLLEPYLSFTLYAPREYLSRAYHDAPKYCATIETVQVKKDEVVFTGEIPARCIQAYRTDLAFYTNGQSVCLTELKGYQAAVGKPVIQPRRPNSRLDKVRHMFSKIT

[0109] Suitablely, the transitional strain of *Bifidobacterium longum* of the present invention may lack the tet(Q) gene. Suitablely, the transitional strain of *Bifidobacterium longum* of the present invention may lack the tet(Q) gene encoding a polypeptide as shown in SEQ ID NO: 2 or a variant sharing at least 80% sequence identity with SEQ ID NO: 2. Suitablely, the variant may share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0110] SEQ ID NO: 2

[0111] MRFDNASNVVYYCLIQMNIINLGILAHIDAGKTSVTENLLFASGATEKCGRVDNGDTITDSMDIEKRRGITVRASTTSIIWNGVKCNIIDTPGHMDFIAEVERTFKMLDGAVLILSAKEGIQAQTKLLFNTLQKLQIPTIIFINKIDRAGVNLERLYLDIKTNL SQDVLCMQTVVDGSVYPVCSQTYIKEEYKEFVCDHDDNILERYLADSEIPPTDYWNTIIALVAKAKVYPVLHGSAMFNIGINELMDAITSFILPPASVSDRLSAYLYKIEHDPKGHKRSFLKIIDGSLRLRDVVRINDSEKSIKIKNLKTIYQGREINVDEVGA NDIAIVEDMEDFRIGDYLGAEPCLIQGLSHQHPALKSSVRPDKPEERSKVISALNTLWIEDPSLSFSINSYSDELEISLYGLTQKEIIQTLLEERFSVKVHFDEIKTIYKERPIKKVNKIIQIEVPPNPYWATIGLTLEPLPLGAGLQIESDISYGYLNHSFQN AVFEGIRMSCQSGLHGWEVTDLKVTFTQAEYYSPVSTPADFRQLTPYVFRLALQQSGVDILEPMLYFELQIPQEASSKAITDLQKMMSEIEDSCNNEWCHIKGKVPLNTSKDYASEVSSYTKGLGIFMVKPCGYQITKDGYSDNIRMNEKDKLLFMFQKSMSLK

[0112] Resistance to erythromycin may be provided by the erm(49) gene encoding an rRNA methyltransferase. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the erm(49) gene. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the erm(49) gene encoding the polypeptide shown in SEQ ID NO: 3 or a variant sharing at least 80% sequence identity with SEQ ID NO: 3. Suitablely, the variant may share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 3.

[0113] SEQ ID NO: 3

[0114] MRNIKDTQNFLHSKELVRHLIGICNIKLDDVVIEIGPGKGIITNELAHKARKVVAIEFDEELYEKLKNKFQSNNKVDIIYGDILNYTPRIPSYCVFSNIPFNITSEILNKFLSDKKNEKMFLIMQYEPFIKYAGNPYGAETLRSMLYKPFFD MDLKYRFDPSDFKPAPQARIVLASFERKQFPDVKKEEEKLYKDFLAYIYTNKGETFFAKIKTLFSSNQIKRVWGQIKIDKTTKISEVPYESILKVFKLFFLYGTDANKQLVVNSFNNMNKQNNKLQKNHRNNSKAKSWNSNRKRKPYHRNNV

[0115] Resistance to erythromycin and clindamycin may be provided by the erm(X) gene encoding a ribosomal protective protein. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the erm(X) gene. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the erm(X) gene encoding a protein comprising SEQ ID NO: 4 or a variant sharing at least 80% sequence identity with SEQ ID NO: 4. Suitablely, the variant may share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 4.

[0116] SEQ ID NO: 4

[0117] MSAYGHGRHENGQNFLTNHKIINSIIDLVKQTSGPIIEIGPGSGALTHPMAHLGRAITAVEVDAKLAAKLTQETSSAAVEVVHDDFLNFRLPATPCVIVGNIPFHLTTAILRKLLHAPAWTDAVLLMQWEVARRRAGVGAST MMTAQWSPWFTFHLGSRVPRTAFRPQPNVDGGILVIRRVGDPKIPIEQRKAFQAMVHTVFTARGRGIGEILRRAGLFSSRSETQSWLRSRGIDPATLPPRLHTNDWIDLFQVTGSSLPHHRPISPSGSSQRPPQQKNRSRRR

[0118] Resistance to streptomycin can be provided by mutations within the rpSL gene encoding the ribosomal S12 protein. More specifically, a mutation replacing an A residue with a G residue at nucleotide position 128 has been shown to provide streptomycin resistance (see Kiwaki & Sato; Int J Food Microbiol. 2009 Sep 15; 134(3):211-5). Suitablely, the present invention's Bifidobacterium longum transitional strain may have an A residue at position 128 of the rpSL gene. Suitablely, the present invention's Bifidobacterium longum transitional strain does not contain the G128A mutation in the rpSL gene. An exemplary rpSL gene sequence containing an A at position 128 is shown as SEQ ID NO: 5.

[0119] SEQ ID NO: 5

[0120] TTGCCTACTATTGAACAGCTCGTCCGTAAGGGACGTCAGGCAAAGCCGAAGAAGTCCAAGACTTTGGCCCTGAAGGGCAGCCCGCTGCGTCGCGGCGTGTGCACCCGTGTCTACACCACCACCCCGAAGAAGCCGAACTCGGCTCTGCGTAAGGTCGCTCGTGTGCGCCTGTCCTCGGGCATCGAA GTCACCGCCTACATTCCGGGCGAGGGCCACAACCTGCAGGAGCACTCCATCGTGCTCGTGCGCGGCGGCCGTGTGAAGGATCTCCCGGGTGTCGTTACCACATCGTGCGTGGCGCGCTCGATACCCAGGGTGTCAAGGACCGTAAGCAGGGTCGTTCCCTGTATGGAGCAAAGAAGGCGAAGTAA

[0121] Resistance to chloramphenicol may be provided by the crmX gene encoding a ribosomal protective protein. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the crmX gene. Suitablely, the *Bifidobacterium longum* transitional strain of the present invention may lack the crmX gene encoding a polypeptide comprising SEQ ID NO: 6 or a variant sharing at least 80% sequence identity with SEQ ID NO: 6. Suitablely, the variant may share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 6.

[0122] SEQ ID NO: 6

[0123] MPFALYMLALAVFVMGTSEFMLAGLLPAIATELDVSVGTAGLLTSAFAVGMVVGAPVMAAFARRWPPRLTLIVCLLVFAGSHVIGAMTPVFSLLLITRVLSALANAGFLAVALSTATTLVPANQKGRALSILLSGTTIATVVGVPAGALLSTALGWRTTFWAIAILCIPAAVGVIRGVTNNVGRSETSATSPRLR VELSQLATPRLILAMALGALNNGGTFAFTFLAPIVTETAGLAEAWVSVALVMFGIGSFLGVTIAGRLSDQRPGLVLAVGGPLLLLTGWIVLAVVASHPVALIVLVQGFLSFGVGSTLITRVLYAASGAPTMGGSYATAALNIGAAAGPVLGALGLATGLGLLAPVWVASVLTAIALVIMLLTRRALTKTAAEAN

[0124] Glycan substrate / carbohydrate-active enzymes (CAZymes)

[0125] The present invention provides a transitional strain of Bifidobacterium longum that encodes a specific spectrum of carbohydrate-active enzymes (CAZyme).

[0126] Carbohydrate active enzymes (CAZymes) are responsible for the synthesis and breakdown of glycoconjugates, oligosaccharides, and polysaccharides. They typically correspond to 1%–5% of the genes in living organisms. Glycoconjugates, oligosaccharides, and polysaccharides play important roles in many biological functions, such as serving as structural and energy storage components, and in many intracellular and intercellular events. The CAZyme classification is a sequence-based family classification system associated with the structure and molecular mechanisms of CAZymes (www.cazy.org).

[0127] CAZyme includes glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and the carbohydrate binding module family (CBM).

[0128] GH catalyzes the hydrolysis of glycosidic bonds between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. In most cases, the hydrolysis of glycosidic bonds is catalyzed by two amino acid residues of the enzyme: the common acid (proton donor) and the nucleophile / base. Depending on the spatial position of these catalytic residues, the hydrolysis occurs via overall retention or overall inversion of the terminal isomer configuration.

[0129] The GH classification system is provided by the CAZy classification. In this paper, GH are divided into families (e.g., GH1, GH2, GH3, GH4, etc.) based on molecular function. These families are then further divided into subfamilies based on subgroups found within each family, which share a more recent ancestor and are generally more consistent in molecular function (e.g., GH13_1, GH13_2, GH13_3, GH13_4, etc.).

[0130] Suitablely, the present invention provides a transitional strain of *Bifidobacterium longum* encoding a glycosyl hydrolase family 43_17 (GH43_17) enzyme. GH43_17 contains the activities of both α-L-arabinofuranase (EC 3.2.1.55) and endonucleo-β-1,4-xylanase (EC 3.2.1.8), and has the ability to break down complexed carbohydrates such as arabinogalactan, arabinogalactan, and arabinoxylan. Suitablely, the GH43_17 gene contains the sequence of SEQ ID NO: 7 or has at least 60% sequence identity with SEQ ID NO: 7. Suitablely, the GH43_17 gene contains a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7.

[0131] SEQ ID NO: 7

[0132] ATGAAACGAACTGACATCCACCTGCGCGATCCGTTCGTCCTGCCTCACGACGGTGTCTATTACCTGTATGGCACCCGCGCTGATAACGTGTGGGGCGCGATGGATGGTTTTGATTGCTACACCAGCCGCGACCTTGACAATTGGGAGGGTCCGTTCGAGGTGTTCCACAAGCCGGATGAATTCACGGCCGACCGTGCTTACTGGGCGCCCGAATGCTACGAGCGAGACGGTGTATTCCACCTGATTGCCACGCTCGGCGAGCCGGACGGGCGCAAAAGCGTGCACATGCTACGCGCTGATAGTCCGCTTGATCCGTTCGAATATGTCTGCCGGCTGACCGATCCGAATCAGTCCTGCATTGACGGAACTCTGCATGGTGAAGGTACCGATATGTGGCTTGTCTACTCGCATTCCTTGGAGGATGTGCCCGCCGGAGACATGGATGCCGTACGTCTGTCCTCCGACCTGACTCGGACGGTGGGGGAGAGCATGACATTGTTCCAGGCCTCGGATGCGCCGTGGGCGGTGCCGGTGCCGTTCGCGAAAGCGGAATTCGGCATCGACGAGGACGCCTACTTCTCCGATGGTCCCTGCCTGTGCAGGCTTTCCAACGGACGGCTGGCGATGCTGTGGTCGAGCTGGTCGACGGAAGGCGGATATGCAGTCGGCCAGGCCATCAGCGAATCAGGGTCGATTGCTGGGCCTTGGACGCAATGCCCCGAGCCTCTGCTTAGCCACGGCGGCCACGGCATGCTGTTCAACGGTCTCGATGGCGTGCTGCGTTACGCGGTCCACTCGCCCAACGACCCCGGCCAGGAACGGCCTACGTTTTTGTGCGTCGAAGAACAAGACGGGCTGCTGACGATTACGGAATAG

[0133] Suitablely, the GH43_17 gene may encode a protein as shown in SEQ ID NO: 8 or a sequence having at least 80% sequence identity with SEQ ID NO: 8. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.

[0134] SEQ ID NO: 8

[0135] MKRTDIHLRDPFVLPHDGVYYLYGTRADNVWGAMDGFDCYTSRDLDNWEGPFEVFHKPDEFTADRAYWAPECYERDGVFHLIATLGEPDGRKSVHMLRADSPLDPFEYVCRLTDPNQSCIDGTLHGEGTDMWLVYSHSLEDVPAG DMDAVRLSSDLTRTVGESMTLFQASDAPWAVPVPFAKAEFGIDEDAYFSDGPCLCRLSNGRLAMLWSSWSTEGGYAVGQAISESGSIAGPWTQCPEPPLLSHGGHGMLFNGLDGVLRYAVHSPNDPGQERPTFLCVEEQDGLLTITE

[0136] Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 43_22 (GH43_22) gene. Suitably, the GH43_22 gene comprises SEQ ID NO: 9 and / or SEQ ID NO: 10, or a sequence having at least 60% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10. Preferably, the *Bifidobacterium longum* transitional strain of the present invention comprises the GH43_22 gene having at least 60% sequence identity with SEQ ID NO: 9 and the GH43_22 gene having at least 60% sequence identity with SEQ ID NO: 10. Suitably, the GH43_22 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10.

[0137] SEQ ID NO: 9

[0138]

[0139] SEQ ID NO: 10

[0140]

[0141] Suitablely, the GH43_22 gene may encode the protein shown in SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence having at least 80% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12. Preferably, the *Bifidobacterium longum* transitional strain comprises the GH43_22 gene encoding the protein shown in SEQ ID NO: 11 or a sequence having at least 80% sequence identity with SEQ ID NO: 11, and the GH43_22 gene encoding the protein shown in SEQ ID NO: 12 or a sequence having at least 80% sequence identity with SEQ ID NO: 12. Suitablely, the protein may comprise a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12.

[0142] SEQ ID NO: 11

[0143]

[0144] SEQ ID NO: 12

[0145]

[0146] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 43_27 (GH43_27) gene. Suitablely, the GH43_27 gene comprises the sequence of SEQ ID NO: 13 or has at least 60% sequence identity with SEQ ID NO: 13. Suitablely, the GH43_27 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13.

[0147] SEQ ID NO: 13

[0148]

[0149] Suitablely, the GH43_27 gene may encode a protein as shown in SEQ ID NO: 14 or a sequence having at least 80% sequence identity with SEQ ID NO: 14. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14.

[0150] SEQ ID NO: 14

[0151]

[0152] Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 43_29 (GH43_29) gene. Suitably, the GH43_29 gene comprises the sequence of SEQ ID NO: 15 or has at least 60% sequence identity with SEQ ID NO: 15. Suitably, the GH43_29 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15.

[0153] SEQ ID NO: 15

[0154]

[0155] Suitablely, the GH43_29 gene may encode a protein as shown in SEQ ID NO: 16 or a sequence having at least 80% sequence identity with SEQ ID NO: 16. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 16.

[0156] SEQ ID NO: 16

[0157]

[0158] Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 121 (GH121) gene. Suitably, the GH121 gene comprises the sequence of SEQ ID NO: 17 or has at least 60% sequence identity with SEQ ID NO: 17. Suitably, the GH121 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17.

[0159] SEQ ID NO: 17

[0160]

[0161] Suitablely, the GH121 gene may encode a protein as shown in SEQ ID NO: 18 or a sequence having at least 80% sequence identity with SEQ ID NO: 18. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18.

[0162] SEQ ID NO: 18

[0163]

[0164] Appropriately, the transitional strain of Bifidobacterium longum contains the GH43_17 gene and one or more genes selected from the GH43_22, GH43_27, GH43_29 and GH121 genes as defined herein.

[0165] Appropriately, the transitional strain of Bifidobacterium longum contains one or more of the following genes: GH43_17, GH43_22, GH43_27, GH43_29, and GH121.

[0166] Suitablely, one or more of the arabinogalactan-degrading GH methods described herein include a signal peptide. A “signal peptide” can refer to a short amino acid sequence typically present at the N-terminus of a polypeptide that allows the polypeptide to be secreted outside sterile cells. Without being bound by theory, this can advantageously allow the present invention’s Bifidobacterium longum transitional strains (typically found in the diet when present at high molecular weight) to act as the primary degrader of the complex structure of arabinogalactan. Suitablely, a “primary degrader” can refer to bacteria capable of depolymerizing a specific polysaccharide into monosaccharides, disaccharides, and oligosaccharides that can absorb themselves and ferment themselves into acidic end products such as acetate or lactate. Suitablely, GH43_22 enzyme, GH43_27 enzyme, GH43_29 enzyme, GH_121 enzyme, GH43_24 enzyme, and / or GH30_5 enzyme may include a signal peptide. Appropriately, each of the enzymes GH43_22, GH43_27, GH43_29, GH_121, GH43_24, and GH30_5 may contain a signal peptide.

[0167] Suitable, the Bifidobacterium longum transitional strain of the present invention contains a gene encoding a glycosyl hydrolase family that targets arabinogalactan.

[0168] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 43_24 (GH43_24) gene. Suitablely, the GH43_24 gene comprises the sequence of SEQ ID NO: 19 or has at least 60% sequence identity with SEQ ID NO: 19. Suitablely, the GH43_24 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19.

[0169] SEQ ID NO: 19

[0170]

[0171] Suitablely, the GH43_24 gene may encode a protein as shown in SEQ ID NO: 20 or a sequence having at least 80% sequence identity with SEQ ID NO: 20. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20.

[0172] SEQ ID NO: 20

[0173]

[0174] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 127 (GH127) gene. Suitablely, the GH127 gene comprises the sequence of SEQ ID NO: 21 or a sequence having at least 60% sequence identity with SEQ ID NO: 21. Suitablely, the GH127 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21.

[0175] SEQ ID NO: 21

[0176]

[0177] Suitablely, the GH127 gene may encode a protein as shown in SEQ ID NO: 22 or a sequence having at least 80% sequence identity with SEQ ID NO: 22. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22.

[0178] SEQ ID NO: 22

[0179] MNVTITSPFWKRRRDQIVESVIPYQWGVMNDEIDTTVPDDPAGNQLADSKSHAVANLKVAAGELDDEFHGMVFQDSDVYKWLEEAAYALAYHPDPELKALCDRTVDLIARAQQPDGYLDTPYQIKSGVWADRPRFSLIQQSHEMYVMGHYIEAAVAYHQVTGNE QALEVAKKMADCLDANFGPEEGKIHGADGHPEIELALAKLYEETGEKRYLTLSQYLIDVRGQDPQFYTKQLKALNGDNIFPDLGFYKPTYFQAAEPVRDQQTADGHAVRVGYLCTGVAHVGRLLGDRGLIDTAKRFWTNIVARRMYVTGAIGSTHVGESFTYDYD LPNDTMYGETCASVAMSMFAQQMLDLEPKGEYADVLEKELFNGSIAGISLDGKQYYYVNALETTPDGLDNPDRHHVLSHRVDWFGCACCPANIARLIASVDRYIYTERDGGKTVLSHQFIANTAEFASGLTVEQRSNFPWDGHVEYTVSLPASATDSSVRFGLR IPGWSRGSYTLTVNGKPAVGSLEDGFVYLVVNAGDTLEIALELDMSVKFVRANSRVRSDAGQVAVMRGPLVYCAEQVDNPGDLWNYRLADGVTGADAAVAFQADLLGGVDTVDLPAVREHADEDDAPLYVDADEPRAGEPATLRLVPYYSWANREIGEMRVFQRR

[0180] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 30_5 (GH30_5) gene. Suitablely, the GH30_5 gene comprises the sequence of SEQ ID NO: 23 or has at least 60% sequence identity with SEQ ID NO: 23. Suitablely, the GH30_5 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23.

[0181] SEQ ID NO: 23

[0182]

[0183] Suitablely, the GH30_5 gene may encode a protein as shown in SEQ ID NO: 24 or a sequence having at least 80% sequence identity with SEQ ID NO: 24. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24.

[0184] SEQ ID NO: 24

[0185]

[0186] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 43_32 (GH42_32) gene. Suitablely, the GH42_32 gene comprises the sequence of SEQ ID NO: 25 or has at least 60% sequence identity with SEQ ID NO: 25. Suitablely, the GH42_32 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25.

[0187] SEQ ID NO: 25

[0188]

[0189] Suitablely, the GH42_32 gene may encode a protein as shown in SEQ ID NO: 26 or a sequence having at least 80% sequence identity with SEQ ID NO: 26. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26.

[0190] SEQ ID NO: 26

[0191] MTATISNGVSASYSPAEDELGAADPTALLAESGDLKPLAERTYTNPVPYADGKSHTAPDPFVLKYRDLYYCYATDEHGILVSTSPDMVHWTSHGFCYTEAGRRNFWAPSVILINGVFHMYFSNMPAEETDTHTEIMRVAVSEDPLGPFEKKAELFNTFAIDSQVVYGDDGQLYLLYAD NQVTGLSDDRRPGTSVMIDRLVTPYSRENKPRPLIVPTMDEEIFARNRFGDGRDWHTVEGATYFAYRDRAFITYSANAYEHEDYFVGYSYAQLPNKQADAHIDQLDWTKQLNENRFDPLLIRSPKVEGTGHNSIVKAPNAVDDWIVYHGRNADDELYVGTEQRVMRIDPLYYAEGGLDT PGPTAAAQSAPLYGTVHDDFADGLNAGWSVISGAAHTESDVDGHALVADESSVFIAVSGKSSATQVIDVWAKAPVTPLGARFGIVVRYQDANNLTKLEVDAGRQVISVVDVIGGVASERVTNADLHDFDSHAWHEYRLERRYCRLEIRIDGRFAASCTISDKPGRAGLFSLRTGAAFS AYAATEHVNLWGAGLRDLGRELHADRRLVIDGGVRSSGVCPVTLELAYPLVSNRFVLDFAGQTSRGQALLSLGEYRLSGTASSVEFMRNGKSLPSTPEPARLRVFEDNVRRDRSGRAVLTIRIEALNGTMRLHLRGKTWQVPFADNAARARITLDRASLTGYERTSLESSIEERSASGN

[0192] Appropriately, the transitional strain of Bifidobacterium longum contains one or more genes selected from the GH43_24 gene, GH127 gene, GH30_5 gene and GH43_32 gene as defined herein.

[0193] Appropriately, the transitional strain of Bifidobacterium longum contains the GH43_17 gene and one or more genes selected from the GH43_24, GH127, GH30_5 and GH43_32 genes as defined herein.

[0194] Appropriately, the transitional strain of Bifidobacterium longum contains one or more of the following genes: GH43_17, GH43_24, GH127, GH30_5, and GH43_32.

[0195] Appropriately, the transitional strain of Bifidobacterium longum includes the GH43_17, GH43_22, GH43_27, GH43_29, GH121, GH43_24, GH127, GH30_5 and GH43_32 genes as defined herein.

[0196] Appropriately, transitional strains of Bifidobacterium longum include GH43_17, GH43_22, GH43_27, GH43_29, GH121, GH43_24, GH127, GH30_5, and GH43_32 as defined herein.

[0197] GH43_17 gene cluster

[0198] Appropriately, transitional strains of Bifidobacterium longum may contain one or more genes encoding family 31 glucosidase (GH31), ABC transporter, Lac-I type regulator, MFS transporter and / or AraC family transcription regulator.

[0199] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 31 (GH31) gene. Suitablely, the GH31 gene comprises the sequence of SEQ ID NO: 27 or has at least 60% sequence identity with SEQ ID NO: 27. Suitablely, the GH31 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27.

[0200] SEQ ID NO: 27

[0201]

[0202] Suitablely, the GH31 gene may encode a protein as shown in SEQ ID NO: 28 or a sequence having at least 80% sequence identity with SEQ ID NO: 28. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28.

[0203] SEQ ID NO: 28

[0204] MTTSFTIDGNALIWTGDGETLRIEPWEENSVRVRATRNRGFGPVDWALLEPKNESGRVADIAVGEDGEHASLTNGSITVKADSNHAPLLSAGYETFRCDLSFWNAEGELLFREYPQGGSLLLKARDYTPVSGESFAVTTSFSADPKERLYGMGEYQQDVLDLKGSTFELAH RNSQASVPFVVSSKGYGFLWHNPAIGRATFGRNRTEWAAQSTDQIDYWVTAGDSYAQIESQYADATGHAPVMPEWGMGFWQCKLRYWNQEQLLDVARGFKSRNIPLDLIVIDFFHWPHLGDYKFEDEFWPDPEAMVAELNSMGVKLMVSVWPQVSVSSENFVEMKRNNYLVS AEAGLNLDMMFEEPCVNYDPTNPGARKFVWDKCKANYWDKGVRAFWLDEAEPEYGVYDFRNYRYHMGSDLNVGNVYPQAYNRGFYEGQIEAGMEGEIVNLTRCAWAGSQRYGSLVWSGDVGSTFADLKSQITCAIHMGMAGIPWFTTDMGGFHDGVIDSDSFKELLARWCAF SCFLPVMRNHGDRSLGESTGKQTITKATGEHRSPSGADNEPWSYGPEMESIFRKYIAVREVMRPYTRELFQSAHEQGQPLVRGLFYEFPTDEHVADIADEYLYGPDILVAPVVEAGAASRSVYLPGDETTTWTDLRDGAVYAGGQSIESSAAIDTVPAFARDGRDHGLIGLL

[0205] Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises one or more ABC transporter genes. Suitably, the ABC transporter gene comprises SEQ ID NO: 29 to SEQ ID NO: 31, or a sequence having at least 60% sequence identity with SEQ ID NO: 29 to SEQ ID NO: 31. Suitably, the ABC transporter gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29 to SEQ ID NO: 31. Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises a gene having at least 60% sequence identity with SEQ ID NO: 29, a gene having at least 60% sequence identity with SEQ ID NO: 30, and a gene having at least 60% sequence identity with SEQ ID NO: 31.

[0206] SEQ ID NO: 29

[0207] ATGACGCATCGTAGCACCTGGTGGAAAACCGCTCTCGGCATCATATTGACGCTCATCATGATGTTTCCTGTCTACTGGATGATCAACATCTCGTTCACTGGTAAGGCATCCATTCGTTCCGGCGACCTGTGGCCCAAGGATTTCACCTTTGACAACTACGCCCGCGTAATCGCCGACCAAATGCCCTATCTGGGCACTTCCATCCTCGTAGCGGTATGCTGCGTGATTCTAACGCTGGTCATCGCACTGCCTGCCGCCTACGCACTGGCTTTGCTGCGCTGTCCAGGCAGCGGCGCGCTCAGCTTCCTGCTCATCGTGGCTCAGATGATTCCCGCCGTCGTGATGTCGCTCGGCTTCTACGAGATTTATAACAACATTGGTCTGCTCGATACGTTGCCCGGCCTGATCCTCGCCGACTCGACCATTGCGGTGCCGTTCGCGGTCATGCTCCTGACTTCTTTCATGGCCGGCATCCCGCGGTCCCTGCTTGAGGCCGCCGAAGTGGATGGAGCCTCACGTACCCGTCGCTTCTTTTCCATTGTCATCCCGTTATCGCGCAATTCGATCGTGACCGTCTCCCTGTTCGCTTTCCTATGGTCTTGGAGCGACTTCCTGTTCGCTTCCACCCTTGACTCCGGCGGCGGCAAGATGCGCCCGATCACTATGGGTCTGTACAACTATATCGGTGCGCAGACCCAGGAATGGGGGCCGATGATGGCCACCGCAGTGCTTGCATCCATTCCCGCGACCATCCTGCTTGTCTTCGCCCAGAAGTACGTCGCCGCAGGCGTGACCGCCGGTGCTGTTAAGGACTAA

[0208] SEQ ID NO: 30

[0209] ATGACAGCCTCAACAACAAGCCCCGTTCGCCGGGCAAAGTCCGGCACTCCGGTCCGGGCCAAACTGGCCATCGCCGGATTCATTGCCCCACTGATTATCTACTTGGTAATCTTTTACGCGTTCCCGCTCATCCAGAACGTGTCAATGAGCCTGCACCGATACACGCGACGAACCTTCGTTACCGGAGATGCGCTGTTCGTGGGTCTCGACATCTACAAGGAAGTCATTTCCTCCGTGGAGTTCTGGCCGGTTGTGGGGCAGACCTTCGTGTTCGTGGTCGTCTCGCTGATATTCCAATATGTAATCGGCTTGGCCCTGGCGGTGTTCTTCAACGATAACTTCAAGCTCTCTGGTGTGCTGCGCGGCATCATGCTGGTTCCGTGGCTGTTGCCGCTGATTGTTTCTGGAACCGTCTGGCAGTGGATGATGGACCCTGACTCCGGCATCCTCAACATGTTCCTCGGTCTGTTTGACATCGAACCCATCTGGTGGCTCCAGGCGGATAACTCGCTGTGGGCCGTCATCATCGCCAACATCTGGCTGGGAATCCCCTTCAACCTCGTGATCCTGTATTCCGGCCTACAGAACATCAGCGGCGACCTGTATGAAGCCGCCTCCCTCGATGGCTGCAACGCCTGGCAGCGCTTCTGGAAGATCACCTTCCCTCTCCTGAAGCCCGTCACTTCGATCACCCTGTTGCTCGGCTTCGTCTATACATTGAAGGTCGTTGACGTGATCTGGATGATGTCCCAGGGAACCGGCACCTCGCGTACCCTCGCCACCTGGGCCTATTCGATGGCATTTGGCAAGGGAACTTCAATGACTATCAAATACTCGGAGGCTTCGGTGCTCGGCACGATTCTCATCATCGTGGCGTTGATTTTCGGACTGATTTACCTGCGGGTCCAGAAGACCCAGGAAACCTGCTAA

[0210] SEQ ID NO: 31

[0211]

[0212] Suitablely, the ABC transporter gene may encode a protein as shown in SEQ ID NO: 32 to SEQ ID NO: 34, or a polypeptide having at least 80% sequence identity with SEQ ID NO: 32 to SEQ ID NO: 34. Suitablely, the gene may encode a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 32. Suitablely, the gene may encode a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 33. Suitablely, the gene may encode a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 34.

[0213] SEQ ID NO: 32

[0214] MTHRSTWWKTALGIILTLIMMFPVYWMINISFTGKASIRSGDLWPKDFFTFDNYARVIADQMPYLGTSILVACCVILTLVIALPAAYALALLRCPGSGALSFLLIVAQMIPAVVMSLGFYEIYNNIGLLDTLPGL ILADSTIAVPFAVMLLTSFMAGIPRSLLEAAEVDGASRTRRFFSIVIPLSRNSIVTVSLFAFLWSWSDFLFASTLDSGGGKMRPITMGLYNYIGAQTQEWGPMMATAVLASIPATILLVFAQKYVAAGVTAGAVKD

[0215] SEQ ID NO: 33

[0216] MTASTTSPVRRAKSGTPVRAKLAIAGFIAPLIIYLVIFYAFPLIQNVSMSLHRYTRRTFVTGDALFVGLDIYKEVISSVEFWPVVGQTFVFVVVSLIFQYVIGLALAVFFNDNFKLSGVLRGIMLVPWLLPLIVSGTVWQWMMDPDSGILNMFL GLFDIEPIWWLQADNSLWAVIIANIWLGIPFNLVILYSGLQNISGDLYEAASLDGCNAWQRFWKITFPLLKPVTSITLLLGFVYTLKVVDVIWMMSQGTGTSRTLATWAYSMAFGKGTSMTIKYSEASVLGTILIIVALIFGLIYLRVQKTQETC

[0217] SEQ ID NO: 34

[0218] MKSNTALKITAALCSCAMLVGVSACGSNSTTDDKVIEWWDDWTRHEDGSEFDKLVKACAPEGYTIERQAIATSDLLNNLTTAIKEDNGPDVAVIDNPMIPSAVDAGLVAGSDETGLDVSAWDENLEAPGVVDGQAYGVPLGGSNTLGLMYNPTIIEAAGVDVSTITDWDSLNAAIKKVVDAGYKGITFSGISGEE GVFQFLPWFWGAGGDLSKLDSQAQKDAEDLLSGWISKGWAPKSATTNTQSASWDLFLAGDYGFAEIGTWMQSEADEAGAKLIPIPAKDGGVATVPTGGEFAMVAYHKKDAESHYKLANQVIECLSEDETLLKVSNALSNLAAKKAVRAEQLAASDGLAQWKESIENAAGRTSDLGLKYEEASASISESLLAALNAA

[0219] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises a Lac-I type regulatory gene. Suitablely, the Lac-I type regulatory gene comprises a sequence of SEQ ID NO: 35 or having at least 60% sequence identity with SEQ ID NO: 35. Suitablely, the Lac-I type regulatory gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 35.

[0220] SEQ ID NO: 35

[0221]

[0222] Suitable, the Lac-I type regulatory gene may encode a protein as shown in SEQ ID NO: 36 or a sequence having at least 80% sequence identity with SEQ ID NO: 36. Suitable, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 36.

[0223] SEQ ID NO: 36

[0224] MVTINDVAREAGVSKTTVSFVLSGSRPVAAATEQRIREAMDRLGYTVNHAARSLSTSKTMTIAVVTSNRQDAYFDIARGTYINGLSRAAAETGYDMLITNDPDGSATENACQSHKADGLVFLDVRQNDPRVPIAAESGIPTVSLGVPVNPMNLDVVDTDFTDMAAST MRTLHDAGHRRVSVITLSSRVIAEQLNDTARFLREIERSGERLGMHATIRHCSTRPGIIDTDIARILDGRGEDTAFVIHNESAVLVFRRAVEHRGLRIPEDISVIAINEKQMSDALYLPYSAYENDVELVTQSAVNTLVDRIEHPELTPRTLIKASYIDRDSVANI

[0225] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises an facilitater superfamily (MFS) gene. Suitablely, the MFS gene comprises a sequence of SEQ ID NO: 37 or having at least 60% sequence identity with SEQ ID NO: 37. Suitablely, the MFS gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 37.

[0226] SEQ ID NO: 37

[0227]

[0228] Suitablely, the MFS gene may encode a protein as shown in SEQ ID NO: 38 or a sequence having at least 80% sequence identity with SEQ ID NO: 38. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 38.

[0229] SEQ ID NO: 38

[0230] MAEFHYAIGHFHCAGHRIGCSGIPGQLALQRADDCFGITLTALVGAWLTGKLASILSRKTVALIGAGGMLLFGLLPYFVHSSLAAVIAFSALMGVCLGFINNVLPTLISVHYEGDERQSIMGQQVAVASIGAMVFMTVAGKLATAQWYHAYLIYLFAAVVLVVCAFTLPTKNGETDEAGRIQGTGPSASIR EVMTGKLWFLVVAGFFFLLANNAYSNNLSLLVEQRGLGDAGTAGLISTIGQFGGLLAGLCVGLMVRFVKNHLLMVGFIVEGLSLLLLGCSASLPLLIIGSFFAGAGLSIYYAQAPFLVTVIEKPYLIPLGIAAMTTANALGGFASPVLVNAINGLFGSHAAGAMFIGAAIALAGAVALGVSGFQKKCLESAK

[0231] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention contains an AraC family transcriptional regulatory gene. Suitablely, the AraC gene contains a sequence of SEQ ID NO: 39 or a sequence having at least 60% sequence identity with SEQ ID NO: 39. Suitablely, the AraC gene contains a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 39.

[0232] SEQ ID NO: 39

[0233] ATGGAGCGCGATGCTTTCCGGCTGCCGGGCCTCACCGCCGGCGATGACAACCAGTATGCCGATCACACGCTCACCGGCATGGCAGCCGATGCGGCGAACGTCATAGCCGCAGGCGGTCCCGCCCCGCTGACTAGCTTCGGCACTGTCGCTCAAGCCGCCCATCTCAATCCAGATGACGGCTTCGGCATCATTGGCCATGATCTTGCACACCCATCGCACCTACACCGGCATGACTATATGGAAATCACGCACGCCATCGCCGGTACGGTACTGGTCTGGGTCGAAGGAGAGACCAACGTGCTGACACAGGGCGGCACCATACTCATCAAGCCTGGAGCCCGTCATCTCATCTCCCCCATCATCGAATACGGGCAAACACCACACGAGGCGGACATCCTGATTAAACCCGAGCTCATCAGGCAATGCCGCATTCCGATTCTGGAAGCAGCCGGCGCCGACCGGATGTTCATTAGCTGGCTTGACGATGACCGGCAGACCCACTGCCTGCTGGCAGCCGGCAAGCACCACGCCGGCGAGGCCGCTATCAGCCGCATGTTCATCGCCTACTGCATCAACGCAACCTACAGGCCAGACTTCACCGTCATCGGCAACCTGCTCGAGCTGTTCCACGAAACGTCCCGAGTCTTGGAACACCAGCCACGTACCGATCCGCTGATCGCCGCCATCATCGAAACCATCACGGCAGATCCCGCCACGGCCCACAACCAGGCCATAGCGGACACACTTGGATACAGCGTGGGATATCTGTCCCGGTACGCGCGCAAGCACAGCGGGCACACACTCGGCCAACTCATCAACGAGGAAAGGCTCCGACTCGGCGCCGAACTGCTCGTCACCACCGACGACACCATTGCCGAAATCACCCGAACCATTGGCTACGAAAGTCCAGCCTATTTCCATAAACTCTTCCGCAGCCGCTACCTCATTACCCCCGACCGCTACCGCAACGACTTCCGTATCGCATTACGTTGCGGATGA

[0234] Suitablely, the AraC gene may encode a protein as shown in SEQ ID NO: 40 or a sequence having at least 80% sequence identity with SEQ ID NO: 40. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 40.

[0235] SEQ ID NO: 40

[0236] MERDAFRLPLGLTAGDDNQYADHTLTGMAADAANVIAAGGPAPLTSFGTVAQAAHLNPDDGFGIIGHDLAHPSHLHRHDYMEITHAIAGTVLVWVEGETNVLTQGGTILIKPGARHLISPIIEYGQTPHEADILIKPELIRQCRIPILEAAGADRMFISWLDDDRQT HCLLAAGKHHAGEAAISRMFIAYCINATYRPDFTVIGNLLELFHETSRVLEHQPRTDPLIAAIIETITADPATAHNQAIADTLGYSVGYLSRYARKHSGHTLGQLINEERLRLGAELLVTDDTIAEITRTIGYESPAYFHKLFRSRYLITPDRYRNDFRIALRCG

[0237] Appropriately, the transitional strain of Bifidobacterium longum contains the MFS transporter and AraC family transcriptional regulatory genes.

[0238] Appropriately, the transitional strain of *Bifidobacterium longum* contains GH43_17, the MFS transporter, and AraC family transcriptional regulatory genes. Appropriately, GH43_17, the MFS transporter, and AraC family transcriptional regulatory genes are contained within a gene cluster.

[0239] As used in this article, a "gene cluster" can refer to a group of genes located adjacent to each other on a chromosome.

[0240] Appropriately, the transitional strain of Bifidobacterium longum contains each of the genes for GH31, ABC transporter, Lac-I type regulator, MFS transporter, and / or AraC family transcription regulator.

[0241] Appropriately, the transitional strain of *Bifidobacterium longum* contains GH43_17, MFS transporter, AraC, GH31, ABC transporter, and Lac-I type regulatory gene. Appropriately, GH43_17, MFS transporter, AraC family transcription regulators, GH31, ABC transporter, and Lac-I type regulatory gene are included in the aa gene cluster as described above.

[0242] Appropriately, the Bifidobacterium longum transitional strain also contains xylulokinase genes and / or xylose isomerase genes. Appropriately, xylulokinase genes and / or xylose isomerase genes are included in the gene clusters defined above.

[0243] Suitablely, the xylulokinase gene comprises SEQ ID NO: 41 or a sequence having at least 60% sequence identity with SEQ ID NO: 41. Suitablely, the xylulokinase gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 41.

[0244] SEQ ID NO: 41

[0245]

[0246] Suitablely, the xylulokine gene may encode a protein as shown in SEQ ID NO: 42 or a sequence having at least 80% sequence identity with SEQ ID NO: 42. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 42.

[0247] SEQ ID NO: 42

[0248] MTRVLVAGVDTSTQSTKVRITDAATGEQVRFGQAKHPDGTSVNPEFWWEAFTKAAEQAGGLDDVAALAVGGQQHGMVILDKQGNVIRDAMLWNDTSSAPQAAALIDKLGATPAEGDEPDDVTARGK QRWVKAVGSSPVASYTLTKVAWVAENEPENAKKIAAVCLPHDWLSWRIAGYGPVAEGEDAHLEALFTDRSDASGTIYYDAAHDEYRRDLIAMVLTPAEGEEAAKAHADAIVLPTVLGPHEAAAVKAD PAIAGKDVEGGCIIGPGGGDNAMASLGLGMAVGDVSVSLGTSGVAAAIAENPVYDLTGAISGFADCTGHYLPLACTINGSRILDAGRAALGVDYDELAELAFKAEPGAGGITLVPYFDGERTPNRP DATASLTGLTLHNTTKENLARAFVEGLLCSQRDCLELIRSLGAEINRILLIGGGAKSVAIRTLAPSILGMDVTRPATDEYVAIGAARQAAWVLSGEAEPLTWQLTIEGVETGEPTEAVYEAYAKARG

[0249] Suitablely, the xylose isomerase gene comprises SEQ ID NO: 43 or a sequence having at least 60% sequence identity with SEQ ID NO: 43. Suitablely, the xylose isomerase gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 43.

[0250] SEQ ID NO: 43

[0251]

[0252] Suitablely, the xylulose isomerase gene may encode a protein as shown in SEQ ID NO: 44 or a sequence having at least 80% sequence identity with SEQ ID NO: 44. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44.

[0253] SEQ ID NO: 44

[0254] MGLWDVDKIEYVGRAKGPKEDFAFHYYDADKVVAGKKMKDWLRFGVAWWHTFNQELVDPFGTGTAHRPYYKYTDPMDQALAKVDYAFELFQKLGVEYFCFHDRDIAPEGDTL RETNANLDKVVDKIDENMKSTGVKLLWNTSSLFTNPRFVSGAATSPFADIYAYAGGQLKKSLEIGKRLGAENYVFWGGREGYENLWNTEMKRETDHIAKFFHMCADYAKEIG FEAQFLIEPKPKEPTLHQYDFDAATAIEEFLRNHDLTDVFKLNLEGNHANLAGHTYQHEIRVARESGFLGSLDANQGDKLIGWDMDEFPTDLYETVAVMWEVLQAGSIGPHGG LNFDAKPRRTSFYEEDLFRSHIAGMDAYAAGLLVADKMNQDGFIQNLQAERYSSYDSGIGKDIDEGNVTLADLEAYSLDKPQSELIAATKSDHLESVKATINNYIIDALAEVE

[0255] Human milk oligosaccharides (HMOs)

[0256] Appropriately, compared with other Bifidobacterium longum transitional strains, the Bifidobacterium longum transitional strains of the present invention preferentially utilize 3-fucosylated lactose (3-FL), as demonstrated by better growth, for example as shown in the embodiments of the present invention.

[0257] Suitablely, when cultured in the presence of 3-FL, the *Bifidobacterium longum* transitional strain of the present invention may have a growth rate of at least 0.6 kJ. Suitablely, when cultured in the presence of 3-FL, the *Bifidobacterium longum* transitional strain of the present invention may have a growth rate of at least 0.7 kJ, at least 0.8 kJ, or at least 0.9 kJ. The growth rate can be calculated by culturing on a given substrate or substrate mixture for a period of time and modeling the growth curve using a logistic growth model to obtain the relative growth rate k. An exemplary method for determining the growth rate is provided in this embodiment. Without being bound by theory, preferential growth on 3-FL is considered advantageous because the level of 3-FL increases in human breast milk during weaning. Preferential growth on 3-FL suggests that the *Bifidobacterium longum* transitional strain of the present invention may be particularly adapted to survive and grow in the microbiome during weaning.

[0258] Suitably, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 25 (GH25) gene. Suitably, the GH25 gene comprises the sequence of SEQ ID NO: 45 or having at least 60% sequence identity with SEQ ID NO: 45. Suitably, the GH25 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 45.

[0259] SEQ ID NO: 45

[0260]

[0261] Suitablely, the GH25 gene may encode a protein as shown in SEQ ID NO: 46 or a sequence having at least 80% sequence identity with SEQ ID NO: 46. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46.

[0262] SEQ ID NO: 46

[0263] MSNPTNDGINLNYLANVRPSSRQLVWQRMEMYAFIHFGMNTMTDREWGLGHEDPALFDPQNVDVEQWMDALVAGGMTGVILTCKHHDGFCLWPSRYTQHTVAASPWRDGKGDLVREVSE SARRHGLKFGVYLSPWDRTEESYGKGKAYDDFYVGQLTELLTQYGPIFSVWLDGANGEGKNGKTQYYDWDRYYNVIRSLQPNAVISVCGPDVRWAGNEAGHVRDNEWSVVPRRLRSAELT MENSQQEDDASFASTVRSQDDDLGSREAVSGYGDDVCWYPAEVDTSIRPGWFYHKYEDDKVMSADQLFDLWLSAVGGNSSLLLNIPPSPEGLFAEPDVESLKGLGSRINEFRKALASSC CEVKTSSADETAMRLLDGNQDTYWSPDANDVAPAVTLTFPQLTTINAVVVEEAIEYGQRIEHMRVTGVLSDGTECVLGQFGTVGYRRILRFDDVEVSSVTLHVDDSRFTPMISRAAAVRI

[0264] Suitablely, the *Bifidobacterium longum* transitional strain of the present invention comprises the glycosyl hydrolase family 95 (GH95) gene. Suitablely, the GH95 gene comprises the sequence of SEQ ID NO: 47 or has at least 60% sequence identity with SEQ ID NO: 47. Suitablely, the GH95 gene comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 47.

[0265] SEQ ID NO: 47

[0266]

[0267] Suitablely, the GH95 gene may encode a protein as shown in SEQ ID NO: 48 or a sequence having at least 80% sequence identity with SEQ ID NO: 48. Suitablely, the protein may contain a sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48.

[0268] SEQ ID NO: 48

[0269] MKLTFDGISSCWEEGIPLGNGRMGAVLCSEPETDVLYLNDDTLWSGYPHAETSPVTPEIVAKARQASLQDDYTAATRIIKEATLQEKDEQIYEPFGTARIQYSTPADGRESMKRQLDLARALAGETFQMGDANVHVDAWCSEPDDLLVYRMSSDAPVDVNISVAGTFLKQSRASLETVSDGHRATLVVMGRMPGL NIGLLPHPSEHPWEDEQDGTGMAYAGAFSLTVTGGDINVDDNSLQCSHITGLSLRFRSMSGFKGSDQQPERSMTVIADHLEKTIDEWSTDLQTMLDRHIADYRRYFDRVAIHLGSAHDDDTELPFSAILRSDENKEPHRLEMLAEAMFDFGRYMLISSSRPHTQPANLQGIWNHKDFPNWYSAYTTNINVEMNYWM TGPCALKELIEPLVSMNEELLAPGHDAADADRILGCRGSAVFHNVDLWRRALPANGDPMWAFWPFGQAWMCRNLFDEYLFNQDASYLARIWPIMRDNARFCMDFLSETEHGLAPSPATSPENCFLVNGEPVSVAQSSENATAIVRNLLDDLIQASHDLENLDEEDRNLVREAESVRSQLAETRLGADGRVLEWNDEFI ESDPQHRHLSHLYELHPGAGITSKTPRLEEAARKSLEVRGDDGSGWSIVWRMIMWARLRDAEHAKRIIGMFLRPVDANAETNLLGGGVYDSGLCAHPPFQIDGNLGFPAALSEMLVQSHDGWIRVLPALPEDWHEGSFHALRARGGIQVDATWTDQTVEYTLRCSKPTEITLNVLGTDMGRVALSPDKPFKGTIRR

[0270] Suitable, the Bifidobacterium longum transitional strains of the present invention may contain the GH25 gene and the GH95 gene as defined herein.

[0271] Embodiments

[0272] This invention provides embodiments according to the following numbered clauses:

[0273] 1. A transitional strain of Bifidobacterium longum deposited at the National Center for Microbial Culture Collection (CNCM) with accession number CNCM I-5942, or the aforementioned transitional strain of Bifidobacterium longum possessing the identification characteristics of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

[0274] 2. A transitional strain of Bifidobacterium longum, wherein the transitional strain of Bifidobacterium longum has at least 99% average nucleotide identity (ANI) with the Bifidobacterium longum strain deposited in CNCM with accession number CNCM I-5942.

[0275] 3. The Bifidobacterium longum transitional strain as described in Clause 1, wherein the Bifidobacterium longum transitional microbial strain has at least 98.1% ANI with the Bifidobacterium longum transitional strain deposited with accession number CNCM I-5942.

[0276] 4. A transitional strain of *Bifidobacterium longum* according to any of the preceding clauses, wherein the transitional strain of *Bifidobacterium longum* is not resistant to either tetracycline or erythromycin.

[0277] 5. A transitional strain of *Bifidobacterium longum* according to any of the preceding clauses, wherein the transitional strain of *Bifidobacterium longum* is not resistant to any of tetracycline, erythromycin, clindamycin and ampicillin.

[0278] 6. A transitional strain of *Bifidobacterium longum* according to any of the preceding clauses, wherein the transitional strain of *Bifidobacterium longum* is not resistant to any of tetracycline, erythromycin, clindamycin, ampicillin, gentamicin, streptomycin, chloramphenicol and vancomycin.

[0279] 7. A transitional strain of *Bifidobacterium longum* according to any of the preceding clauses, wherein:

[0280] (i) The lack of resistance to tetracycline is due to the absence of a tetracycline resistance gene, preferably the tetW gene encoding a protein having at least 80% sequence identity with SEQ ID NO: 1 and / or the tetQ gene encoding a protein having at least 80% sequence identity with SEQ ID NO: 2;

[0281] (ii) The lack of resistance to erythromycin is due to the absence of an erythromycin resistance gene, appropriately the Erm49 gene, which encodes a protein having at least 80% sequence identity with SEQ ID NO: 3;

[0282] (iii) The lack of resistance to erythromycin and / or clindamycin is due to the absence of a corresponding resistance gene, suitably the Erm(X) gene encoding a protein having at least 80% sequence identity with SEQ ID NO: 4; and / or

[0283] (iv) The lack of resistance to chloramphenicol is due to the absence of a chloramphenicol resistance gene, appropriately the CrmX gene which encodes a protein having at least 80% sequence identity with SEQ ID NO: 6.

[0284] 8. A transitional strain of Bifidobacterium longum according to any of the preceding clauses, wherein the transitional strain of Bifidobacterium longum comprises the glycosyl hydrolase family 43_17 (GH43_17) gene; suitably wherein the GH43_17 gene comprises SEQ ID NO: 7 or a sequence having at least 60% sequence identity with SEQ ID NO: 7.

[0285] 9. The Bifidobacterium longum transitional strain according to any of the preceding clauses, wherein the Bifidobacterium longum transitional strain further comprises a major facilitater superfamily (MFS) gene; suitably wherein the MFS gene comprises SEQ ID NO: 37 or a sequence having at least 60% sequence identity with SEQ ID NO: 37.

[0286] 10. The Bifidobacterium longum transitional strain according to any of the preceding clauses, wherein the Bifidobacterium longum transitional microbial strain further comprises the AraC gene; suitably wherein the AraC gene comprises SEQ ID NO: 39 or a sequence having at least 60% sequence identity with SEQ ID NO: 39.

[0287] 11. The transitional strain of Bifidobacterium longum as described in Clause 10, wherein the GH43_17 gene, the MFS gene, and the AraC gene are contained in a gene cluster.

[0288] 12. A transitional strain of Bifidobacterium longum according to any one of clauses 8 to 11, wherein the transitional strain of Bifidobacterium longum further comprises one or more of the GH31 gene and the LacI gene; preferably it further comprises the xylulose kinase gene and the xylose isomerase gene.

[0289] 13. The Bifidobacterium longum transition strain according to Clause 12, wherein the GH43_17 gene, the MFS gene, the AraC gene, the GH31 gene, and the LacI gene are contained in a gene cluster; preferably wherein the GH43_17 gene, the MFS gene, the AraC gene, the GH31 gene, the LacI gene, the xylulose kinase gene, and the xylose isomerase gene are contained in a gene cluster.

[0290] 14. The Bifidobacterium longum transitional strain according to any of the preceding clauses, wherein the Bifidobacterium longum transitional strain further comprises one or more genes encoding one or more glycoside hydrolases selected from the group consisting of: GH43_17, GH43_22, GH43_27, GH43_29, GH121, GH43_24, GH127, GH30_5, GH43_32 and GH30.

[0291] 15. The Bifidobacterium longum transitional strain according to any of the preceding clauses, wherein the Bifidobacterium longum transitional strain further comprises the GH29 gene and the GH95 gene.

[0292] 16. A transitional strain of Bifidobacterium longum according to any of the preceding clauses, wherein the transitional strain of Bifidobacterium longum preferentially utilizes 3-fucosylated lactose (3-FL).

[0293] 17. A transitional strain of Bifidobacterium longum according to any of the preceding clauses, wherein the transitional strain of Bifidobacterium longum has a growth rate of at least 0.6k when cultured in the presence of 3-FL.

[0294] 18. A composition comprising a transitional strain of Bifidobacterium longum as described in any of the preceding clauses; preferably, the composition is a probiotic composition.

[0295] 19. A composition comprising a transitional strain of Bifidobacterium longum as described in any of the preceding clauses, and at least one additional probiotic and / or prebiotic.

[0296] 20. Use as a dietary supplement of the Bifidobacterium longum transitional strain or composition according to any of the preceding clauses; suitably, the Bifidobacterium longum transitional strain or composition is administered to the subject in combination with a fiber-containing diet or fiber-containing food.

[0297] 21. The use of any of the transitional strains or compositions of Bifidobacterium longum according to any one of Clauses 1 to 19 for promoting and / or assisting a subject in the metabolism of a fiber-containing diet or food.

[0298] 22. A method for promoting and / or assisting a subject in the metabolism of a fiber-containing diet or food; said method comprising administering to said subject a transitional strain or composition of Bifidobacterium longum according to any one of clauses 1 to 19.

[0299] 23. The use of any Bifidobacterium longum transitional strain or composition according to any one of Clauses 1 to 19 for promoting and / or assisting the transition of infants and / or young children from a milk-based diet to solid foods.

[0300] 24. A method for promoting and / or assisting the transition of infants and / or young children from a milk-based diet to solid foods, the method comprising administering to the infants and / or young children a transitional strain or composition of Bifidobacterium longum according to any one of clauses 1 to 19.

[0301] 25. The use according to any one of Clauses 20, 21 or 23 or the method according to Clauses 22 or 24, wherein the Bifidobacterium longum transitional strain is administered in combination with a prebiotic.

[0302] 26. The composition according to Clause 19 or the use or method according to Clause 25, wherein the prebiotic is fiber and / or human milk oligosaccharides (HMOs); suitably, wherein the HMO is 3-FL.

[0303] Examples

[0304] The invention is further described with reference to the following embodiments. It should be understood that the invention protected by the claims is not intended to be limited in any way by these embodiments.

[0305] Example 1 - Isolation and phylogenetic identity of NCC 5025 strain

[0306] A transitional strain of *Bifidobacterium longum*, NCC 5025, was isolated from the feces of weaned infants (6 to 12 months old) at Nestlé Research. This transitional strain was obtained from fecal samples by culturing on Eugon tomato agar, followed by preliminary identification using MALDI-ToF MS (Biotyper, Bruker Scientific Instruments, Billerica, USA) and confirmation by sequencing. The isolate was deposited as NCC 5025 at the Nestlé Culture Collection (Lausanne, Switzerland) and further deposited as CNCM I-5942 at the Collection Nationalede Culture Microorganisms (CNCM, Paris, France). PacBio sequencing of NCC 5025 was performed according to the supplier's recommendations. Sequencing data were further assembled using the de novo assembly analysis application of the Stratified Genome Assembly Process (HGAP4), available through the SMRT Link portal (Pacific Biosciences, Menlo Park, USA). The obtained sequences were compared with publicly available Bifidobacterium longum genomes using the average nucleotide identity (ANI) calculated using OrthoANIu v1.2 (Yoon et al., 2017). The resulting pairwise genome similarity matrices were further used to construct a UPGMA phylogenetic tree using BioNumerics software (v8.0, bioMérieux SA, Massey Itrel, France). The analysis revealed that NCC 5025 belongs to the transition group of Bifidobacterium longum because it clusters with other strains of this presumed new descriptive subspecies (Vatanen et al.; Cell; 10 Nov 2022; 185(23):4280-4297.e12). Phylogenetically, NCC5025 is located between strains isolated from China (e.g., JDM301; CMCC P001) and strains isolated from Bangladesh (NCC5000-NCC5004), sharing 98.4% average nucleotide identity (ANI) with the latter group of strains (see [link to article]). Figure 1 ).

[0307] Example 2 - Antibiotic resistance profiling

[0308] Phenotypic antibiotic testing of *Bifidobacterium longum* transitional strains was performed according to the official method ISO 10932, following the recommendations of EFSA (EFSA J 16, e05206, doi:10.2903 / j.efsa.2018.5206(2018)). *Bifidobacterium longum* ATCC 15707 was used as an internal control, as required by ISO method 10932. The minimum inhibitory concentration (MIC) obtained against this control strain was within the range determined for this strain (see Annex to the ISO method). The obtained MICs were compared with the applicable EFSA thresholds (EFSA Journal 2012 Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance) to determine the sensitivity or resistance phenotype to the relevant antibiotic list. The MICs obtained against *Bifidobacterium longum* transitional strains NCC 5000, 5001, 5002, 5003, 5004, and 5025 are depicted in Table 1.

[0309] The results showed that most strains were considered resistant to several antibiotics considered important for EFSA. *Bifidobacterium longum* transitional strains NCC 5000 and NCC 5001 were considered resistant to erythromycin and clindamycin. NCC 5003 was considered resistant to tetracycline, erythromycin, and clindamycin. NCC 5004 was considered resistant to tetracycline, erythromycin, clindamycin, and ampicillin. Similarly, *Bifidobacterium longum* transitional strain NCC 5002 showed resistance to tetracycline.

[0310] Bifidobacterium longum transitional strain NCC 5025 is the only strain sensitive to all antibiotics considered relevant by EFSA, namely gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, ampicillin, and vancomycin.

[0311]

[0312] Example 3 - Carbohydrate-active enzymes (CaZy) profiling

[0313] Transitional strains of *Bifidobacterium longum* were annotated using a combination of the dbCAN3 tool (Yin et al., 2012; Zhang et al., 2018) and the databases HMMdb (v10) and DIAMOND (v 2.0.14). Query sequences with >0.50 coverage and e-value <1e-15 were annotated using HMMER based on the dbCAN CAZyme domain HMM database. DIAMOND was also used to annotate query sequences in the CAZy database (Lombard et al., 2014) (http: / / www.cazy.org / ) with hits with >0.90 identity and e-value <1e-100. In cases where CAZyme annotations for query sequences did not match between the HMMER and DIAMOND tools, HMMER annotations were preferred. Only the CAZyme family and subfamilies encoding glycoside hydrolases (GH) and polysaccharide lyases (PL) were used for comparative analysis of *Bifidobacterium longum* clades.

[0314] The putative extracellular activity of all genes predicted as CAZyme by dbCAN3 was further analyzed. For this purpose, signal peptides were predicted using DeepSig (Savojardo et al.; Bioinformatics, 34(10), 2018, 1690–1696), a deep convolutional neural network (v4.0) trained on the well-known SignalP (Petersen et al.; Nat Methods; Sep 29, 2011; 8(10):785-6), and tested on UniproKB. The network retrieved the N-terminus of the input sequence with a threshold of 21 residues. The input was then forwarded to a feature extraction module, which output binary (not present, present) for signal peptide prediction.

[0315] Analysis revealed that NCC 5025 possesses a unique enzyme setting compared to other genomes in the Bifidobacterium longum transitional clade. Specifically, it is the only strain to possess glycosyl hydrolase family 43 subfamily 17 (GH43_17), which encodes an enzyme with the combined activities of α-L-arabinofuranosylase (EC 3.2.1.55) and endo-β-1,4-xylanase (EC 3.2.1.8), capable of breaking down complexed carbohydrates such as arabinogalactan, arabinogalactan, and arabinoxylan. To date, the only characterized enzyme activities of GH43_17 have come from Enterobacter (Pereira et al.; Nat Commun. Jan 2021; 12(1):459.) and Bacillus faecium (Helbert et al.; 2019; 116(13); 6063-6068).

[0316] In summary, among transitional Bifidobacterium longum, NCC 5025 possesses a unique CAZyme profile. Its genome encodes five distinct CAZymes targeting arabinogalactan (GH43_22, GH43_27, GH43_29, GH121, and the exclusive GH43_17), compared to four encoded by UCD399 and BSM11-5, while the remaining transitional Bifidobacterium longum genomes encode three or fewer of these CAZymes. Figure 2 Furthermore, four of the five arabinogalactan-degrading CAZyme species present in NCC 5025 possess signal peptides, giving this bacterium an advantage as a primary degrader of the complex structure of arabinogalactan when present at high molecular weights (typically in the diet). Figure 2 ).

[0317] The transitional form of *Bifidobacterium longum*, NCC5025, also encodes five different CAZymes (GH43_24, GH127, GH30_5, GH43_32, and GH43_17) that target arabinogalactan, two of which have signal peptides. Additionally, NCC5025 contains three genes encoding inulin-degrading CAZyme (GH32), similar to NCC5000 and UCD399. Another transitional form of *Bifidobacterium longum* has two or fewer genes encoding GH32, resulting in lower efficiency in utilizing inulin from the environment. Figure 2 ).

[0318] Bifidobacterium longum transitional strain NCC 5025 also contains several enzymes involved in the degradation and metabolism of human milk oligosaccharides (HMOs). It contains GH20 (lactose-N-bioglycosidase) and GH112 (lactose-N-disaccharide phosphorylase), as well as several GH42 (β-galactosidase) enzymes involved in the degradation and metabolism of lactose-N-tetrasaccharides (LNTs) and their constituents. This strain also possesses GH29 and GH95 (fucosidase) genes encoding fucosylated human milk oligosaccharides (such as 2'FL, 3'FL, or diFL). In summary, Bifidobacterium longum transitional strain NCC 5025 can act as a major degrader of arabinogalactan, arabinogalactan, and inulin due to its unique CAZyme library (including the exclusive presence of GH43_17). Carbohydrate blends containing combinations of arabinogalactan, inulin, or fucoidylated HMOs can give NCC 5025 an advantage in growth and the production of beneficial metabolites.

[0319] Example 4 - GH43_17-encoding gene cluster of NCC 5025

[0320] By comparing all available genomes of transitional Bifidobacterium longum using BioNumerics software (v8.0, bioMérieux SA, Massey-Italie, France), the GH43_17 gene of NCC 5025 was identified as being located in a strain-specific genetic region. This unique region contains family 31 glucosidase (GH31; NCC5025_001581), followed by ABC transporters (NCC5025_001580-001578), Lac-I type regulators (NCC5025_001577), GH43_17 enzyme (NCC5025_001576), MFS transporters (NCC5025_001575), and AraC family transcription regulators (NCC5025_001574) (see [link to original text]). Figure 3 (and Table 2).

[0321]

[0322] Further BLASTn analysis of all genes contained in this region using BioNumerics software (v8.0, bioMérieux SA, Massey-Italie, France) revealed that the Lac-I type regulator (CDS_000417), GH43_17 enzyme (CDS_000418), MFS transporter (CDS_000419), and AraC family transcriptional regulator (CDS_000420) are not homologous to other species of *Bifidobacterium longum* juvenile subspecies. Homologous to the gene encoding family 31 glucosidase (GH31; CDS000413) and ABC transporter (CDS_000414-00416) genes with relatively low similarity (maximum 80% coverage / 60% identity) was found in three closely related isolates, JDM301, BXY01, and CMCC P001.

[0323] Example 5 - NCC 5025 has high growth rate on 3-FL

[0324] Transitional strains of *Bifidobacterium longum* were obtained from the Nestlé Culture Collection and reactivated from lyophilized stock in MRS supplemented with 0.05% cysteine ​​(MRSc) using two consecutive culture steps (16 h, 37 °C, anaerobic). The reactivated cultures were then centrifuged, washed, and resuspended in 1 volume of PBS. The washed cells were then inoculated into carbon-free MRS-based medium (MRSc-C) (10 g L⁻¹ bactoproteose peptone n°3, 5 g L⁻¹ bacterial yeast extract, 1 g L⁻¹ Tween 80, 2 g L⁻¹ diammonium citrate, 5 g L⁻¹ sodium acetate, 0.1 g L⁻¹ magnesium sulfate, 0.05 g L⁻¹ manganese sulfate, 2 g L⁻¹ disodium phosphate, 0.5 g L⁻¹ cysteine), with 3-FL added as the sole carbon source at a final concentration of 0.5%. Growth was then carried out in 96-well microplates with a volume of 200 µl per well. Incubation was performed anaerobically for 46 h, during which the optical density was measured at 580 nm using a spectrophotometer. Growth curves were then modeled using a logistic growth model to obtain the relative growth rate k for each variant.

[0325] Among all tested Bifidobacterium longum transitional strains, NCC 5025 exhibited the highest growth rate on 3FL, indicating that this strain is most adapted to this substrate (see [link]). Figure 4 It has been shown that 3-FL is a human milk oligosaccharide, which shows the greatest increase in human breast milk during the transition period between a milk-based diet and a solid diet (Plows, JF, et al., Longitudinal Changes in Human Milk Oligosaccharides (HMOs) Over the Course of 24 Months of Lactation. J Nutr, 2021.151(4): pp.876-882), and therefore these results demonstrate the advantage of applying NCC 5025 during this period.

[0326] Example 6 - Growth of NCC 5025 on high molecular weight dietary fiber

[0327] The inventors tested whether strain NCC 5025 possessed the ability to grow on relevant high molecular weight fibers. For this purpose, selected transitional strains of *Bifidobacterium longum* (NCC 5002, NCC 5004, and NCC 5025, respectively) were grown on the aforementioned sugar-free MRSc medium, to which 5 g / L% arabinogalactan (from beet pulp in Megazyme) or inulin (from Orafti HSI in Beneo) was added. Growth assays were performed in a BioLector XT microbial reactor system (m2p-labs GmbH, Basweller, Germany) using 48-cell plates inserted into an anaerobic chamber for up to 50 hours (2 ml per well, stirred at 600 rpm, CO2 atmosphere, 37°C). Growth over time was tracked by continuously measuring scattered light at 620 nm.

[0328] Surprisingly, the results demonstrated that among the tested strains, *Bifidobacterium longum* transitional strain NCC 5025 possessed a specific ability to grow on inulin (average size DP6–8, Tsatsaragkou et al.; Foods 2021, 10(5), 951) and high molecular weight arabinogalactan. Compared to other *Bifidobacterium longum* transitional strains NCC 5002 and NCC 5004, *Bifidobacterium longum* transitional strain NCC 5025 grew faster (faster doubling time) and ultimately yielded higher yields. On high molecular weight arabinogalactan, strain NCC 5025 was the only strain among all tested to grow (see [link to article]). Figure 5 ).

[0329] Conclusion

[0330] The data provided proves:

[0331] a) The transitional form of Bifidobacterium longum, NCC 5025, is significantly different from previously isolated juvenile strains of Bifidobacterium longum and shares 98.4% of the ANI with strains previously isolated from infants in Bangladesh (Vatanen et al. 2022; see above).

[0332] b) This is the only Bifidobacterium longum transitional strain to date that is not resistant to a group of antibiotics that are considered relevant by EFSA;

[0333] c) Bifidobacterium longum transitional NCC 5025 has a unique carbohydrate-active enzyme (CaZy) spectrum, including the presence of GH43 subfamily 17 enzymes, which have not been characterized in Bifidobacterium longum species to date.

[0334] d) Bifidobacterium longum transitional NCC 5025 grows particularly well on 3-FL;

[0335] e) Bifidobacterium longum transitional NCC 5025 grows well on a group of food-derived fibers (e.g., inulin and arabinogalactan).

[0336] Overall, the data suggest that this strain is particularly well-adapted to the weaning period and may perform better than other Bifidobacterium longum transitional strains in this environment. Similarly, the data indicate that this strain may also perform better than other Bifidobacterium longum transitional strains in diets containing food-derived fiber (e.g., in adulthood).

[0337] Although the invention has been described by way of example, it should be understood that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, if known equivalents exist for specific features, such equivalents should be incorporated as expressly mentioned in this specification.

[0338]

[0339]

Claims

1. A transitional strain of Bifidobacterium longum deposited at the National Center for Microbial Culture Collection (CNCM) with accession number CNCM I-5942, or the aforementioned transitional strain of Bifidobacterium longum possessing the identification characteristics of the transitional strain of Bifidobacterium longum deposited with accession number CNCM I-5942.

2. A transitional strain of Bifidobacterium longum, wherein the transitional strain of Bifidobacterium longum has at least 99% average nucleotide identity (ANI) with the Bifidobacterium longum strain deposited in CNCM with accession number CNCMI-5942.

3. The Bifidobacterium longum transitional strain according to claim 1, wherein the Bifidobacterium longum transitional strain has at least 98.1% ANI with the Bifidobacterium longum transitional strain deposited with accession number CNCM I-5942.

4. The Bifidobacterium longum transitional strain according to any of the preceding claims, wherein the Bifidobacterium longum transitional strain is not resistant to any one of tetracycline, erythromycin, clindamycin and ampicillin; preferably, wherein the Bifidobacterium longum transitional strain is not resistant to any one of tetracycline, erythromycin, clindamycin, ampicillin, gentamicin, streptomycin, chloramphenicol and vancomycin.

5. The Bifidobacterium longum transitional strain according to any of the preceding claims, wherein the Bifidobacterium longum transitional strain comprises the glycosyl hydrolase family 43_17 (GH43_17) gene; suitably wherein the GH43_17 gene comprises SEQ ID NO: 7 or a sequence having at least 60% sequence identity with SEQ ID NO:

7.

6. The Bifidobacterium longum transitional strain according to any of the preceding claims, wherein the Bifidobacterium longum transitional strain further comprises a major facilitater superfamily (MFS) gene and / or an AraC gene; suitably wherein the MFS gene comprises a sequence of SEQ ID NO: 37 or having at least 60% sequence identity with SEQ ID NO: 39; suitably wherein the AraC gene comprises a sequence of SEQ ID NO: 39 or having at least 60% sequence identity with SEQ ID NO: 39; preferably wherein the GH43_17 gene, the MFS gene, and the AraC gene are contained in a gene cluster.

7. The Bifidobacterium longum transitional strain according to claim 6, wherein the Bifidobacterium longum transitional strain further comprises one or more of the GH31 gene and the LacI gene; preferably, wherein the Bifidobacterium longum transitional strain further comprises a xylulose kinase gene and a xylose isomerase gene; and even more preferably, wherein the GH43_17 gene, the MFS gene, the AraC gene, the GH31 gene, the LacI gene, the xylulose kinase gene and the xylose isomerase gene are contained in a gene cluster.

8. The Bifidobacterium longum transitional strain according to any of the preceding claims, wherein the Bifidobacterium longum transitional strain further comprises one or more genes encoding one or more glycoside hydrolases selected from the group consisting of: GH43_17, GH43_22, GH43_27, GH43_29, GH121, GH43_24, GH127, GH30_5, GH43_32 and GH30.

9. A transitional strain of *Bifidobacterium longum* according to any of the preceding claims, wherein the transitional strain of *Bifidobacterium longum* preferentially utilizes 3-fucosyllactose (3-FL); preferably wherein, when cultured in the presence of 3-FL, the transitional strain of *Bifidobacterium longum* has a growth rate of at least 0.6 kJ.

10. A probiotic composition comprising a transitional strain of Bifidobacterium longum according to any of the preceding claims.

11. A composition comprising a transitional strain of Bifidobacterium longum according to any of the preceding claims and at least one additional probiotic and / or prebiotic.

12. Use of the Bifidobacterium longum transitional strain or composition according to any of the preceding claims as a dietary supplement; suitably, wherein the Bifidobacterium longum transitional strain or composition is administered to a subject in combination with a fiber-containing diet or fiber-containing food.

13. The use of any one of the Bifidobacterium longum transitional strains or compositions according to claims 1 to 9 for promoting and / or assisting a subject in the metabolism of a fiber-containing diet or food.

14. The use of any one of the Bifidobacterium longum transitional strains or compositions according to claims 1 to 9 for promoting and / or assisting the transition of infants and / or young children from a milk-based diet to solid foods.

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