Application of bifidobacterium longum transition type microorganism

Abstract

Treating and / or preventing allergy and / or allergic sensitization. The present invention relates to a Bifidobacterium longum transition microorganism for use in the treatment and / or prevention of allergies and / or allergic sensitization in infants or young children.

Description

Technical Field

[0001] The present invention relates to probiotics and prebiotics, in particular Bifidobacterium longum transitional microorganisms or prebiotics promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms for use in treating and / or preventing allergies and / or allergic sensitization in infants or young children. Background Art

[0002] Allergy is one of the most common health problems, affecting patients of all ages. Over the past few decades, the prevalence of allergies has increased rapidly. It is estimated that more than one-third of the world's population is affected, so much so that allergies have been considered a new epidemic in industrialized countries. The reasons for the steady increase in allergic diseases are not fully understood. The genetic background of the host is a prominent factor, and recently discovered genes have been shown to be associated with respiratory allergies / asthma and skin symptoms. Environmental factors, such as lifestyle, pollution, smaller family size, and reduced stimulation of the immune system by microorganisms in the early life stage due to improved hygiene, seem to play an important role in the high prevalence and higher severity of allergic manifestations.

[0003] Allergic sensitization (especially to food allergens) that occurs during childhood, especially in early childhood, is dangerous because the development of an “allergic phenotype” or “atopy” has been shown to promote subsequent sensitization to other allergens. Therefore, an allergic reaction during childhood may be the first step in an allergic cascade that triggers multiple allergic reactions later in life (a process often referred to as the “atopic progression”). For example, children who have persistent food hypersensitivity in early life have a significantly increased risk of developing allergic rhinitis (hay fever) or asthma later in childhood. Children with mild food hypersensitivity also have an increased risk of developing upper respiratory allergies, but the increase is less than that of children with persistent food hypersensitivity. Therefore, reducing the severity of food hypersensitivity may be crucial to slowing the “atopic progression”. In this context, managing the onset of allergies and preventing allergic reactions during childhood and infancy is extremely important.

[0004] During the first few years of life, the immune system of an infant is in an active state of development. Intervention, prevention, avoidance, management, mitigation or regulation of allergic reactions in early childhood can affect not only short-term allergic profiles, but also long-term allergic profiles later in life.

[0005] Food allergens are among the first allergens encountered by infants early in life: infants who are not exclusively breastfed are often likely to encounter cow's milk proteins. Indeed, milk proteins are one of the most commonly observed causes of food allergies in infants, followed by egg and wheat proteins. In general, food allergies in infants and young children can present with skin symptoms (rashes, eczema, and others) and gastrointestinal symptoms (abdominal cramps; pain, especially in the abdomen; vomiting). Food allergies are the most common trigger for severe allergic reactions that may lead to life-threatening anaphylaxis. Other sensitization and allergic episodes may also occur when infants / young children are exposed to new foods (such as grains, vegetables, fruits, nuts, or fish) and airborne allergens (such as pollens, household dust mites, and animal dander). Adults are largely affected by contact allergies and respiratory allergies. Data from the WHO [Clark, MJ and Million, RP (2009) Allergic rhinitis: market evolution, Nature Reviews, Drug Discovery, 8, pp. 271-272] indicate that up to 30% to 40% of the world's population suffers from some form of respiratory allergy.

[0006] Animals, particularly small animals such as pets (and especially companion animals such as dogs and cats), may also suffer from food allergies and food intolerances, as well as environmental allergens. These often manifest themselves as symptoms similar to those of humans, such as gastrointestinal disturbances such as diarrhea, vomiting, and abdominal discomfort, as well as dermatitis or pruritus. In small animals (particularly dogs), the most common cause of chronic diarrhea is food-responsive enteropathy (diet-responsive enteropathy or food-responsive diarrhea).

[0007] There remains a need to develop new strategies for the treatment and / or prevention of allergic reactions and / or allergic sensitization. Summary of the invention

[0008] The present inventors have determined that microorganisms of the subspecies of Bifidobacterium longum of the clade present in the intestinal microbiome of mammals, particularly humans, during the transitional feeding period (B. longum transitional microorganisms) may have a beneficial effect on reducing the risk of developing allergic reactions and / or allergic sensitization. For example, the present inventors have shown that Bifidobacterium longum transitional microorganisms may be able to modulate intestinal barrier permeability and / or promote an anti-inflammatory and / or tolerogenic environment in the intestinal microbiota during the weaning period.

[0009] Therefore, in a first aspect, the present invention provides a Bifidobacterium longum transitional microorganism for use in treating and / or preventing allergies and / or allergic sensitization in infants or young children.

[0010] The present invention also provides a prebiotic for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children by promoting the growth and / or survival of transitional microorganisms of Bifidobacterium longum in the intestinal tract of infants or young children, wherein the prebiotic is: i. a polysaccharide substrate, which is appropriately selected from the group listed in any one of Tables 1 to 3; and / or ii. a human milk oligosaccharide (HMO), which is appropriately selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) and any combination thereof.

[0011] The present invention also provides a combination of a Bifidobacterium longum transitional microorganism and a prebiotic, which is used for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children; wherein the prebiotic is: i. a polysaccharide substrate, which is appropriately selected from the group listed in any one of Tables 1 to 3; and / or ii. a human milk oligosaccharide (HMO), which is appropriately selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3-O-fucosyllactose (3-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) and any combination thereof.

[0012] The present invention also provides a prebiotic for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children by promoting the growth of transitional microorganisms of Bifidobacterium longum in the intestinal tract of infants or young children.

[0013] The present invention also provides a combination of a Bifidobacterium longum transitional microorganism and a prebiotic, which is used for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children.

[0014] In another aspect, the present invention relates to the use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined herein for promoting immune tolerance in an infant or young child, preferably by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the intestine of the infant or young child.

[0015] In another aspect, the present invention relates to the use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined herein for modulating the intestinal barrier permeability of an infant or young child, preferably by promoting the growth of Bifidobacterium longum transitional microorganisms in the intestine of the infant or young child. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 - Phylogenetic tree of strains belonging to the species Bifidobacterium longum based on average nucleotide identity (ANI) UPGMA. The scale indicates the percentage of identity at each branch point.

[0017] Figure 2 -Using 2×10 6 Transepithelial electrical resistance (TEER) of Caco-2 monolayers after apical treatment with CFU probiotic strains. TEER was measured at 2, 4, 6 and 24 hours after treatment, and each value was normalized to its corresponding 0 hour value and shown as a percentage of the initial value. Data are plotted as mean ± SEM. For each concentration, differences between complete medium (CM) control and treatment at each time point were assessed using a two-way ANOVA with Dunnett's multiple comparison test, and statistical differences are indicated by (*). (*) = p < 0.05; (**) = p < 0.01; (***) = p < 0.001 and (****) = p < 0.0001. NCC5000-5004: Bifidobacterium longum transition strain, NCC2818: Bifidobacterium animalis subsp. lactis and NCC3001: Bifidobacterium longum subsp. longum

[0018] Figure 3 -Peripheral blood mononuclear cells (PBMCs) were stimulated for 36 h in the presence of different bacterial strains, including 10 7 CFU / ml of all transitional B. longum isolates and probiotic strains. Cell culture supernatants were collected to evaluate cytokine expression of IL-10 and IL-12p40 by ELISA. Standard curves for each cytokine were used to calculate absolute amounts (pg / ml) from optical density readings. NCC5000-5004: Bifidobacterium longum transitional strains, NCC2818: Bifidobacterium animalis subsp. lactis, NCC3089: Bifidobacterium longum subsp. infantis, NCC4007: Lactobacillus rhamnosus and NCC2705: Bifidobacterium longum subsp. longum

[0019] Figure 4 - Graphical representation of glycoside hydrolases (GH) and polysaccharide lyases (PL) in the genomes of the Bifidobacterium longum clade. The heatmap shows the presence (light colors) and absence (dark colors) of GH and PL genes, and the size of the circles represents the number of these genes in each genome of a particular strain.

[0020] Figure 5 - Pectin (sugar beet) and arabinogalactan (larch wood) promote the growth of Bifidobacterium longum transition strain NCC 5001 in a complex gut microbiota community. P****<0.0001, ***<0.001, **<0.01, *<0.05, one-way ANOVA with uncorrected Fisher's LSD.

[0021] Figure 6 - Arabinogalactan (larch wood) and starch (potato) promote the growth of Bifidobacterium longum transition strain NCC 5002 in a complex gut microbiota community. P****<0.0001, ***<0.001, **<0.01, *<0.05, one-way ANOVA with uncorrected Fisher's LSD.

[0022] Figure 7 - Representative CAZyme sequences

[0023] Figure 8 - Schematic representation of the organization of genes involved in the degradation and metabolism of fucosylated human milk oligosaccharides in Bifidobacterium longum transitional strains compared to Bifidobacterium longum subsp. infantis ATCC 15697 and Bifidobacterium carinii DSM 21854. Values ​​represent the percentage (%) of identity between different genes.

[0024] Fig. 9 - Growth of B. longum transition strain and B. longum subsp. infantis LMG 11588 on glucose, 2'-FL or 3-FL as sole carbon source (0.5% final). Significant differences between 2'-FL and 3-FL growth of each strain were calculated using one-way ANOVA followed by Sidak's multiple comparison test (ns = not significant, *p value < 0.05, **p value < 0.01).

[0025] Fig.10 - Growth ratio of 3-FL relative to 2'-FL of Bifidobacterium longum transition strain and Bifidobacterium longum subsp. infantis LMG 11588.

[0026] Fig.11 - Riboflavin biosynthesis genes present in transitional strains of Bifidobacterium longum. Panel A shows the organization of the riboflavin biosynthesis operon as found in Bifidobacterium longum NCC 5000. Panel B depicts a heat map of these genes in closely related strains of Bifidobacterium longum. Genes are colored according to their % identity found by Blast to the NCC 5000 gene.

[0027] Fig.12- In vitro, the transitional strain of Bifidobacterium longum transitional reduced IL-5 expression by type 2 skewed helper T cells after 48 hours of stimulation to a similar or even greater extent than the probiotic Bifidobacterium lactis. NCC5000-5004: Bifidobacterium longum transitional strain, NCC2818: Bifidobacterium animalis subsp. lactis, NCC3089: Bifidobacterium longum subsp. infantis

[0028] Fig.13 -Peripheral blood mononuclear cells (PBMCs) were stimulated for 36 h in the presence of different bacterial strains, including 10 7 CFU / ml of all transitional longum B. longum isolates and probiotic strains. Cell culture supernatants were collected to evaluate cytokine expression of IL-10 by ELISA. Standard curves for each cytokine were used to calculate absolute amounts (picograms / ml) from optical density readings. NCC5000-5004: transitional longum B. longum strains, NCC2818: animal B. lactis subsp., NCC3089: longum B. infantis subsp., NCC4007: Lactobacillus rhamnosus and NCC2705: longum B. longum subsp.

[0029] Fig.14 - In vitro batch fermentation of infant microbiota containing 3-fucosyllactose (3FL), with or without supplementation of infant B. longum species or transitional B. longum. Total SCFA corresponds to the sum of the peak integrations of acetate, butyrate and propionate. The bar graphs indicate the dynamics of consumption and production of total SCFA between 0 and 24 hours (blue bars) and between 24 and 48 hours (red bars). DETAILED DESCRIPTION

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

[0031] As used herein, the terms "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., are intended to encompass variations of the specified value and variations from the specified value, such as variations of the specified value and variations from the specified value of 1 / -10% or less, 1 / -5% or less, 1 / -1% or less, and + / 0.1% or less, as long as such variations are suitable for making in the disclosed invention. It should be understood that the value referred to by the modifier "about" or "approximately" itself is also specifically and preferably disclosed.

[0032] The terms "subject", "individual" and "patient" are used interchangeably to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports animals and pets.

[0033] The term "infant" means a human subject under 12 months of age or a non-human animal of comparable age.

[0034] As used herein, the term "infant" or "toddler" may refer to a human subject between the ages of 12 months and 5 years. Appropriately, "infant" may refer to a non-human animal of comparable age.

[0035] The expressions "supplementary feeding period", "supplementary period", "transitional period", "transitional feeding period" and "weaning period" can be used interchangeably, and refer to the period when milk (breast milk or formula food) is replaced by other foods in the diet of an infant or young child. Usually an infant or young child is gradually transferred or transitioned to a mixed diet comprising milk and / or solid food from exclusive milk feeding (breast milk feeding or formula food feeding). The transitional period depends on the infant or young child, but is usually about 4 months old to about 18 months old, such as about 6 months old to about 18 months old, but can be extended to about 24 months or longer in some cases. For humans, the weaning period usually begins between 4 months and 6 months old, and once an infant and / or young child is no longer fed with breast milk or infant formula food, usually at about 24 months old, it is considered that the weaning period is complete. In some embodiments, the weaning period is 4 months to 24 months.

[0036] The expression "composition" or "nutritional composition" refers to any kind of composition or formulation that provides nutritional benefits to an individual and can be safely consumed by humans or animals. The nutritional composition can be in solid (e.g., powder), semi-solid or liquid form, and can include one or more macronutrients, micronutrients, food additives, water, etc. For example, the nutritional composition can include the following macronutrients: protein source, lipid source, carbohydrate source, and any combination thereof. In addition, the nutritional composition can include 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 include food additives, such as stabilizers (when provided in liquid or solid form) or emulsifiers (when provided in liquid form). The amount of various ingredients (e.g., oligosaccharides) can be expressed as g / 100 g of composition on a dry weight basis when the composition is in solid form (e.g., powder), or as a concentration of g / L of composition when the composition refers to a liquid form (the latter also encompasses liquid compositions that can be obtained after reconstitution of the powder in a liquid (such as milk, water), such as reconstituted infant formula or larger / second stage infant formula or infant cereal products or any other formulation designed to provide nutrition for infants or young children). Typically, the nutritional composition can be formulated for enteral, oral, parenteral or intravenous intake, and it typically includes one or more nutrients selected from the following: a lipid or fat source, a protein source, and a carbohydrate source. Preferably, the nutritional composition is for oral use.

[0037] In a particular embodiment, the nutritional composition is a “synthetic nutritional composition.” The expression “synthetic nutritional composition” means a mixture obtained by chemical and / or biological methods.

[0038] As used herein, the expression "infant formula" refers to a food intended for specific nutritional use in the first few months after the birth of an infant and which itself meets the nutritional needs of such a person (in accordance with Article 2(c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formula and follow-up infant formula). It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and Special Infants (including foods for special medical purposes). The expression "infant formula" covers both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0039] "Second infant formula" or "follow-on formula" is given from the 6th month onwards. Infant formula constitutes the main liquid element in the gradually diversified diet of such persons.

[0040] The expression "baby food" refers to a foodstuff intended for specific nutritional use by infants or young children during the first few years of life.

[0041] The expression "infant cereal composition" refers to a foodstuff intended for specific nutritional use by infants or young children during the first years of life.

[0042] The expression "growing-up milk" (or GUM) refers to a milk-based beverage, usually with added vitamins and minerals, which is intended for infants or children.

[0043] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying or admixing human milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients. Thus, the fortifier may be administered after being dissolved in human breast milk, infant formula, growing-up milk, or human breast milk fortified with other nutrients, or it may be administered as a separate composition. When administered as a separate composition, a milk fortifier may also be identified as a "supplement."

[0044] The term "metabolism" is used herein to mean that a substrate can be broken down, adsorbed and / or utilized by a microorganism. For example, a substrate can promote and / or contribute to the growth and / or survival of a microorganism.

[0045] Suitably, the term "capable of metabolizing a glycan substrate" may mean that the transitional strain of Bifidobacterium longum encodes at least one CAZyme that is capable of utilizing a glycan substrate. For example, the CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within a glycan substrate. Suitably, the transitional strain of Bifidobacterium longum may encode at least one, at least two, at least three, at least four or at least five CAZymes that are capable of utilizing a glycan substrate. Suitably, the term "capable of metabolizing a glycan substrate" may mean that a glycan substrate (or a fiber or component comprising a glycan substrate) is capable of promoting the growth and / or survival of the transitional strain of Bifidobacterium longum (e.g., when added to an anaerobic culture of a transitional strain of Bifidobacterium longum). The growth and / or survival of the transitional strain of Bifidobacterium longum may be determined, for example, by measuring the abundance of 16S rDNA using a PCR method. An exemplary test for measuring the growth of a transitional strain of Bifidobacterium longum in the presence of a glycan substrate (e.g., in the form of fiber) is provided in this example.

[0046] Suitably, the polysaccharide substrate can be metabolized by the transitional microorganism of Bifidobacterium longum. Suitably, the polysaccharide substrate may be able to promote the growth and / or survival of the transitional strain of Bifidobacterium longum. The polysaccharide substrate that can promote the growth and / or survival of the transitional strain of Bifidobacterium longum can be determined by the anaerobic culture of the transitional strain of Bifidobacterium longum and the polysaccharide substrate to be tested, for example. The growth and / or survival of the transitional strain of Bifidobacterium longum can be determined by measuring the abundance of bacterial cell number, cell density (for example, by optical density measurement) and / or 16S rDNA, for example, using PCR method. An exemplary test for measuring the growth of the transitional strain of Bifidobacterium longum in the presence of a polysaccharide substrate is provided in the embodiment. The polysaccharide substrate capable of promoting the growth and / or survival of the Bifidobacterium longum transition-type strain may increase the number of Bifidobacterium longum transition-type bacteria in the anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% compared to the number of Bifidobacterium longum transition-type bacteria in the control anaerobic culture not comprising the HMO. Suitably, the polysaccharide substrate capable of promoting the growth and / or survival of the Bifidobacterium longum transition-type strain may increase the number of Bifidobacterium longum transition-type bacteria in the anaerobic culture by a statistically significant amount (e.g., p-value <0.05 determined by one-way ANOVA) compared to the number of Bifidobacterium longum transition-type bacteria in the control anaerobic culture not comprising the polysaccharide substrate.

[0047] "Glycan substrate" refers to a polysaccharide that can be metabolized by a microorganism. A polysaccharide substrate can be, for example, a glycoconjugate, an oligosaccharide or a polysaccharide. Glycoconjugate polysaccharides may include N-linked polysaccharides or O-linked polysaccharides within glycoproteins and proteoglycans or glycolipids. For example, O-linked polysaccharides may include proteins or peptides in which the oxygen atom of a serine or threonine residue is connected to a monosaccharide, an oligosaccharide or a polysaccharide, such as in the case of glycosaminoglycans (GAGs). Other examples of "polysaccharide substrates" are cellulose, which is a polysaccharide composed of β-1,4-linked D-glucose, and chitin, which is a polysaccharide composed of β-1,4-linked N-acetyl-D-glucosamine. Polysaccharides may be homopolymers or heteropolymers of monosaccharide residues, and may be linear or branched. "Glycan substrates" as used herein include, for example, oligosaccharides and polysaccharides.

[0048] "Oligosaccharides" may refer to carbohydrates having more than 2 but relatively few monosaccharide units, typically 3, 4, 5, 6, and up to 10. Exemplary oligosaccharides include, but are not limited to, fructooligosaccharides, galacto-oligosaccharides (raffinose, stachyose, verbascose), maltooligosaccharides, gentio-oligosaccharides, cellooligosaccharides, milk oligosaccharides (e.g., those present in mammary secretions), isomaltooligosaccharides, lactofructooligosaccharides, manno-oligosaccharides, melibiose-derived oligosaccharides, pecto-oligosaccharides, xylo-oligosaccharides.

[0049] The term "polysaccharide" may refer to a carbohydrate having more than ten monosaccharide units. Exemplary polysaccharides include, but are not limited to, starch, arabinogalactan, laminarin, chrysophycean, xylan, arabinoxylan, mannan, fucoidan, and galactomannan. It should be understood that there is no precise boundary or distinction between the terms oligosaccharide and polysaccharide, nor is such a distinction required to practice the present invention.

[0050] 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 an aldose and an amino sugar, except for keratan, which has a galactose in place of the aldose. GAGs are divided into four groups based on the core disaccharide structure.

[0051] As used herein, "mucin" may refer to a family of high molecular weight, heavily glycosylated proteins (glycoconjugates). A key feature 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.

[0052] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, which suggests that they perform important functions that may not be directly related to their caloric value. It has been specifically noted in the art that these carbohydrates play a key role in the early development of infants and young children, such as the maturation of the immune system. Many different types of HMO are found in human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine with a variety of bonds between these molecules, so human milk contains a large number of different types of oligosaccharides, and more than 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the terminal position of the non-reducing end is occupied by sialic acid and / or fucose (if any). Depending on the presence of fucose and sialic acid in the oligosaccharide structure, HMOs can be divided into non-fucosylated (neutral) or fucosylated (neutral), sialylated (acidic) and non-sialylated molecules, respectively.

[0053] The expression "fucosylated oligosaccharides" refers to oligosaccharides with fucose residues. Such oligosaccharides are neutral. Some examples are 2'-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllactose-N-hexaose, fucosyllactose-N-neohexaose, difucosyllactose-N-hexaose I, difucosyllactose-N-neohexaose II and any combination of these substances. Fucosylated oligosaccharides represent the largest part of human milk, with 2'-FL accounting for up to 30% of the total HMOs. Fucosylated oligosaccharides are thought to reduce the risk of infection and inflammation and promote the growth and metabolic activity of specific commensal microorganisms, thereby reducing inflammatory responses.

[0054] The expression "N-acetylated oligosaccharide" encompasses "N-acetyllactosamine" and "oligosaccharides comprising N-acetyllactosamine". Such oligosaccharides are neutral oligosaccharides having an N-acetyl-lactosamine residue. Suitable examples are LNT (lactose-N-tetraose), p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neotetraose), DSLNT (disialyl lactose-N-tetraose) and any combination thereof. Other examples are lactose-N-hexose, lactose-N-neohexose, p-lactose-N-hexose, p-lactose-N-neohexose, lactose-N-octose, lactose-N-neooctose, iso-lactose-N-octose, p-lactose-N-octose and lactose-N-decasaccharide.

[0055] The expressions "at least one fucosylated oligosaccharide" and "at least one N-acetylated oligosaccharide" are to be understood as "at least one type of fucosylated oligosaccharide" and "at least one type of N-acetylated oligosaccharide".

[0056] The term "sialylated oligosaccharide" refers to an oligosaccharide with charged sialic acid residues. Such oligosaccharides are acidic. Some examples are 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyllactose-N-tetraose (Lst, such as Lst-a, Lst-b or Lst-c).

[0057] Suitably, the term "capable of metabolizing an HMO" may mean that the Bifidobacterium longum transitional strain encodes at least one CAZyme that is capable of utilizing an HMO. For example, the CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within an HMO. Suitably, the Bifidobacterium longum transitional strain may encode at least one, at least two, at least three, at least four or at least five CAZymes that are capable of utilizing an HMO. Suitably, the term "capable of metabolizing an HMO" may mean that the HMO is capable of promoting 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 may be determined, for example, by measuring the abundance of 16S rDNA using a PCR method.

[0058] The term fiber is used herein to refer to carbohydrates that are indigestible to humans or animals. This article also discusses such fibers related to carbohydrates. Suitably, the fiber can be fermented by one or more Bifidobacterium longum transitional microorganisms provided in the uses or compositions of the present invention and / or fermented in one or more regions in the gastrointestinal tract of an organism (such as a human or non-human animal). In the context of the present invention, as used herein, the expression "fiber" or "multiple fibers" or "dietary fiber" or "multiple dietary fibers" refers to the indigestible part of food derived from plants in the small intestine, which includes two main components: soluble fiber, which dissolves in water; and insoluble fiber. A mixture of fibers is included within the scope of the above-mentioned terms. Soluble fiber is easily fermented into gas and physiologically active byproducts in the colon, and can be prebiotics and viscous. Insoluble fiber is insoluble in water, is metabolically inert and swells, or it can be a prebiotic and is metabolized and fermented in the large intestine. Chemically, dietary fiber consists of carbohydrate polymers with three or more monomer units that are not hydrolyzed by endogenous enzymes in the small intestine, such as arabinoxylan, cellulose, and many other plant components, such as resistant starch, resistant dextrin, inulin, lignin, chitin, pectin, arabinan, arabinogalactan, galactan, xylan, beta-glucan and oligosaccharides. Non-limiting examples of dietary fiber are: prebiotic fibers such as oligofructose (FOS), inulin, galacto-oligosaccharides (GOS), fruit fibers, plant fibers, cereal fibers, resistant starches such as high amylose corn starch.

[0059] As used herein, "added fiber" or "added dietary fiber" refers to an ingredient consisting primarily or entirely of fiber that is added to a supplemental nutritional composition, and its content in the fiber contributes to the total fiber content of the composition. The total fiber content of the supplemental nutritional composition is provided by the sum of the amount of fiber naturally present in the ingredients used in the formula (e.g., from whole grain cereal flour) plus the amount of added fiber.

[0060] The term "prebiotic" refers to non-digestible carbohydrates that have beneficial effects on the host by selectively stimulating the growth and / or activity of beneficial 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).

[0061] The term "probiotic" means a microbial cell preparation or microbial cell component having 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: 10107-10). The microbial cells according to the present invention are typically bacteria.

[0062] The term "cfu" is to be understood as colony forming units.

[0063] The “gut microbiome” is the composition of microorganisms (including bacteria, archaea, and fungi) that live in the digestive tract.

[0064] The term "gut microbiome" may include "gut microbiota" and their "activity sites", which may include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolites (signaling molecules, toxins, organic and inorganic molecules), and molecules produced by coexisting hosts and structured by surrounding environmental conditions (Berg, G. et al., 2020. Microbiome, 8(1), pp. 1-22).

[0065] Bifidobacterium longum transitional microorganism

[0066] Previously, it has been identified that the longum subspecies microorganism of the evolutionary branch is present in the intestinal microbiome of mammals, especially humans during the transition feeding period. The longum microorganisms belonging to this evolutionary branch are referred to as longum transitional in this article. Longum transitional strains NCC 5000, NCC 5001, NCC5002, NCC 5003 and NCC 5004 are produced by Nestle ( DES PRODUITS SA) was deposited in the French National Collection of Microorganisms (CNCM), Pasteur Institute on May 11, 2021 in accordance with the Budapest Treaty, with the deposit numbers CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, respectively. It is shown in U.S. provisional patent application WO2023 / 278441 that the relative abundance of Bifidobacterium longum transitional microorganisms during the transition feeding period (e.g., weaning period) is greater than that of Bifidobacterium longum infantis subspecies (Bifidobacterium infantis) or any of the long subspecies of Bifidobacterium longum. In fact, the relative abundance of Bifidobacterium longum infantis subspecies decreases at the beginning of the transition feeding period until the end of the transition feeding period, while the abundance of the long subspecies of Bifidobacterium longum begins to increase. Vatanen et al. demonstrated that this unique Bifidobacterium longum evolutionary branch expanded with the introduction of solid foods and possesses enzymes for utilizing breast milk and solid food substrates (Vatanen et al.; 2022, Cell 185, 1-18; published online November 1, 2022; https: / / doi.org / 10.1016 / j.cell.2022.10.011).

[0067] Suitably, the Bifidobacterium longum transitional microorganism may encode one or more CAZymes selected from the group listed in Table 1. Suitably, the Bifidobacterium longum transitional microorganism may encode one or two CAZymes selected from the group listed in Table 1.

[0068] Suitably, the Bifidobacterium longum transitional microorganism encodes at least one CAZyme selected from the group listed in Table 1 and one or more of the CAZymes selected from the groups listed in Table 2 and Table 3. For example, the Bifidobacterium longum transitional microorganism may encode at least 2, at least 5, at least 10, at least 20 or at least 30 CAZymes selected from the groups listed in Table 2 and Table 3.

[0069] Suitably, the Bifidobacterium longum transitional microorganism encodes (i) at least one CAZyme selected from the group listed in Table 1 and (ii) each of the CAZymes listed in Table 3 or each of the CAZymes listed in Table 3 except GH5_44.

[0070] Suitably, the Bifidobacterium longum transitional microorganism encodes (i) at least one CAZyme selected from the group listed in Table 1 and (ii) each of the CAZymes listed in Table 3 or each of the CAZymes listed in Table 3 except GH25.

[0071] Suitably, the Bifidobacterium longum transitional microorganism does not encode one or more of the CAZymes listed in Table 4. Suitably, the Bifidobacterium longum transitional microorganism does not encode any of the CAZymes listed in Table 4.

[0072] The Bifidobacterium longum transitional microorganism of the present invention advantageously carries a gene encoding a CAZyme, thereby allowing sialic acid residues to be cleaved from polysaccharides such as sialylated oligosaccharides, glycoproteins and glycolipids. This allows for the effective utilization of sialylated oligosaccharides present in breast milk at the time of weaning, and thus can participate in the proper development of the intestinal microbiome of infants and / or young children. It may also help prevent the presence of intestinal pathogens.

[0073] In some embodiments, the Bifidobacterium longum transitional microorganism comprises a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene that is at least 60% identical to the BLON_2348 gene present in Bifidobacterium longum subsp. infantis ATCC15697.

[0074] In some embodiments, the transitional microorganism of Bifidobacterium longum comprises about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, about 148%, about about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% identity to a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene.

[0075] In some embodiments, the transitional microorganism of Bifidobacterium longum comprises a BLON_2348 gene that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 74%, at least 73%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 98%, at least 99%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%, at least 100%,

[0076] In some embodiments, the transitional microorganism of Bifidobacterium longum used according to the present invention comprises: a glycosyl hydrolase family 95 (GH95, α-L-galactosidase; α-L-fucosidase; α-1,2-L-fucosidase) gene, which is at least 60% identical to the BLON_2335 gene present in Bifidobacterium longum subsp. infantis ATCC 15697; and / or a glycosyl hydrolase family 29 (GH29, α-L-fucosidase; α-1,3 / 1,4-L-fucosidase; α-1,2-L-fucosidase), which is at least 60% identical to the BLON_2336 gene present in Bifidobacterium longum subsp. infantis ATCC 15697.

[0077] In some embodiments, the Bifidobacterium longum transitional microorganism used according to the present invention comprises a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having at least 60% identity to the BLON_2348 gene present in Bifidobacterium longum subsp. infantis ATCC 15697.

[0078] In some embodiments, the Bifidobacterium longum transitional microorganism preferentially utilizes 3-fucosyllactose (3-FL) over 2'-fucosyllactose (2'-FL). Suitably, the Bifidobacterium longum transitional microorganism may preferentially utilize 3-FL over 2'-FL at a ratio between 0.1:5, preferably at a ratio between 0.1:4, more preferably at a ratio between 0.2:2.

[0079] In some embodiments, the Bifidobacterium longum transitional microorganism used in the present invention utilizes 3-FL more efficiently than 2'-FL, as evidenced by better growth, e.g., as shown in this example. In some embodiments, the Bifidobacterium longum transitional microorganism has at least 98% average nucleotide identity (ANI) with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, and CMCC-P0001 (ATCC BAA-2753), and any combination thereof. Suitably, the Bifidobacterium longum transitional microorganism encodes one or more CAZymes selected from the group listed in Table 1 and has an ANI of at least 98% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753) and any combination thereof. Suitably, the Bifidobacterium longum transitional microorganism encodes one or more GH31 CAZymes and has an ANI of at least 98% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753) and any combination thereof. In some embodiments, the Bifidobacterium longum transitional microorganism has an ANI of about 98% to 100% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687, and CMCC-P0001 (ATCC BAA-2753), and any combination thereof. In some embodiments, the Bifidobacterium longum transitional microorganism has an ANI of at least 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753), and any combination thereof.In some embodiments, the Bifidobacterium longum transitional microorganism has an ANI of 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%, at least 99.9% or at least 100% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753), and any combination thereof.

[0080] Methods for sequencing microbial genomes are known in the art (see, e.g., Segerman; Front. Cell. Infect. Microbiol.; 2020; 10; Article 527102 & Donkor; Genes; 2013; 4(4); 556-572). By way of example, metagenomics methods can be used. For example, suitable metagenomics methods can be performed using shotgun sequencing data. Suitable metagenomics methods are known in the art and include, for example, MetaPhlAn3.0 (see Beghini et al.; eLife 2021; 10: e65088; https: / / huttenhower.sph.harvard.edu / metaphlan) for metagenomics sequencing.

[0081] "Average nucleotide identity (ANI)" is a term in the art that refers to a distance-based method for describing species based on paired comparisons of genomic sequences, and is a computer alternative to the traditional DNA-DNA hybridization (DDH) technology that has been used for 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 greater than 70% correlation will be 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 and can be used for species description. ANI has been evaluated in multiple laboratories and has become the gold standard for species demarcation (see, e.g., 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)).

[0082] The ANI of common genes between two known strains is a powerful means of comparing genetic relatedness between strains, and an ANI value of about 95% corresponds to a 70% DNA-DNA hybridization standard for defining species. See, e.g., 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 comparison of all sequences common between 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 of two genomes are known in the art. See, e.g., 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 a specific embodiment, the ANI between two bacterial genomes can be determined, for example, by averaging the nucleotide identities of homologous genes identified as bidirectional best hits (BBHs). The protein-coding genes of 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 retain only BBHs that show at least 70% sequence identity over at least 70% of the length of the shorter sequence in each BBH pair. The ANI of genome A to genome B is defined as the percent identity multiplied by the sum of the aligned lengths of all BBHs, divided by the sum of the lengths of the BBH genes. These and ANI determination techniques are known in the art.

[0083] Suitably, the Bifidobacterium longum transitional-type microorganism selected from the group consisting of CNCM 1-5683, CNCM 1-5684, CNCM 1-5685, CNCM 1-5686, CNCM 1-5687 and CMCC-P0001 (ATCC BAA-2753) represents a reference genome to which the microorganism genome is compared.

[0084] The genome sequences of Bifidobacterium longum transition strains NCC 5000 (CNCM I-5683), NCC 5001 (CNCM I-5684), NCC 5002 (CNCM I-5685), NCC 5003 (CNCM I-5686) and NCC 5004 (CNCM I-5687) are available through the Joint Genome Initiative (JGI) research number Gs0156595 ( https: / / genome.jgi.doe.gov / portal / ). The analysis plan number and taxonomic unit number of each genome are as follows:

[0085] strains JGI Analysis Project JGI Taxa Bifidobacterium longum NCC 5000 Ga0527908 2951181949 Bifidobacterium longum NCC 5001 Ga0529016 2951184202 Bifidobacterium longum NCC 5002 Ga0529017 2951186501 Bifidobacterium longum NCC 5003 Ga0529018 2951188792 Bifidobacterium longum NCC 5004 Ga0529019 2951191018

[0086] In some embodiments, the Bifidobacterium longum transitional microorganism used in the present invention is isolated from a human.

[0087] In some other embodiments, the Bifidobacterium longum transitional microorganism is not the subspecies Bifidobacterium longum subspecies longum or Bifidobacterium longum subspecies infantis.

[0088] Suitably, the Bifidobacterium longum transitional microorganism is provided in the form of a probiotic. Suitably, the Bifidobacterium longum transitional microorganism is provided in a composition.

[0089] Treatment and / or prevention of allergic reactions and / or allergic sensitization

[0090] As used herein, "allergy" may refer to an allergic disease or an allergic reaction (including symptoms thereof).

[0091] Bifidobacterium longum transitional microorganisms and / or prebiotics can be used for the treatment and / or prevention of allergies and / or allergic sensitization in infants or young children.

[0092] As used herein, "treatment" may refer to administering a Bifidobacterium longum transitional microorganism and / or a prebiotic to a subject with an existing disease or condition in order to reduce, alleviate or ameliorate at least one symptom associated with the disease and / or slow, mitigate or block progression of the disease.

[0093] As used herein, "prevention" may refer to the administration of a Bifidobacterium longum transitional microorganism and / or a prebiotic to a subject who has not yet contracted a disease and / or has not developed any symptoms of the disease, to prevent or attenuate the cause of the disease or to reduce or prevent the development of at least one symptom associated with the disease. The subject may have a predisposition to develop the disease, or the subject is considered to be at risk of developing the disease. Suitably, the Bifidobacterium longum transitional microorganism and / or a prebiotic may be administered to a subject to reduce the likelihood of allergic reactions and / or allergic sensitization in infants or young children.

[0094] Allergic sensitization (especially sensitization to food allergens) that occurs during childhood, especially in early childhood, is dangerous because it has been shown that the development of an "allergic phenotype" or "atopy" promotes subsequent sensitization to other allergens. Therefore, allergic reactions in childhood may be the first step in an allergic cascade that triggers multiple allergic reactions in later life (this process is often referred to as the "atopic progression"). For example, children who suffer from persistent food hypersensitivity in early childhood have a significantly increased risk of developing allergic rhinitis (hay fever) or asthma in late childhood (Ostblom, E. et al., 2008, Phenotypes of food hypersensitivity and development ofallergic diseases during the first 8years of life, Clinical and ExperimentalAllergy, 38(8), pp. 1325-1332). Children with mild food hypersensitivity also have an increased risk of developing upper respiratory tract allergies, but the increase is less than that of children with persistent food hypersensitivity. Therefore, reducing the severity of food hypersensitivity may be crucial to slowing the “atopic progression.” In this setting, managing allergic episodes and preventing allergic reactions during childhood and infancy are extremely important.

[0095] In one embodiment, prevention and / or reduction of the risk of allergic reactions and / or allergic sensitization is by primary prevention. "Primary prevention" refers to the effect of preventing or reducing the risk of sensitization of a patient to an allergen, characterized by the absence or reduction of allergen-specific IgE antibodies. The result of preventing or reducing sensitization can be that when contacting the same allergen, allergic symptoms do not occur or allergic symptoms are alleviated. By regulating the way in which a patient is sensitized to an allergen or a group of allergens (primary prevention), subsequent allergic responses can also be regulated.

[0096] Food allergens are among the first allergens encountered by infants early in life, and infants who are not exclusively breastfed are most likely to encounter cow's milk proteins. Indeed, milk proteins are one of the most commonly observed causes of food allergies in infants, followed by egg and wheat proteins. In general, food allergies in infants and young children can present with skin symptoms (rashes, eczema, and others) and gastrointestinal symptoms (abdominal cramps; pain, especially in the abdomen; vomiting). Food allergies are the most common trigger for severe allergic reactions, which can lead to life-threatening anaphylaxis.

[0097] Other sensitization and allergic reactions may also occur when infants / young children are exposed to new foods (such as grains, vegetables, fruits, nuts or fish) and airborne allergens (such as pollen, household dust mites and animal dander). Adults are greatly affected by contact allergies and respiratory allergies. Recent data from WHO (Clark, MJ and Million, RP, 2009, Allergic rhinitis: market evolution, Nature Reviews, Drug Discovery, 8, pp. 271-272) indicate that up to 30% to 40% of the world's population suffers from some form of respiratory allergy.

[0098] Animals, particularly small animals such as pets (and especially companion animals such as dogs and cats), may also suffer from food allergies and food intolerances, as well as environmental allergens. These often manifest themselves as symptoms similar to those of humans, such as gastrointestinal disturbances such as diarrhea, vomiting, and abdominal discomfort, as well as dermatitis or pruritus. In small animals (particularly dogs), the most common cause of chronic diarrhea is food-responsive enteropathy (diet-responsive enteropathy or food-responsive diarrhea).

[0099] The compounds and compositions of the present invention are useful for preventing or treating food allergies, respiratory allergies, and skin allergies by treating and / or preventing or reducing the risk of a subject being sensitized to an allergen.

[0100] In some embodiments, the allergic response is a specific IgE-related immune response and / or a T cell-dependent hypersensitivity reaction. Therefore, in some embodiments, the risk of treating and / or preventing and / or reducing the occurrence of allergic reactions and / or allergic sensitization includes reducing or preventing specific IgE-related immune responses and / or T cell-dependent hypersensitivity reactions. In some embodiments, allergic inflammation is alleviated and / or tolerance (e.g., oral tolerance) is enhanced.

[0101] Suitably, the allergic disease is selected from one or more of the group consisting of food allergy, respiratory allergy and skin allergy.

[0102] In one embodiment, the allergic disease is selected from one or more of the group consisting of rhinitis, asthma, dermatitis, atopic dermatitis, contact dermatitis, eczema, atopic eczema, urticaria, psoriasis, eosinophilic esophagitis and eosinophil-related gastrointestinal diseases.

[0103] In one embodiment, the allergen in the allergic disease is selected from one or more of the following: food allergens, dust mites, pollen, mold or mold spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers, pollution or pet dander.

[0104] In one embodiment, the allergen in the allergic disease is a food allergen. Suitably, the food allergen is selected from the group consisting of nuts, tree nuts, peanuts, fish, shellfish, molluscs, crustaceans, milk, eggs, soy, gluten, cereals, wheat, oats, barley, rye, celery, corn, lupins, sulfites, sesame, mustard, rice, poultry and meat.

[0105] In one embodiment, the allergen is an aeroallergen. Suitably, wherein the aeroallergen is selected from dust mites, pollen, mould or mould spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers, pollution or pet dander.

[0106] Suitably, treating, preventing or reducing the risk of an allergic reaction and / or allergic sensitization may refer to reducing or ameliorating one or more symptoms as described herein.

[0107] As used herein, "food allergy" refers to an abnormal immune response to one or more food allergens, usually an IgE response caused by the release of histamine, but non-IgE immune responses are also encompassed. Symptoms of food allergy may include itching, swelling of the tongue, vomiting, diarrhea, urticaria, difficulty breathing, or low blood pressure. When symptoms are severe, they are called anaphylaxis.

[0108] As used herein, the term "food allergen" refers to a protein or derivative thereof that causes an abnormal immune response. The purified food allergens can be named using the systematic nomenclature of the Allergen Nomenclature Sub-Committee of the World Health Organization and the International Union of Immunology. The allergen name consists of the abbreviation of the scientific name of its source (genus: 3 to 4 letters; species: 1 to 2 letters) and Arabic numerals, for example, Der p 1 refers to the first allergen to be described from the household dust mite Dermatophagoides pteronyssinus. Food allergens are derived from proteins with a variety of biological functions, including proteases, ligand binding proteins, structural proteins, pathogen-related proteins, lipid transfer proteins, exoproteins, and calcium-binding proteins. A list of food allergens is provided on the official website of the WHO / IUIS Allergen Nomenclature Database at http: / / www.allergen.org / index.php. (Radauer, C. et al., 2014, Allergy, 69(4), pp. 413-419, and Pomés, A. et al., 2018. Molecular immunology).

[0109] As used herein, "respiratory allergy" or "aeroallergens" refers to an abnormal immune response to one or more airborne allergens. Airborne allergens can include pollen, mold or mold spores, weed pollen, tree pollen, grass pollen, and dander. Respiratory allergy can include, for example, allergic rhinitis and allergic asthma. Symptoms of allergic rhinitis (hay fever) include runny or stuffy nose, sneezing, red, itchy and tearing eyes, and swelling around the eyes. Symptoms of allergic asthma include wheezing attacks, coughing, chest tightness, and shortness of breath.

[0110] As used herein, "skin allergy" refers to an abnormal immune response caused by contact with one or more environmental allergens. Environmental allergens may include food allergens, dust mites, pollen, mold or mold spores, weed pollen, tree pollen, grass pollen, fleas, pet hair, feathers, or pet dander. Skin allergies may include, for example, dermatitis, atopic dermatitis, contact dermatitis, eczema, atopic eczema, urticaria, and psoriasis. These are generally a group of diseases that cause inflammation of the skin, and symptoms include itching, redness of the skin, and rash.

[0111] Allergic diseases may also include other allergic inflammatory conditions, such as eosinophilic esophagitis and eosinophilic-related gastrointestinal diseases. Eosinophilic esophagitis is an allergic inflammatory condition of the esophagus involving eosinophils (a type of white blood cell). Symptoms are difficulty swallowing, food impaction, vomiting, and heartburn.

[0112] Suitably, the Bifidobacterium longum transitional microorganism and / or prebiotics of the present invention can increase the level of anti-inflammatory cytokines in infants or young children. Suitably, the Bifidobacterium longum transitional microorganism and / or prebiotics can increase the IL-10 level in infants or young children. IL-10 is a key immunomodulatory cytokine secreted by several immune cells including regulatory T cells, and has been shown to attenuate the response mediated by proinflammatory IL-12p40 / helper T cell type 1. This cytokine is critical in the establishment and maintenance of intestinal homeostasis between host immune cells and intestinal microbiota. Therefore, abnormal IL-10 signaling has been reported in chronic inflammatory bowel diseases such as inflammatory bowel disease (IBD). Changes in intestinal microbiota during weaning induce a strong immune response, while inducing a regulatory immune response (weaning response). The interference of this weaning response may increase the susceptibility to immunopathology (such as allergic diseases) in the future.

[0113] Suitably, the Bifidobacterium longum transitional microorganism and / or the prebiotic may increase the IL-10 / IL-12 ratio in the infant or young child.

[0114] Suitably, Bifidobacterium longum transitional microorganisms and / or prebiotics can reduce the level of proinflammatory cytokines, particularly cytokines associated with allergic and / or atopic responses. For example, Bifidobacterium longum transitional microorganisms and / or prebiotics can reduce the level of IL-5 in infants or young children. IL-5 is an interleukin produced by type 2 helper T cells and mast cells, which stimulates B cell growth and increases immunoglobulin secretion (mainly IgA). It is also a key regulator in eosinophil activation. IL-5 is associated with the cause of several allergic diseases (including allergic rhinitis and asthma). Reducing IL-5 levels has been shown to reduce allergic symptoms in diseases (such as severe asthma), for example (Casale et al.; 2021; Annals of Allergy, Asthma & Immunology; 127 (3); 354-362 and Sposato et al.; 2021; Int Arch Allergy Immunol; 182 (4): 311-318).

[0115] The cytokine effects mediated by the probiotics and / or prebiotics of the present invention can be systemic. Thus, the cytokine effects (e.g., an increase in IL-10 levels, an increase in the IL-10 / IL-12 ratio, and / or a decrease in IL-5) can systemically reduce the risk of allergic reactions and / or allergic sensitization and / or promote immune tolerance, as described herein. The cytokine effects can occur locally in the intestines, lungs, and / or skin of infants or young children. Suitably, the cytokine effects can occur in the intestines of infants or young children. Thus, the cytokine effects can reduce the risk of allergic reactions and / or allergic sensitization and / or promote immune tolerance in specific organs or systems.

[0116] Suitably, the Bifidobacterium longum transitional microorganism and / or the prebiotic may modulate the permeability of the intestinal epithelial barrier of an infant or young child. Suitably, the Bifidobacterium longum transitional microorganism and / or the prebiotic may reduce the permeability of the intestinal epithelial barrier. Increased permeability of the intestinal epithelial barrier may be associated with, for example, increased passage of haptens and antigens across the intestinal epithelium.

[0117] The commensal flora of the gastrointestinal tract constitute the earliest and most abundant stimulators of the development of the gut-associated lymphoid tissue and the associated immune system.

[0118] Epidemiological studies have demonstrated that Western living conditions (e.g., less consumption of fermented foods, greater use of antibiotics and other drugs, and improved hygiene) are associated with an increase in allergic diseases. This is related to the so-called "hygiene hypothesis," which proposes that lack of exposure to microbial stimuli in early childhood is a major factor influencing the prevalence of allergic diseases. Epidemiological studies have indeed demonstrated a correlation between the development of allergic diseases and disturbances in the gastrointestinal flora. Epidemiological data have demonstrated that children with atopic reactions have different intestinal flora compared to children without atopic reactions.

[0119] Such changes in intestinal flora may also have a negative impact on intestinal barrier integrity. Impaired barrier function, known as "leaky gut", has long been considered a susceptibility factor for gastrointestinal diseases (Heyman, M. Eur. J. Gastroenterol. Hepatol. 17: 1279-1285; Odenwald M., Nature Reviews Gastroenterology & Hepatology, (2017), (14), 9 21). Therefore, changes in intestinal barrier integrity / function have multiple consequences, depending on other accidental findings and genetic and epigenetic conformations that promote the occurrence of multiple diseases. Food allergy patients often show increased intestinal permeability, which is related to the severity of their clinical symptoms (Ventura, MT et al., 2006. Dig. Dis. Sci. 38: 732-736). Preclinical animal models also provide supporting evidence to support the role of intestinal barrier dysfunction and leaky gut, i.e. susceptibility to oral sensitization and subsequent development into food allergy. In addition, changes in intestinal permeability induced by a Western diet promote food allergen sensitization and clinical allergic symptoms in mice in response to dietary antigens (Hussain M. et al., J. Allergy Clin. Journal of Immunology, (2019)). Probiotics represent a nutritional attempt to improve / enhance intestinal barrier integrity and / or function (Ewaschuk JB et al., Am J Physiol Gastrointest Liver Physiol. 2008 Nov; 295(5): G1025-34). Therefore, enhancing intestinal barrier integrity by supplementing with probiotics can prevent at-risk individuals from being sensitized to oral allergens. (Tulyeu J, Microorganisms. 2019 October 16; 7(10)). In addition to the established role of intestinal barrier function in allergen sensitization, uncontrolled immune responses to dietary or environmental antigens promote the development of type 2 immune-mediated allergic diseases. Probiotic cultures or probiotic mixtures have well-known immunomodulatory properties that can prevent or alleviate allergic responses.

[0120] The epithelial barrier of human neonates is not fully mature at birth. The transfer of macromolecules or antigens through the intestinal epithelium of infants or young children induces the differentiation of regulatory T cells (Treg), and is necessary for inducing tolerance and preventing allergic diseases. However, the passage through the epithelium of antigens is important in determining immune response / outcome. Compared with uncontrolled paracellular passage (leakage), the transcellular passage with intestinal epithelial cell processing may be preferred, which may cause inflammation and / or sensitization instead.

[0121] As demonstrated in this example, the Bifidobacterium longum transitional microorganism encodes genes involved in the production of riboflavin and folate.

[0122] Riboflavin (vitamin B2) is an essential metabolite for host physiological processes. Mammalian hosts are unable to produce riboflavin and are therefore strictly dependent on external supplies from the diet and from intestinal microbial production. Once converted into active forms (FAD and FMN), riboflavin participates in a variety of metabolic pathways, including energy metabolism, fatty acid oxidation, and purine catabolism.

[0123] Preclinical and clinical studies of acute and chronic riboflavin deficiency lead to mucosal damage (EFSA Journal. 2010; 8 (10): 1814.). The gastrointestinal tract has one of the largest mucosal epithelial cells, which forms a direct barrier, allowing the absorption of nutrients and immune sensing, while limiting the transport of potentially harmful antigens and microorganisms. Riboflavin was found to maintain the structure and function of the small intestine, including the size and cellularity of the duodenal crypts in a rat model (Yates et al., Br J Nutr. 2001; 86 (5): 593-9) and the length and number of villi (Williams et al., Gut. 1996; 39: 220-225). Yates et al. also confirmed the importance of luminal riboflavin in the early development of the gastrointestinal tract in rats after birth, because even when the systemic riboflavin status was maintained via injection, the lack of luminal riboflavin also led to crypt hypertrophy and reduced crypt bifurcation (Yates et al., Dig Dis Sci 2003; 48 (6): 1159-64.). Riboflavin depletion affects the proliferation and proliferation potential of adult enterocytes, which may impair gastrointestinal function (Nakano et al., Dig Dis Sci 2011; 56(4): 1007-19.). These results highlight the role of riboflavin in supporting the normal development or maintenance of mucosal barriers, including the gastrointestinal mucosa. Therefore, during the transition from breast milk to solid foods, Bifidobacterium longum transitional microorganisms may maintain intestinal epithelial integrity through riboflavin production, thereby promoting a healthy imprint of the immune system.

[0124] Folic acid (vitamin B9) is another essential vitamin for nucleic acid synthesis and protein synthesis, and insufficient folic acid levels have been associated with immune response changes. In particular, folate receptor 4 is highly expressed in regulatory T (Treg) cells, and folic acid participates in maintaining Treg cell survival (Kunisawa et al., 2013, Front Immunol 4: 189). Mice fed with a folic acid-deficient diet have a reduced number of intestinal Treg cells, leading to an increase in inflammatory susceptibility (Kunisawa et al., 2012; PLoS One 7 (2): e32094). Treg is essential in controlling pro-allergic TH2 cell responses and producing anti-inflammatory IL-10. Bifidobacterium longum transitional microorganisms can affect tolerance immune responses via the production of folic acid.

[0125] The present invention also provides the use of a prebiotic as described herein to increase riboflavin and / or folate levels in the intestinal tract of an infant or child; wherein the riboflavin and / or folate are increased because the prebiotic promotes the growth and / or survival of Bifidobacterium longum transitional microorganisms in the intestinal tract of the infant or child.

[0126] The present invention also provides a method for increasing riboflavin and / or folic acid levels in the intestinal tract of an infant or child; wherein the method comprises administering a prebiotic as described herein to the infant or child so as to promote the growth and / or survival of Bifidobacterium longum transitional microorganisms in the intestinal tract of the infant or child.

[0127] Bifidobacterium longum transitional microorganisms and / or prebiotics can promote immune tolerance in infants and / or young children. For example, Bifidobacterium longum transitional microorganisms and / or prebiotics can promote immune tolerance in the intestinal tract of infants and / or young children. Immune tolerance can be promoted by mechanisms as described herein, such as increased IL-10 production, reduced IL-5 production, and / or reduced permeability of the intestinal epithelial barrier.

[0128] Prebiotics

[0129] The present invention also provides a prebiotic for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children by promoting the growth of transitional microorganisms of Bifidobacterium longum in the intestinal tract of infants or young children, wherein the prebiotic is:

[0130] i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3;

[0131] and / or

[0132] ii. human milk oligosaccharides (HMO), which are suitably selected from 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-

[0133] The group consisting of O-sialyllactose (6'-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and any combination thereof.

[0134] In another aspect, the present invention provides a prebiotic for treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children by promoting the growth of transitional microorganisms of Bifidobacterium longum in the intestinal tract of infants or young children.

[0135] Glycan Substrates / Carbohydrate-Active Enzymes (CAZymes)

[0136] Bifidobacterium longum transitional microorganisms encode a spectrum of carbohydrate-active enzymes (CAZymes). Without wishing to be bound by theory, it is believed that targeting these CAZymes by, for example, providing suitable glycan substrates in the form of prebiotics may promote the growth and / or survival of Bifidobacterium longum transitional microorganisms in the intestinal microbiota of infants or young children.

[0137] Suitably, promoting the growth and / or survival of Bifidobacterium longum transition-type microorganisms may refer to increasing the number and / or concentration of Bifidobacterium longum transition-type microorganisms in the intestinal microbiota.

[0138] In particular, the CAZyme encoded by each of the transitional strains of Bifidobacterium longum NCC 5000, NCC 5001, NCC 5002, NCC5003 and NCC 5004, which were deposited at the Pasteur Institute under the Budapest Treaty on May 11, 2021, with the deposit numbers CNCM I-5683, CNCM I-5684, CNCM I.5685, CNCM I-5686 and CNCM I-5687, respectively, has been determined.

[0139] Carbohydrate-active enzymes (CAZymes) are responsible for the synthesis and degradation of glycoconjugates, oligosaccharides and polysaccharides. They generally correspond to 1%-5% of the genes in living organisms. Glycoconjugates, oligosaccharides and polysaccharides play an important role in many biological functions, for example as structural and energy reserve components, and in many intracellular and intercellular events. The carbohydrate-active enzyme (CAZy) classification is a sequence-based family classification system related to the structure and molecular mechanism of CAZymes (www.cazy.org).

[0140] CAZymes include glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and carbohydrate binding module families (CBM).

[0141] Suitably, a CAZyme can be a glycoside hydrolase (GH). 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: a common acid (proton donor) and a nucleophile / base. Depending on the spatial position of these catalytic residues, hydrolysis occurs via overall retention or overall inversion of the anomeric configuration.

[0142] The GH classification system is provided by the CAZy classification. Herein, GHs 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 the family, which share a closer ancestor and are generally more consistent in molecular function (e.g., GH13_1, GH13_2, GH13_3, GH13_4, etc.).

[0143] Table 1 provides detailed information on CAZymes unique to Bifidobacterium longum transitional strains (i.e., not encoded by Bifidobacterium longum suis / suillum, Bifidobacterium longum longum, or Bifidobacterium longuminfantis strains). Table 1 also provides a summary of glycan substrates metabolized by each CAZyme and exemplary dietary fiber sources / ingredients.

[0144] Table 1

[0145]

[0146]

[0147] Table 2 provides information on the presence of at least one transitional strain of Bifidobacterium longum but not present in the Figure 4 Detailed information on a CAZyme in at least one of the groups of Bifidobacterium longum subsp. suis, Bifidobacterium longum subsp. longum, or Bifidobacterium longum subsp. infantis strains shown. Table 2 also provides a summary of glycan substrates metabolized by each CAZyme and exemplary dietary fiber sources / ingredients.

[0148] Table 2

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Table 3 provides detailed information on the CAZymes present in all B. longum strains analyzed (i.e., B. longum transitional, B. longum suis, B. longum ssp. longum, and B. longum infantis). Table 3 also provides a summary of the glycan substrates metabolized by each CAZyme and exemplary dietary fiber sources / ingredients.

[0157] Table 3

[0158]

[0159]

[0160]

[0161] Table 4 provides detailed information on CAZymes that are not encoded by the Bifidobacterium longum transitional strain but are encoded by one or more of Bifidobacterium longum subsp. suis, Bifidobacterium longum subsp. longum, and Bifidobacterium longum subsp. infantis.

[0162] Table 4

[0163]

[0164] Representative sequences of the CAZymes listed in Tables 1 to 4 are shown in Figure 7 Suitably, the CAZyme referred to in any one of Tables 1 to 4 may include or consist of Figure 7 Suitably, a CAZyme may comprise or consist of Figure 7 The variants are composed of variants of the corresponding sequences shown in Tables 1 to 4, which retain at least one function of the corresponding CAZyme listed in Tables 1 to 4. Suitably, the variants can provide each functional activity of the corresponding CAZyme listed in Tables 1 to 4. Suitably, the variants can include or consist of Figure 7The sequences listed in Table 1 have an amino acid sequence composition of at least 70% sequence identity and retain at least one functional activity, preferably each functional activity, of the corresponding CAZyme listed in Table 1 to Table 4. Suitably, the variant may comprise Figure 5 The variants may be selected from the group consisting of or having amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the corresponding sequences listed in Tables 1 to 4. The variants may retain at least one functional activity, preferably each functional activity, of the corresponding CAZyme listed in Tables 1 to 4.

[0165] Suitably, the prebiotic for use in the present invention may include a polysaccharide substrate selected from the group listed in any one of Tables 1 to 3.

[0166] Suitably, the prebiotic for use in the present invention may include a combination of polysaccharide substrates selected from the group listed in any one of Tables 1 to 3.

[0167] The combination of glycan substrates may include at least 2, at least 4, at least 10, at least 20, at least 30, at least 40, or at least 50 glycan substrates selected from the groups listed in Tables 1 to 3. The combination may include each of the glycan substrates listed in Tables 1 to 3.

[0168] Suitably, the prebiotic may include one or more polysaccharide substrates selected from those listed in Table 1 or Table 2.

[0169] The prebiotic may include at least 2, at least 4, at least 10, at least 20, or at least 30 of the polysaccharide substrates listed in Tables 1 and 2. The prebiotic may include each of the polysaccharide substrates listed in Tables 1 and 2.

[0170] Suitably, the polysaccharide substrate may comprise or consist of pectin, arabinogalactan and / or starch.

[0171] Suitably, the polysaccharide substrate may comprise or consist of pectin.

[0172] Suitably, the polysaccharide substrate may comprise or consist of arabinogalactan.

[0173] Suitably, the polysaccharide substrate may comprise or consist of starch.

[0174] Suitably, the polysaccharide substrate is provided in the form of dietary fiber.For example, the dietary fiber may be a prebiotic fiber.

[0175] Suitably, the glycan substrate may be included in an ingredient, such as a dietary ingredient.

[0176] The ingredient contains one or more polysaccharide substrates which may be selected from the group consisting of purified polysaccharides or purified oligosaccharides, dietary fiber ingredients, semi-purified food ingredients, raw food ingredients, food additives, HMOs, semi-purified or purified peptidoglycans.

[0177] Semi-purified food ingredients can be fruit, vegetable or grain extracts.

[0178] Raw food ingredients can be fruits, vegetables, grains, seaweed or microalgae.

[0179] The food additive may be guar gum or gum arabic.

[0180] Suitably, the peptidoglycan may be a GAG.

[0181] Suitably, the polysaccharide substrate may be included in the purified fibers.

[0182] Exemplary ingredients and / or purified fibers comprising suitable glycan substrates are provided in Tables 1 to 3. In particular, the dietary fibers and / or ingredients comprising a given glycan substrate are identified in the same row as the glycan substrate.

[0183] Pectin may be included in fruit or vegetable pectin. Thus, suitable ingredients comprising pectin include, but are not limited to: fruits (e.g., apples, pears), vegetables, legumes (peas), and roots (e.g., beets). Suitable purified fibers comprising arabinogalactans include peach pectin. Suitably, pectin extracted from beets contains arabinan, galactan, and arabinogalactan and may be provided as an ingredient.

[0184] Arabinogalactan may be included in fruit or vegetable pectin. Exemplary suitable ingredients including arabinogalactan include, but are not limited to, fruit, vegetable, whole grain, and seaweed dietary fibers. Suitable purified fibers including arabinogalactan include peach pectin, larch wood arabinogalactan, and gum arabic. Suitably, arabinogalactan may be provided in larch wood arabinogalactan.

[0185] Starch can be included in resistant starch from cereals (whole grain), legumes, vegetables (e.g. corn) and roots (e.g. potato). Exemplary suitable ingredients including starch include, but are not limited to corn. Suitable purified fibers including starch include high amylose starch and resistant dextrins. Suitably, starch can be provided in potato, corn or other ingredients. Suitably, starch can be included in potato ingredients.

[0186] Human milk oligosaccharides (HMO)

[0187] Suitably, the prebiotic comprises an HMO.

[0188] Suitably, HMO can be metabolized by Bifidobacterium longum transitional microorganisms. Suitably, HMO may be able to promote the growth and / or survival of Bifidobacterium longum transitional strains. HMOs that can promote the growth and / or survival of Bifidobacterium longum transitional strains can be determined by, for example, the anaerobic culture of Bifidobacterium longum transitional strains and HMO to be tested. The growth and / or survival of Bifidobacterium longum transitional strains can be determined by measuring the abundance of bacterial cell number, cell density (e.g., by optical density measurement) and / or 16S rDNA, such as using PCR methods. An exemplary test for measuring the growth of Bifidobacterium longum transitional strains in the presence of HMO is provided in this embodiment 6. Compared with the number of Bifidobacterium longum transitional bacteria in the control anaerobic bacteria culture not comprising HMO, the HMOs that can promote the growth and / or survival of Bifidobacterium longum transitional strains can increase the number of Bifidobacterium longum transitional bacteria in the anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100%. Suitably, the HMO capable of promoting the growth and / or survival of a Bifidobacterium longum transitional-type strain may increase the number of Bifidobacterium longum transitional-type bacteria in an anaerobic culture by a statistically significant amount (e.g. p-value <0.05 as determined by one-way ANOVA) compared to the number of Bifidobacterium longum transitional-type bacteria in a control anaerobic culture not comprising the HMO.

[0189] The HMO can be a fucosylated oligosaccharide (i.e., an oligosaccharide having a fucose residue; for example, 2'-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lactose-N-fucosylpentaose (e.g., lactose-N-fucosylpentaose I, lactose-N-fucosylpentaose II, lactose-N-fucosylpentaose III, lactose-N-fucosylpentaose V), lactose-N-fucohexose, lactose-N-difucohexose I, fucosyllactose-N-hexose, fucosyllactose-N-neohexose, difucosyllactose-N-hexose I, difucosyllactose-N-neohexose II, and any combination thereof), N-acetylated oligosaccharide. oligosaccharides (e.g., LNT (lactose-N-tetraose), para-lactose-N-neohexose (para-LNnH), LNnT (lactose-N-neotetraose), DSLNT (disialyl lactose-N-tetraose), lactose-N-hexose, lactose-N-neohexose, para-lactose-N-hexose, para-lactose-N-neohexose, lactose-N-octaose, lactose-N-neooctaose, allolactose-N-octaose, para-lactose-N-octaose and lactose-N-decaose and any combination thereof) and / or sialylated oligosaccharides (e.g., 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL) or Lst (sialyllactose-N-tetraose), Lst-a, Lst-b or Lst-c)).

[0190] The prebiotic may comprise at least one prebiotic oligosaccharide selected from the group consisting of 2'-O-fucosyllactose (2'FL), 3'-O-fucosyllactose (3FL), lactose difucotetraose / difucosyllactose (DFL), 3'-O-sialyllactose (3-SL), 6'-O-sialyllactose (6-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT); and any combination thereof.

[0191] The prebiotic may comprise 34 to 85 wt% 2'-FL, 10 to 40 wt% LNT, 4 to 14 wt% DFL, and 9 to 31 wt% 3-SL and 6-SL combined.

[0192] In some embodiments, the prebiotic comprises

[0193] - 26 to 65% by weight, preferably 32 to 54% by weight, of 2'-FL;

[0194] - 10 to 40% by weight, preferably 11 to 20% by weight, of LNT;

[0195] - 4 to 14% by weight, preferably 4 to 8% by weight, of DFL;

[0196] - 9 to 31 wt. %, preferably 8 to 22 wt. % of 3'-SL and 6'-SL combined; and

[0197] - 12 to 38% by weight, preferably 17 to 31% by weight, of 3-FL.

[0198] The prebiotic may comprise between 0.001 g / L and 12 g / L 2'-FL, preferably between 0.002 g / L and 10 g / L 2'-FL, more preferably between 0.005 g / L and 5 g / L 2'-FL.

[0199] The prebiotic may comprise between 0.001 g / L and 5 g / L DFL, preferably between 0.002 g / L and 4 g / L DFL, more preferably between 4 g / L and 3 g / L DFL.

[0200] The prebiotic may comprise between 0.01 g / L and 6 g / L of LNT, preferably between 0.025 g / L and 5 g / L of LNT, more preferably between 0.05 g / L and 1 g / L of LNT.

[0201] The prebiotic may comprise between 0.001 g / L and 2 g / L 6'-SL, preferably between 0.002 g / L and 1.5 g / L 6'-SL, more preferably between 0.005 g / L and 1 g / L 6'-SL.

[0202] The prebiotic may comprise between 0.01 g / L and 2 g / L 3'-SL, preferably between 0.025 g / L and 1.5 g / L 3'-SL, more preferably between 0.05 g / L and 1 g / L 3'-SL.

[0203] The prebiotic may comprise between 0.01 g / L and 7 g / L 3-FL, preferably between 0.025 g / L and 6 g / L 3-FL, more preferably between 0.05 g / L and 5 g / L 3-FL.

[0204] Suitably, 3'-O-fucosyllactose (3'FL) and lacto-N-tetraose (LNT) contained in the prebiotic promote the growth of Bifidobacterium longum transitional microorganisms which preferentially utilize 3-fucosyllactose (3-FL) over 2'-fucosyllactose (2'-FL).

[0205] Combination of Bifidobacterium longum transitional microorganism and prebiotics

[0206] The present invention also provides a combination of a Bifidobacterium longum transitional microorganism for use according to the invention and a prebiotic.

[0207] The Bifidobacterium longum transitional microorganism and the prebiotic may be administered separately, simultaneously or sequentially.

[0208] Suitably, the Bifidobacterium longum transitional microorganism and the prebiotic may be administered in the form of a combined composition.

[0209] Suitably, the combination of the Bifidobacterium longum transitional microorganism and the prebiotic may be referred to as a "symbiotic preparation".

[0210] In aspects of the invention using a combination of a Bifidobacterium longum transitional microorganism and a prebiotic (e.g., a glycan substrate), each aspect can be selected so that the Bifidobacterium longum transitional microorganism is capable of metabolizing the glycan substrate provided in the combination. Such selection can be performed, for example, by selecting a Bifidobacterium longum transitional microorganism encoding a CAZyme as a glycan substrate (or selecting an ingredient comprising the glycan substrate) from the same row of Tables 1 to 3.

[0211] The combination of the present invention is not limited to requiring that the Bifidobacterium longum transitional microorganism is able to metabolize the glycan substrate provided in the combination. Thus, the present invention includes any combination of the Bifidobacterium longum transitional microorganism and the glycan substrate disclosed herein.

[0212] Suitably, the composition comprises one or more glycan substrates as described herein.

[0213] Suitably, the composition comprises a transitional form of Bifidobacterium longum that preferentially utilizes 3-fucosyllactose (3-FL) over 2'-fucosyllactose (2'-FL) mixed with 3'-O-fucosyllactose (3-FL) and lacto-N-tetraose (LNT). On a dry weight basis, the composition may comprise between 10 3 Up to 10 12 Probiotic strains between 10 cfu, more preferably between 10 7 with 10 12 cfu, such as between 10 8 with 10 10 cfu, the probiotic strain is mixed with 3-Fl, the amount of which is between 0.01g / L and 7g / L 3-FL, preferably between 0.025g / L and 6g / L 3-FL, more preferably between 0.05g / L and 5g / L 3-FL, and mixed with LNT, the amount of which is between 0.01g / L and 6g / L LNT, preferably between 0.025g / L and 5g / L LNT, more preferably between 0.05g / L and 1g / L LNT.

[0214] Composition

[0215] The Bifidobacterium longum transitional microorganism, prebiotic or symbiotic preparation used in the present invention may be provided in the form of a composition.

[0216] The composition can be appropriately applied to an individual, such as an infant or young child, in any suitable form such as a nutritional composition of a dosage unit (e.g., tablet, capsule, powder sachet, etc.). The composition can be in powder, semi-liquid or liquid form. The composition can be added to a nutritional composition, infant formula, food composition, supplement, baby food, second stage infant formula, growing milk, infant cereal or fortifier for an infant or young child. In some embodiments, the composition of the present invention is an infant formula, baby food, infant cereal, growing milk, supplement or fortifier that can be used for an infant or young child.

[0217] By way of example, the composition may comprise further components which may be beneficial in reducing the risk of developing an allergic reaction and / or an allergic reaction. Additionally or alternatively, the composition may comprise other components which may be beneficial during the weaning period.

[0218] For example, the composition may comprise additional probiotics, such as probiotics known to have an effect on reducing the risk of developing allergic reactions and / or allergic reactions (e.g., Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium longum subsp. infantis), formula (e.g., partially hydrolyzed formula, extensively hydrolyzed formula, amino acid-based formula or complete formula), baby food (with or without milk fat), milk fat, cereals, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), butyrate and / or gamma-linolenic acid (GLA).

[0219] Based on dry weight, the Bifidobacterium longum transitional microorganism can be present in an amount of about 10 3 cfu to 10 12 cfu of probiotic strains, preferably between 10 7 cfu with 10 12 cfu, such as between 10 8 cfu with 10 10 cfu of the probiotic strain is included in the composition. In one embodiment, the Bifidobacterium longum transitional microorganism is alive. In another embodiment, the Bifidobacterium longum transitional microorganism is non-replicating or inactivated. In some other embodiments, there may be live and inactivated Bifidobacterium longum transitional microorganisms.

[0220] Suitably, the composition comprises one or more glycan substrates as described herein.

[0221] In some embodiments, the composition comprises at least one prebiotic oligosaccharide selected from the group consisting of 2'-O-fucosyllactose (2FL), 3'-O-fucosyllactose (3FL), lactose difucotetraose / difucosyllactose (DFL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL) and lacto-N-tetraose (LNT), and any combination thereof.

[0222] In some embodiments, the composition comprises

[0223] - 26 to 65% by weight, preferably 32 to 54% by weight, of 2'-FL;

[0224] - 10 to 40% by weight, preferably 11 to 20% by weight, of LNT;

[0225] - 4 to 14% by weight, preferably 4 to 8% by weight, of DFL;

[0226] - 9 to 31 wt. %, preferably 8 to 22 wt. % of 3'-SL and 6'-SL combined; and

[0227] - 12 to 38% by weight, preferably 17 to 31% by weight, of 3-FL.

[0228] The composition may comprise between 0.001 g / L and 12 g / L of 2'-FL, preferably between 0.002 g / L and 10 g / L of 2'-FL, more preferably between 0.005 g / L and 5 g / L of 2'-FL.

[0229] The composition may comprise between 0.001 g / L and 5 g / L DFL, preferably between 0.002 g / L and 4 g / L DFL, more preferably between 4 g / L and 3 g / L DFL.

[0230] The composition may comprise between 0.01 g / L and 6 g / L of LNT, preferably between 0.025 g / L and 5 g / L of LNT, more preferably between 0.05 g / L and 1 g / L of LNT.

[0231] The composition may comprise between 0.001 g / L and 2 g / L 6'-SL, preferably between 0.002 g / L and 1.5 g / L 6'-SL, more preferably between 0.005 g / L and 1 g / L 6'-SL.

[0232] The composition may comprise between 0.01 g / L and 2 g / L 3'-SL, preferably between 0.025 g / L and 1.5 g / L 3'-SL, more preferably between 0.05 g / L and 1 g / L 3'-SL.

[0233] The composition may comprise between 0.01 g / L and 7 g / L of 3-FL, preferably between 0.025 g / L and 6 g / L of 3-FL, more preferably between 0.05 g / L and 5 g / L of 3-FL.

[0234] In some embodiments, the composition comprises a transitional type microorganism of Bifidobacterium longum that preferentially utilizes 3-fucosyllactose (3-FL) over 2'-fucosyllactose (2'-FL) mixed with 3'-O-fucosyllactose (3-FL) and lactose-N-tetraose (LNT). The composition may comprise between 10 3 Up to 10 12 Probiotic strains between 10 cfu, more preferably between 10 7 with 10 12 cfu, such as between 10 8 with 10 10cfu, the probiotic strain is mixed with 3-Fl, the amount of which is between 0.01g / L and 7g / L 3-FL, preferably between 0.025g / L and 6g / L 3-FL, more preferably between 0.05g / L and 5g / L 3-FL, and mixed with LNT, the amount of which is between 0.01g / L and 6g / L LNT, preferably between 0.025g / L and 5g / L LNT, more preferably between 0.05g / L and 1g / L LNT.

[0235] Suitably, 3'-O-fucosyllactose (3'FL) and lacto-N-tetraose (LNT) included in the composition promote the growth of Bifidobacterium longum transitional microorganisms that preferentially utilize 3-fucosyllactose (3-FL) over 2'-fucosyllactose (2'-FL).

[0236] method

[0237] In another aspect, the present invention provides a method for treating and / or preventing allergies and / or allergic sensitization in infants or young children; wherein the method comprises administering an effective amount of a transitional microorganism of Bifidobacterium longum, a prebiotic, or a combination of a transitional microorganism of Bifidobacterium longum and a prebiotic to a subject in need thereof.

[0238] In another aspect, the present invention relates to the use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination of a Bifidobacterium longum transitional microorganism and a prebiotic for the preparation of a medicament for treating and / or preventing allergies and / or allergic sensitization in infants or young children.

[0239] The Bifidobacterium longum transitional microorganism may be a Bifidobacterium longum transitional microorganism as described herein.

[0240] The prebiotic may be a prebiotic as described herein.

[0241] The combination of a Bifidobacterium longum transitional microorganism and a prebiotic may be provided in any form as described herein. For example, the combination may be provided in a composition as described herein.

[0242] Implementation

[0243] The present invention provides embodiments according to the following numbered clauses:

[0244] 1. A Bifidobacterium longum transitional microorganism for use in treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children.

[0245] 2. A prebiotic for treating and / or preventing a condition in an infant or young child by promoting the growth and / or survival of transitional microorganisms of Bifidobacterium longum in the intestinal tract of the infant or young child.

[0246] Allergic reactions and / or allergic sensitization in infants and children, wherein the prebiotic is:

[0247] i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or

[0248] ii. Human milk oligosaccharides (HMO), suitably selected from 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lactose-N-tetraose (LNT)

[0249] and lacto-N-neotetraose (LNnT) and any combination thereof.

[0250] 3. A Bifidobacterium longum transitional microorganism for use according to clause 1, wherein the Bifidobacterium longum transitional microorganism is used in combination with a prebiotic, and wherein the prebiotic is:

[0251] i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or

[0252] ii. Human milk oligosaccharides (HMO), suitably selected from 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lactose-N-tetraose (LNT)

[0253] and lacto-N-neotetraose (LNnT) and any combination thereof.

[0254] 4. A prebiotic for use according to clause 2, wherein the prebiotic is used in combination with a Bifidobacterium longum transitional microorganism.

[0255] 5. A combination of a Bifidobacterium longum transitional microorganism and a prebiotic for use in the treatment and / or prevention of allergic reactions and / or allergic sensitization in infants or young children; wherein the prebiotic is:

[0256] i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or

[0257] ii. human milk oligosaccharides (HMOs), which are suitably selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and any combination thereof.

[0258] 6. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any preceding clause, wherein preventing allergic reactions and / or allergic sensitization comprises reducing the risk of developing allergic reactions and / or allergic sensitization.

[0259] 7. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of clauses 2 to 6, wherein the prebiotic is a polysaccharide substrate selected from the group listed in any one of Tables 1 to 3.

[0260] 8. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of clauses 2 to 6, wherein the prebiotic is a HMO selected from the group consisting of 2'-FL, 3'-FL, di-FL, 3'-SL, 6'-SL, LNT and LNnT and any combination thereof.

[0261] 9. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of clauses 2 to 8, wherein the HMO is 3'-FL.

[0262] 10. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing the HMO and / or the glycan substrate.

[0263] 11. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism preferentially utilizes 3'-fucosyllactose (3'-FL) over 2'-fucosyllactose (2'-FL).

[0264] 12. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing a polysaccharide substrate selected from the group listed in any one of Tables 1 to 3.

[0265] 13. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism encodes one or more CAZymes selected from the group listed in Table 1, preferably wherein the Bifidobacterium longum transitional microorganism also encodes one or more CAZymes selected from Table 2 and Table 3.

[0266] 14. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any of the preceding clauses, wherein the Bifidobacterium longum microorganism has an average nucleotide identity (ANI) of at least 98% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753) and any combination thereof.

[0267] 15. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism and / or prebiotic increases IL-10 levels in the infant or young child.

[0268] 16. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism and / or prebiotic increases the IL-10 / IL-12 ratio in the infant or young child.

[0269] 17. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism and / or prebiotic reduces IL-5 in the infant or young child.

[0270] 18. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism or prebiotic modulates the permeability of the intestinal epithelial barrier.

[0271] 19. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism or prebiotic reduces the permeability of the intestinal epithelial barrier.

[0272] 20. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism or prebiotic increases riboflavin and / or folate levels in the infant or young child.

[0273] 21. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination according to any of the preceding clauses for use in treating allergies and / or allergic sensitization in an infant or young child.

[0274] 22. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination according to any of the preceding clauses for use in preventing allergies and / or allergic sensitization in infants or young children.

[0275] 23. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination according to any one of clauses 1 to 21 for use in reducing the risk of allergy and / or allergic sensitization in an infant or young child.

[0276] 24. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any preceding clause; wherein the allergen is a food allergen or a respiratory allergen or a skin allergen.

[0277] 25. Use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined in any one of the preceding clauses for promoting immune tolerance in an infant or young child, preferably by promoting the growth of a Bifidobacterium longum transitional microorganism in the intestinal tract of said infant or young child.

[0278] 26. Use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined in any one of the preceding clauses for modulating the intestinal barrier permeability of an infant or young child, preferably by promoting the growth of Bifidobacterium longum transitional microorganisms in the intestinal tract of said infant or young child.

[0279] Those skilled in the art will appreciate that they are free to incorporate all features of the present invention disclosed herein. Specifically, features described for product of the present invention may be combined with method of the present invention, and vice versa. In addition, features described for different embodiments of the present invention may be combined. If known equivalents exist for specific features, such equivalents are included, as are clearly mentioned in this specification.

[0280] Further advantages and features of the invention will become apparent upon reference to the accompanying drawings and non-limiting examples.

[0281] Example

[0282] Example 1: Transitional Bifidobacterium longum increases intestinal epithelial barrier resistance

[0283] In vitro experiments using a human colorectal adenocarcinoma cell line (Caco-2) have shown that transitional B. longum strains are able to increase transmembrane electrical resistance (TEER) when incubated with epithelial cells for 24 h (see Figure 2 ).

[0284] These results suggest that transitional B. longum plays a role in reducing paracellular permeability during the transition from breast milk to solid foods, which is associated with an increase in dietary antigen uptake and, therefore, contributes to the barrier balance required for optimal maturation of the immune system.

[0285] Example 2: Transitional Bifidobacterium longum increases the anti-inflammatory cytokine IL-10, improves the IL-10 / IL-12 ratio and reduces Low IL-5 expression by type 2-skewed helper T cells

[0286] In vitro, the transitional strain of Bifidobacterium longum increased the immunomodulatory response in human peripheral blood mononuclear cells, characterized by the expression of interleukin 10 (IL-10) ( Fig.13 ) and the IL-10 / IL-12p40 ratio increased to that of the probiotic Lactobacillus rhamnosus (NCC4007) ( Figure 3 ) to a similar degree or even to a greater extent.

[0287] Furthermore, in vitro, the transitional strain of Bifidobacterium longum reduced IL-5 expression by type 2-skewed helper T cells after 48 h stimulation to a similar or even greater extent than the probiotic Bifidobacterium lactis (which has known anti-inflammatory properties) ( Fig.12 IL-5 was measured according to the following method: peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of blood clots obtained from healthy adults by density gradient. PBMCs were then incubated at 1.5 × 10 6 Cells / ml were seeded in complete Isocove modified Dulbecco's medium (cIMDM) containing 10% fetal bovine serum, 1% glutamine, 1% penicillin / streptomycin and 0.1% gentamicin in 48-well bottom plates. Cells were cultured in the presence of 50ng / ml IL-4 and 1ug / ml anti-CD40. After 3 days of culture, different bacterial strains including all transitional long B. longum isolates were added at specified concentrations. Cell culture supernatants were collected to evaluate cytokine expression of IL-5 by ELISA. Standard curves for each cytokine were used to calculate absolute amounts (picograms / ml) from optical density readings.

[0288] Example 3: Analysis of carbohydrate active enzyme (CAZyme) genes of Bifidobacterium longum transitional microorganisms

[0289] Combining the dbCAN2 (Zhang et al., Nucleic Acids Res. 46(W1):W95-W101(2018)) tool and the databases HMMdb (v9) and Diamond (v2.0.8), Figure 4The genomes of Bifidobacterium longum subspecies listed in were annotated as CAZymes. Query sequences with >0.50 coverage and e-value <1e-15 were annotated with HMMER according to the dbCAN CAZyme domain HMM database. Diamond was also used to annotate query sequences with hits in the CAZy database (Drula et al., Nucleic Acids Res. 50(D1):D571-D577(2022))(http: / / www.cazy.org / ), identities >0.90, and e-values ​​<1e-102. In case of mismatches in the CAZyme annotations of the query sequences between the HMMER and DIAMOND tools, the HMMER annotation was used preferentially. Only CAZyme families and subfamilies encoding glycoside hydrolases (GH) and polysaccharide lyases (PL) were used for comparative analysis of Bifidobacterium longum subspecies (see Figure 4 ).

[0290] Example 4: Utilization of polysaccharide substrates

[0291] The crushed or homogenized fecal samples were mixed 10 times by adding PBS / glycerol (1 / 10) (w / v) and then centrifuged at 2000g for 2 minutes. The slurry and pellets were then stored at -80°C. The frozen fecal samples were thawed from storage at -80°C before centrifugation at 2000g for 2 minutes. The resulting supernatant was inoculated with a culture medium based on the culture medium disclosed in the following literature: Daguet et al., (Journal of Functional Foods; 2016; 20; 369-379). The culture medium was supplemented with 5g / L of the specific fiber to be tested and 5E07 CFU / ml of bifidobacterium supplement.

[0292] The cultures were established at 37°C under a flow of N2 gas to ensure anaerobic conditions and gentle agitation. Aliquots were taken at designated time points and analyzed.

[0293] Growth of Bifidobacterium longum transition strain NCC 5001 was promoted by pectin (sugar beet) and arabinogalactan (larch wood) ( Figure 5 ).

[0294] Growth of Bifidobacterium longum transition strain NCC 5002 was promoted by arabinogalactan (larch wood) and starch (potato) ( Figure 6 ).

[0295] Example 5: Characterization of Bifidobacterium longum transitional microorganisms

[0296] Bifidobacterium longum transition strains were isolated from the feces of breastfed infants using Eugon tomato agar (ETA). The obtained isolates were sequenced using PacBio to obtain a completely closed assembled genome for each strain. Each strain, together with their genome sequence data, was deposited at the National Collection of Microorganisms (CNCM) at the Nestlé Internal Culture Collection (NCC, Lausanne, Switzerland) and the Pasteur Institute (Paris, France). The genomes of the strains were compared with other publicly available genomes (Table 5) representing the overall diversity of Bifidobacterium longum species by average nucleotide identity (ANI) using OrthoAni (https: / / www.ezbiocloud.net / tools / orthoani), and compared with the metagenome assembly genome (MAG) obtained from the metagenome sequences published in the feces of infants from the same cohort.

[0297] Table 5 - List of genomes used for ANI analysis and their publicly available references. (T) represents the model strain .

[0298]

[0299]

[0300] The analysis showed that the newly described strains grouped together with MAGs obtained from the same cohort, defining a well-defined clade belonging to the species Bifidobacterium longum. Two previously isolated strains, BSM11-5 and 3_mod, were found to be grouped within this newly described clade. This clade was genetically distinct from the subspecies Bifidobacterium longum subsp. longum (96.40% ANI). This clade was related to, but still distinct from, the group of strains previously shown to be new subspecies Bifidobacterium longum (JDM301, CMCC_P0001 and BXY01) (O'Callaghan et al., 2015), sharing 98.260% identity with this group of strains. Figure 1 A phylogenetic tree based on ANI UPGMA is shown. The scale indicates the percent identity (%) at each branch point.

[0301] The dbCAN annotation pipeline (http: / / bcb.unl.edu / dbCAN / ) was used to annotate the above selection of genomes representing the diversity of B. longum subsp. for carbohydrate active enzymes (CAZY). The results showed that B. longum subsp. longum, B. longum subsp. Suis and B. longum subsp. suillum strains contained GH20 (lacto-N-biosidase) involved in the degradation and metabolism of lactose-N-tetraose (LNT). Similar to B. longum subsp. infantis strains, B. longum transition strains also have similar enzymes and additionally carry GH29 (fucosidase) encoding genes involved in the degradation and metabolism of fucosylated human milk oligosaccharides (such as 2'FL, 3'FL or diFL). In addition, three strains (CNCM 1-5684, BSM1-15 and 3_mod) also carried the GH 33 (sialidase) encoding gene involved in the degradation and metabolism of sialylated HMOs such as 3'SL or 6'SL (Table 6).

[0302] Table 6 - The genomes of each representative encode GH20 (lacto-N-biosidase), GH29 (α-fucosidase), Genes for GH95 (α-fucosidase / α-galactosidase) and GH33 (sialidase) glucose hydroxyhydratase family enzymes quantity .

[0303]

[0304]

[0305] All newly obtained genomes were compared and aligned with the genomes of two strains (Bifidobacterium longum subsp. infantis ATCC15697 and Bifidobacterium carinii DSM 21854), which belong to the species where the genes responsible for fucosylated HMO utilization were elucidated (James et al., 2019).

[0306] result

[0307] like Figure 8 As shown, all newly described strains contain the gene responsible for the utilization of fucosylated HMOs. While the NCC5001 organization reflects the one of Bifidobacterium longum subsp. infantis ATCC 15697, all other strains (NCC 5000, NCC5002, NCC 5003, NCC 5004) carry a gene organization closer to Bifidobacterium carinii DSM 21854. Overall, the similarity to the well-described fucosidase of Bifidobacterium longum subsp. infantis ATCC 15697 was higher than 77% (for BLON_2334) and 88% (BLON_2335) in all newly described strains.

[0308] Example 6: Utilization of fucosylated HMO

[0309] All strains obtained from the Nestle Culture Collection were reactivated from freeze-dried stock solutions using two continuous cultivation steps (16 h, 37 ° C, anaerobic) in MRS supplemented with 0.05% cysteine ​​(MRSc). The reactivated cultures were then centrifuged, washed and resuspended in 1 volume of PBS. The washed cells were used to inoculate a MRS-based medium (MRSc-C) (10 g l-1 bacto proteose peptone n ° 3, 5 g l-1 bacterial yeast extract, 1 g l-1 Tween 80, 2 g l-1 diammonium hydrogen 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) without a carbon source, wherein glucose, 2'FL or 3'FL were added at a concentration of 0.5% as the sole carbon source. Growth was then performed in 96-well microplates with a volume of 200 μl per well. Incubation was performed under anaerobic conditions for 48 h and the optical density was measured at 600 nm in a spectrophotometer. Fig. 9 As shown, all B. longum transition strains grew on fucosylated HMOS.

[0310] result

[0311] All B. longum transition strains grew better on 3'FL than on 2'FL and reached higher cell densities on this carbohydrate. This behavior indicates that 3'FL is superior to 2'FL (ratios of 1.8 to 2.8 - see Fig.10 ), which was not observed in Bifidobacterium longum subsp. infantis LMG 11588.

[0312] Table 7 - List of strains (and corresponding numbers) used for growth studies of individual fucosylated HMOs

[0313]

[0314] method

[0315] CACO-2 cell culture and transmembrane resistance measurements

[0316] Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 24-well semipermeable inserts. The Caco-2 monolayer was cultured for 14 days, with the culture medium replaced three times a week until a functional cell monolayer with transmembrane electrical resistance (TEER) was obtained. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing glucose and glutamine and supplemented with HEPES and 20% (v / v) heat-inactivated fetal bovine serum. The TEER of the Caco-2 monolayer was measured (=0 hour time point) before the bacteria were added to the apical compartment. The TEER of the empty insert was subtracted from all readings to calculate the remaining resistance of the insert. The probiotic strain (taken directly from the glycerol stock) was then diluted in Caco-2 complete medium and added to the Caco-2-containing insert toward the top at 2×10E6 colony forming units. Cells were also exposed to Caco-2 complete medium (CM) in both chambers as controls and 0.75% glycerol in the apical compartment as a vehicle control. Cells were treated for 24 hours and TEER was measured at several time points (2 hours, 4 hours, 6 hours and 24 hours). After subtracting the TEER of the empty insert, all time point values ​​were normalized to their own 0 hour value (to account for differences in the initial TEER of the different inserts) and expressed as a percentage of the initial value ( Figure 1 ).

[0317] Immune profiling with PBMCs

[0318] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy adults by density gradient. PBMCs were then cultured at 1.5 × 10 6 10 cells / ml were plated in complete Isocove's modified Dulbecco's medium (cIMDM) containing 10% fetal bovine serum, 1% glutamine, 1% penicillin / streptomycin, and 0.1% gentamicin in 48-well plates. PBMCs were stimulated for 36 h in the presence of different bacterial strains, including 10 7 CFU / ml of all transitional B. longum isolates and probiotic strains. Cell culture supernatants were collected to assess cytokine expression of IL-10 and IL-12p40 by ELISA. Standard curves for each cytokine were used to calculate absolute amounts (picograms / ml) from optical density readings.

[0319] Example 7: Bifidobacterium longum transitional microorganisms produce precursors of folic acid and riboflavin, i.e., maintain intestinal barrier integrity Two metabolites of integrity

[0320] Genetic characterization of transitional isolates of B. longum revealed that they possess a specific genetic region containing a set of six genes ( Fig.11). As shown in the figure, the transitional strain of Bifidobacterium longum and the infantile strain of Bifidobacterium longum are the only strains that carry this set of genes. The entire operon is absent in strains belonging to B. longum subsp. longum, B. longum subsp. Suis, and B. longum subsp. suillum. This region covers two genes (PabA and PabB) that are involved in the conversion of chorismate to 4-amino-4-deoxychorismate (precursor of p-aminobenzoate and folic acid) in different microorganisms. In addition, this region also contains another group of 4 genes (RibD, RibE, RibAB, RibH) related to the biosynthesis of riboflavin, as recently demonstrated in B. longum infantile subsp. (Solopova et al., 2020, Front Microbiol, 2020.11: p.573335).

[0321] The presence of these genes suggests that B. longum transitional microorganisms may be able to produce riboflavin and folate.

[0322] Example 8 Bifidobacterium longum transitional microorganisms produce SCFAs that are beneficial for the prevention and control of allergies .

[0323] method : Infant microbiomes containing 3-fucosyllactose (3FL) supplemented or not supplemented with infant Bifidobacterium longum species or transitional Bifidobacterium longum were subjected to in vitro batch fermentation for 48 hours. Acetate, butyrate, and propionate in the supernatant collected at T0 hours, T24 hours, and T48 hours were measured by 1H-NMR technology. Total SCFA corresponds to the sum of the peak integrations of acetic acid, butyric acid, and propionic acid. The bar graph indicates the dynamics of total SCFA consumption and production between 0 hours and 24 hours (blue bars) and 24 hours and 48 hours (red bars).

[0324] result :

[0325] Fig.14Transitional Bifidobacterium longum was shown to be more metabolically active against weaning-associated HMOs (i.e., 3FL) than Bifidobacterium infantis and to produce more total SCFAs, which is beneficial for allergy prevention and management, as shown in a number of studies (Trompette et al., Mucosal Immunology 15, 908-926 (2022); Roduit et al., Allergy, Apr; 74(4): 799-809 (2019); Canani et al., Sci Rep Aug 21; 8(1): 12500 (2018): Gio-Batta et al., Sci Rep 10, 22449 (2020): Cait et al., 2019 J Allergy Clin Immunol. 2019 Dec; 144(6): 1638-1647.

[0326] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the method and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the present invention as protected by the claims should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes for carrying out the present invention that are apparent to those skilled in the art of molecular biology or related fields are intended to fall within the scope of the following claims.

Claims

1. A Bifidobacterium longum transitional microorganism for use in treating and / or preventing allergic reactions and / or allergic sensitization in infants or young children.

2. A prebiotic for treating and / or preventing allergic reactions in infants or young children by promoting the growth and / or survival of transitional microorganisms of Bifidobacterium longum in the intestinal tract of the infant or young child, wherein the prebiotic is: i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or ii. human milk oligosaccharides (HMOs), which are suitably selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and any combination thereof.

3. The Bifidobacterium longum transitional microorganism for use according to claim 1, wherein the Bifidobacterium longum transitional microorganism is used in combination with a prebiotic, and wherein the prebiotic is: i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or ii. human milk oligosaccharides (HMOs), which are suitably selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and any combination thereof.

4. The prebiotic for use according to claim 2, wherein the prebiotic is used in combination with a transitional microorganism of Bifidobacterium longum.

5. A combination of a Bifidobacterium longum transitional microorganism and a prebiotic for use in the treatment and / or prevention of allergic reactions and / or allergic sensitization in infants or young children; wherein the prebiotic is: i. a polysaccharide substrate, which is suitably selected from the group listed in any one of Tables 1 to 3; and / or ii. human milk oligosaccharides (HMOs), which are suitably selected from the group consisting of 2'-O-fucosyllactose (2'-FL), 3'-O-fucosyllactose (3'-FL), lactose difucotetraose / difucosyllactose (di-FL), 3'-O-sialyllactose (3'-SL), 6'-O-sialyllactose (6'-SL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) and any combination thereof.

6. The Bifidobacterium longum transitional microorganism, prebiotic or combination for use according to any one of claims 2 to 5, wherein the prebiotic is a polysaccharide substrate selected from the group listed in any one of Tables 1 to 3.

7. Bifidobacterium longum transitional microorganism, prebiotic or combination for use according to any one of claims 2 to 5, wherein the prebiotic is a HMO selected from the group consisting of 2'-FL, 3'-FL, di-FL, 3'-SL, 6'-SL, LNT and LNnT and any combination thereof.

8. Bifidobacterium longum transitional microorganism, prebiotic or combination for use according to any one of claims 2 to 5 or 7, wherein the HMO is 3'-FL.

9. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding claims, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing the HMO and / or the glycan substrate.

10. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding claims, wherein the Bifidobacterium longum transitional microorganism preferentially utilizes 3'-fucosyllactose (3'-FL) over 2'-fucosyllactose (2'-FL).

11. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding claims, wherein the Bifidobacterium longum transitional microorganism: (a) is capable of metabolizing a glycan substrate selected from the group listed in any one of Tables 1 to 3; and / or (b) encodes one or more CAZymes selected from the group listed in Table 1, preferably wherein the Bifidobacterium longum transitional microorganism also encodes one or more CAZymes selected from Tables 2 and 3.

12. A Bifidobacterium longum transitional microorganism, a prebiotic or a combination for use according to any one of the preceding claims, wherein the Bifidobacterium longum microorganism has an average nucleotide identity (ANI) of at least 98% with at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753) and any combination thereof.

13. Bifidobacterium longum transitional microorganism, prebiotic or combination for use according to any one of the preceding claims, wherein the Bifidobacterium longum transitional microorganism and / or prebiotic: (a) increasing the level of IL-10 in the infant or young child; (b) increasing the IL-10 / IL-12 ratio in said infant or young child; (c) reducing the level of IL-5 in the infant or young child; (d) modulating the permeability of the intestinal epithelial barrier; preferably wherein said Bifidobacterium longum transitional microorganism and / or prebiotic reduces said permeability of said intestinal epithelial barrier; and / or (e) increasing riboflavin and / or folate levels in said infant or young child.

14. Use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined in any one of the preceding claims for promoting immune tolerance in an infant or young child, preferably by promoting the growth of a Bifidobacterium longum transitional microorganism in the intestinal tract of said infant or young child.

15. Use of a Bifidobacterium longum transitional microorganism, a prebiotic or a combination as defined in any one of the preceding claims for modulating the intestinal barrier permeability of an infant or young child, preferably by promoting the growth of Bifidobacterium longum transitional microorganisms in the intestinal tract of said infant or young child.

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