HMO mixtures and bifidobacterium longum transition microorganisms

Abstract

A mixture of HMO and a transition microorganism of Bifidobacterium longum. The present invention relates to a composition comprising a transition microorganism of Bifidobacterium longum and a mixture of human milk oligosaccharides (HMO) consisting of 2 '-fucosyllactose (2'-FL), difucosyllactose (DFL), lactose-N-tetraose (LNT), 6 '-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3-FL) and / or lactose-N-neotetraose (LNnT). The invention also relates to the use of the composition for preventing, reducing the risk of, and / or treating an infection in an individual. The present invention also provides compositions for promoting long term immune benefit in an individual, preventing and / or reducing the risk of allergen sensitization, preventing and / or reducing the risk of developing a respiratory condition in an individual and / or preventing and / or reducing the risk of developing asthma in an individual.

Description

Technical Field

[0001] This invention relates to synbiotics, particularly combinations of transitional Bifidobacterium longum with human milk oligosaccharides (HMOs), the HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT). The HMO mixture promotes the growth and / or survival of the transitional Bifidobacterium longum. This invention also relates to synbiotics for preventing infection in individuals, reducing the risk of infection in individuals, and / or treating infection in individuals. The present invention also provides synbiotics for promoting long-term immune benefits in individuals, preventing allergen sensitization and / or reducing the risk of allergen sensitization, preventing individuals from developing respiratory illnesses and / or reducing the risk of developing respiratory illnesses and / or preventing individuals from developing asthma and / or reducing the risk of developing asthma. Background Technology

[0002] Viral infection occurs when a virus replicates within a host cell, thus utilizing the host's resources to promote its own proliferation. Viral infection can also interfere with the host's normal functions and can lead to more serious infection-related disorders, including long-term changes in the immune system (such as inflammatory responses) and subsequent allergic or inflammatory diseases.

[0003] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children under two years of age (Karpinnen et al., Clin Microbiol Infect, 2016; 22; 208.e1-e6) and frequently lead to bronchiolitis, an inflammatory bronchial response in infants and young children (Pickles et al., J Pathol, 2015; 235; 266-276). Specifically, severe RSV-induced bronchiolitis is a leading cause of infant morbidity and mortality worldwide (Nair et al., Lancet, 2010; 375; 9725; 2545-1555). Respiratory viruses primarily infect airway epithelial cells. Higher viral loads are associated with increased severity of bronchiolitis, and conversely, rapid reductions in viral load in infants are associated with faster disease resolution (Pickles et al., J Pathol, 2015; 235; 266-276). Well-documented in the literature, during RSV infection, infected and necrotic epithelial cells lead to airway obstruction and inflammation (Pickles et al., J Pathol, 2015; 235; 266-276), thus epithelial cell shedding is a characteristic feature of viral bronchiolitis and is associated with disease severity (Johnson et al., Mod Pathol, 2007; 20; 108–119). Plasma-like dendritic cells (pDCs) are known to play a defensive role in the lesions during RSV infection, and adaptive immune responses, including CD4+ and CD8+ T cells, are important in clearing the virus from the respiratory tract (Openshaw et al., Annu Rev Immunol, 2017; 35; 501-532).

[0004] If the immune defense response to such viral respiratory infections is dysregulated, inflammatory granulocytes, such as neutrophils, along with CD4+ and CD8+ T cell responses, can also contribute to the immunopathological features following respiratory viral infection (Newton et al., Semin Immunopathol, 2016; 38; 471-482). Furthermore, such uncontrolled inflammatory responses can lead to pathological airway smooth muscle remodeling, a hallmark feature of asthma reportedly beginning early in life (O'Reilly et al., JACI, 2013; 131; 1024-1032), and in chronic obstructive pulmonary disease (COPD; Yan F et al., J Transl Med, 2018; 16; 262-270). Therefore, severe viral airway infections early in life represent a major independent risk factor for subsequent respiratory diseases such as allergic airway diseases (e.g., asthma; Feldman et al., Am J Respir CritCare Med, 2015; 191; 34–44) and later chronic obstructive pulmonary disease (Savran O et al., Int J ChronObstruct Pulmon Dis. 2018; 13: 683–693).

[0005] The interaction between the immune system and the microbiome plays a crucial role in human health. These interactions begin in the prenatal period and are essential for the maturation of the immune system in newborns and infants. Several factors influence the composition of the infant's microbiome and the subsequent development of the immune system. These include maternal infection, antibiotic treatment, environmental exposure, delivery pattern, breastfeeding, and food introduction.

[0006] Breastfeeding is a recognized factor that reduces the severity of respiratory viral infections in infants, either directly through milk bioactive substances (such as human milk oligosaccharides) or indirectly through microbiome-mediated immune benefits.

[0007] In recent years, human milk oligosaccharides (HMOs) have become a topic of great interest due to their role in numerous biological processes occurring in human organisms. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al., Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1).

[0008] Infancy, especially the first few weeks, the first 3 months, the first 6 months, or the first 12 months, is a crucial period for establishing a balanced gut microbiota. The regulation of the gut microbiota during infancy is known to have a significant predictable impact on future health. For example, the gut microbiota can influence the development of a robust immune system, normal growth, and even the later development of obesity. However, during infant development, the gut microbiota and its evolution represent a delicate balance between the presence and proliferation (quantity) of many gut bacterial populations. Regarding the impact of gut bacteria on overall infant health, some gut bacteria are classified as "generally positive," while others are "generally negative" (or pathogenic).

[0009] Probiotics, particularly those from the genera *Lactobacillus* and *Bifidobacterium*, are known to support protection against respiratory infections. The role of probiotics in viral respiratory infections was reviewed by Lehtoranta et al. (Lehtoranta et al., *Eur J Clin Microbiol Infect Dis, 2014; 33; 1289-1302*).

[0010] The weaning period has been described as a non-redundant window for immune imprinting (Cahenzli et al., Cell HostMicrobe, 2013, 14(5), 559-70; Olszak et al., Science, 2012, 336(6080): 489-93; Nabhani et al., Immunity, 2019, 50(5), 1276-1288). Healthy immune imprinting promotes appropriate immune responses to environmental stimuli, including infection.

[0011] Due to the loss of Bifidobacterium species in the infant gut and low breastfeeding rates, there is a need to provide infants with HMOs and bacteria that utilize HMOs (such as Bifidobacterium longum transitional microbes and / or Bifidobacterium longum infantis subsp.) to support a healthy microbiome for long-term health.

[0012] Furthermore, the means of preventing or treating viral infections are limited. The number of effective antiviral drugs is limited, such as those used to treat HIV and influenza, and the primary method for controlling viral diseases is vaccination, which aims to prevent outbreaks by building immunity against the virus or a family of viruses.

[0013] There is still a need to develop new strategies for preventing and / or reducing the risk of infection in infants or young children, more specifically preventing viral infections (such as respiratory viral infections) and / or reducing the risk of viral infections (such as respiratory viral infections). Summary of the Invention

[0014] The inventors have determined that *Bifidobacterium longum* subspecies (*Bifidobacterium longum* transitional form), an evolutionary branch present in the gut microbiome of mammals, particularly humans, during the transitional feeding period, can have beneficial effects in preventing and / or reducing the risk of infection. For example, the inventors have shown that *Bifidobacterium longum* transitional form can regulate the levels of protective cytokines (e.g., IL-6) and / or short-chain fatty acids (SCFAs); and regulate intestinal barrier permeability, for example, after damage to or deterioration of intestinal barrier permeability.

[0015] The inventors have also surprisingly discovered that early-life synbiotic intervention (a mixture of Bifidobacterium longum transitional microorganisms and human milk oligosaccharides) has beneficial effects in preventing and / or reducing the risk of infection.

[0016] Furthermore, the inventors have surprisingly discovered that early-life synbiotic intervention (a mixture of infantile bifidobacteria and human milk oligosaccharides) provides defense against virus-induced bronchiolitis and promotes immune benefits that persist into adulthood (e.g., assessed by reducing susceptibility to pollution-induced allergic airway inflammation). Specifically, the inventors have demonstrated that synbiotic intervention leads to rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling following virus clearance.

[0017] These findings confirm the use of synbiotics in the protection and treatment of viral infections, particularly viral bronchiolitis, early in life, and reveal the functional benefits of synbiotics in establishing an effective antiviral immune response associated with faster disease resolution. Given that early-life viral respiratory infections represent a major independent risk factor for subsequent asthma, recurrent wheezing, and chronic obstructive pulmonary disease, dietary synbiotic supplementation may also prevent long-term complications associated with early-life viral respiratory infections.

[0018] Therefore, in a first aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0019] In another aspect, the present invention provides a combination of a Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0020] In another aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual. Suitably, the infection is a viral infection.

[0021] In another aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition for promoting long-term immune benefits in an individual.

[0022] In another aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used for i) preventing allergen sensitization and / or reducing the risk of allergen sensitization and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0023] In some embodiments, the composition or combination further comprises *Bifidobacterium longum* subsp. infantis and / or *Bifidobacterium lactis* microorganisms.

[0024] The inventors also surprisingly found that combinations of six HMOs with three probiotics (Bifidobacterium adolescentis (B. l. iuvenis) + Bifidobacterium lactis + Bifidobacterium infantis) significantly increased the levels of indole-3-propionic acid compared to 6 HMOs alone with Bifidobacterium adolescentis or in combination with Bifidobacterium lactis + Bifidobacterium infantis. Indole-3-propionic acid is a microbial-derived metabolite associated with immune benefits (Li et al., Front. Pharmacol., 2021, 12: 769501).

[0025] Therefore, in one aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0026] In another aspect, the present invention provides a combination of a transitional microorganism of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0027] In another aspect, the present invention provides a composition comprising a transitional form of *Bifidobacterium longum*, *Bifidobacterium longum* subsp. *infantica*, and a mixture of HMOs, the HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual. Suitably, the infection is a viral infection.

[0028] In another aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being intended to promote long-term immune benefits in an individual.

[0029] In another aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used for i) preventing allergen sensitization and / or reducing the risk of allergen sensitization and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0030] In some embodiments, the composition is used to prevent an individual from becoming sensitized to an allergen and / or to reduce an individual's risk of becoming sensitized to an allergen.

[0031] In some embodiments, the composition is used to prevent an individual from developing respiratory illnesses and / or to reduce the risk of an individual developing respiratory illnesses.

[0032] In another aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition for preventing an individual from developing asthma and / or reducing the individual's risk of developing asthma.

[0033] In another aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), for preventing an individual from developing asthma and / or reducing the individual's risk of developing asthma.

[0034] The present invention also provides a prebiotic for preventing and / or reducing the risk of infection in an individual by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the gut of an infant or young child, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0035] This invention also provides a combination of a transitional Bifidobacterium longum microorganism and an HMO mixture for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual; wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT). Suitablely, the infection is a viral infection.

[0036] In another aspect, the present invention provides a combination of a Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), for promoting long-term immune benefits in an individual.

[0037] In another aspect, the present invention provides a combination of a transitional Bifidobacterium longum microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), for i) preventing allergen sensitization and / or reducing the risk of allergen sensitization and / or ii) preventing an individual from developing respiratory illnesses and / or reducing the risk of an individual developing respiratory illnesses.

[0038] This invention also provides a combination of a transitional form of *Bifidobacterium longum*, *Bifidobacterium longum* subsp. *infantica*, and an HMO mixture for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual; wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT). Suitably, the infection is a viral infection.

[0039] In another aspect, the present invention provides a combination of a transitional Bifidobacterium longum microorganism, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), which is intended to promote long-term immune benefits in an individual.

[0040] In another aspect, the present invention provides a combination of a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), which is used for i) preventing allergen sensitization and / or reducing the risk of allergen sensitization and / or ii) preventing and / or reducing the risk of respiratory illness in an individual.

[0041] In some implementations, this combination is used to prevent an individual from becoming sensitized to an allergen and / or to reduce an individual's risk of becoming sensitized to an allergen.

[0042] In some implementations, this combination is used to prevent an individual from developing respiratory illnesses and / or to reduce the individual's risk of developing respiratory illnesses.

[0043] In another aspect, the present invention provides a combination of a Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), for the purpose of preventing an individual from developing asthma and / or reducing the individual's risk of developing asthma.

[0044] In another aspect, the present invention provides a combination of a transitional Bifidobacterium longum microorganism, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), for preventing and / or reducing the risk of developing asthma in an individual.

[0045] In some implementations, Bifidobacterium longum infantis subspecies is Bifidobacterium longum infantis subspecies LMG 11588.

[0046] In some implementations, Bifidobacterium longum infantis is a strain that has at least 99.9% average nucleotide identity (ANI) with Bifidobacterium longum infantis LMG 11588.

[0047] In some implementations, Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446.

[0048] In some implementations, Bifidobacterium lactis is a strain with an ANI of at least 99.9% average nucleotide identity (ANI) with Bifidobacterium lactis CNCM 1-3446.

[0049] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, and 3SL.

[0050] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, and 3-FL.

[0051] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, and LNnT.

[0052] In some embodiments, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, 3-FL, and LNnT.

[0053] In some implementations, the HMO mixture is essentially composed of the following substances:

[0054] i. 31% to 82% by weight, preferably 41% to 70% by weight of 2'-FL;

[0055] ii. 10% to 27% by weight, preferably 14% to 23% by weight of LNT;

[0056] iii. 4% to 11% by weight, preferably 6% to 10% by weight of DFL; and

[0057] iv. 9% to 34% by weight, preferably 11% to 29% by weight of a combination of 6SL and 3SL.

[0058] In some implementations, the HMO mixture is essentially composed of the following substances:

[0059] i. 16% to 69% by weight, preferably 22% to 59% by weight of 2'-FL;

[0060] ii. 9% to 24% by weight, preferably 12% to 21% by weight of LNT;

[0061] iii. 2% to 10% by weight, preferably 3% to 8% by weight of DFL;

[0062] iv. 8% to 26% by weight, preferably 11% to 22% by weight of a combination of 6SL and 3SL; and

[0063] v. 8% to 50% by weight, preferably 11% to 43% by weight of 3-FL

[0064] In some implementations, the HMO mixture is essentially composed of the following substances:

[0065] i. 34% to 85% by weight, preferably 40% to 71% by weight of 2'-FL;

[0066] ii. 10% to 40% by weight, preferably 12% to 26% by weight of LNT;

[0067] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL;

[0068] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL, and

[0069] v. 6% to 30% by weight, preferably 7% to 22% by weight of LNnT.

[0070] In some implementations, the HMO mixture is essentially composed of the following substances:

[0071] i. 20% to 60% by weight, preferably 22% to 55% by weight of 2'-FL;

[0072] ii. 4% to 30% by weight, preferably 6% to 20% by weight of LNT;

[0073] iii. 1% to 12% by weight, preferably 2% to 8% by weight of DFL;

[0074] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL;

[0075] v. 10 wt% to 50 wt%, preferably 13 wt% to 46 wt% of 3-FL, and

[0076] vi. 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0077] In some embodiments, the Bifidobacterium longum transitional microorganism: (i) is capable of metabolizing HMO; (ii) preferentially utilizes 3-FL over 2'-FL; (iii) is capable of metabolizing glycan substrates selected from any of the groups listed in Tables 1 to 3; and / or (iv) encodes one or more CAZymes selected from the groups listed in Table 1, preferably wherein the Bifidobacterium longum transitional microorganism also encodes one or more CAZymes selected from Tables 2 and 3.

[0078] In some embodiments, the transitional microorganism of Bifidobacterium longum 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, CMCC-P0001 (ATCC BAA-2753) and any combination thereof.

[0079] In some embodiments, the composition is in the form of a nutritional composition.

[0080] In some implementations, the nutritional composition is selected from infant formula, stage 1 infant formula, stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers or supplements.

[0081] In some implementations, the individual is an infant, toddler, or child. Preferably, the individual is an infant or toddler.

[0082] In some implementations, the individual is an infant.

[0083] In some implementation schemes, the individual is a young child.

[0084] In some implementation schemes, the individual is a child. Attached Figure Description

[0085] Figure 1 - A phylogenetic tree of strains belonging to the Bifidobacterium longum species based on average nucleotide identity (ANI) UPGMA. The scale indicates the percentage of identity at each branch point.

[0086] Figure 2 - Short-chain fatty acids (SCFAs) (i.e., acetate, butyrate, and propionate) were produced by batch fermentation with 3-fucosyllactose (3-FL) over 48 h. A. Thermographs show the z-fractions of NMR peak densities for 3-FL, the TCA cycle, SCFA intermediates, and SCFAs. Fermentation was tested under three conditions: no supplementation, supplementation with a Bifidobacterium longum transitional strain (NCC5004), or supplementation with Bifidobacterium longum subsp. infantis (NCC3089). Each condition was performed using a triplet of infant fecal inoculum. Samples were collected at the start of fermentation (T0), at 24 h (T24), and at the end of fermentation (T48). B. Abundance of Bifidobacterium longum transitional strain (NCC5004) and Bifidobacterium longum subsp. infantis (NCC3089) at T0, T24, and T48 of batch fermentation with 3-FL, measured by strain-specific qPCR.

[0087] Figure 3- Short-chain fatty acids (SCFAs) (i.e., acetate, butyrate, and propionate) were produced by batch fermentation of pea fiber rich in arabinogalactan over 48 hours. A. Thermographs show the z-fractions of the TCA cycle, SCFA intermediates, and NMR peak densities of SCFAs. Fermentation was tested under three conditions: no supplementation, supplementation with a Bifidobacterium longum transition strain (NCC5002), or supplementation with Bifidobacterium longum subsp. infantis (NCC3089). Each condition was performed in triplicate using one infant fecal inoculum. Samples were collected at the start of fermentation (T0), at 24 hours (T24), and at the end of fermentation (T48). B. Abundance of Bifidobacterium longum transition strain (NCC5002) and Bifidobacterium longum subsp. infantis (NCC3089) at T0, T24, and T48 of batch fermentation with pea fiber, measured by strain-specific qPCR.

[0088] Figure 4 - Interleukin-6 (IL-6) was produced by monocytes after training with different probiotics and subsequent stimulation with LPS. The bars represent the median IL-6 response, and the dashed lines represent the IL-6 levels in untrained monocytes.

[0089] Figure 5 - Incubate Caco-2 monolayers with transitional Bifidobacterium longum NCC5002 (black line), Bifidobacterium lactis NCC2818 (gray line) or a vector (dashed line) for 24 hours, then stimulate with pro-inflammatory cytokines to measure transmembrane resistance (TEER).

[0090] Figure 6 - After incubation with transitional Bifidobacterium longum NCC5002 (black line), Bifidobacterium lactis NCC2818 (gray line), or a vector (dashed line), followed by stimulation with pro-inflammatory cytokines, the micromolecular flux through the Caco-2 cell monolayer was measured.

[0091] Figure 7 - A diagram of glycoside hydrolases (GH) and polysaccharide lyases (PL) in the genome of the Bifidobacterium longum clade. The heatmap shows the presence (light color) and absence (dark color) of GH and PL genes, and the size of the circles represents the number of these genes in each genome of a specific strain.

[0092] Figure 8 - Pectin (sugar beet) and arabinogalactan (larch wood) promote the growth of Bifidobacterium longum transitional strain NCC5001 in a complex gut microbiota community. P **** < 0.0001, *** <0.001, ** <0.01, * <0.05, one-way ANOVA with uncorrected Fisher LSD.

[0093] Figure 9-Arabinogalactan (larch wood) and starch (potato) promote the growth of Bifidobacterium longum transitional strain NCC5002 in a complex gut microbiota community. P **** < 0.0001, *** <0.001, ** <0.01, * <0.05, one-way ANOVA with uncorrected Fisher LSD.

[0094] Figure 10 - Representative CAZyme sequences

[0095] Figure 11 - A schematic diagram of the organization of genes involved in the degradation and metabolism of fucosylated human milk oligosaccharides in transitional strains of Bifidobacterium longum compared to Bifidobacterium longum subsp. infantum ATCC 15697 and Bifidobacterium kanamycin DSM 21854. Values ​​represent the percentage (%) of identity between different genes.

[0096] Figure 12 - Growth of Bifidobacterium longum transitional strains and Bifidobacterium longum infant subspecies LMG 11588 on glucose, 2'-FL, or 3-FL as the sole carbon source (0.5% final). Significant differences in 2'-FL and 3-FL growth between each strain were calculated using one-way ANOVA followed by Sidak multiple comparison tests (ns = not significant, *p < 0.05, **p < 0.01).

[0097] Figure 13 - The growth ratio of 3'-FL to 2'-FL of Bifidobacterium longum transitional strain and Bifidobacterium longum infant subspecies LMG 11588.

[0098] Figure 14 - A schematic diagram of a model of allergic airway inflammation exacerbated by viral airway infection and pollution in early life.

[0099] Figure 15 - Early life nutritional intervention using synbiotics (Bifidobacterium infantis + 6HMO) can reduce virus-induced lung lesions.

[0100] Figure 16 and Figure 17 - Early life nutritional interventions using synbiotics (Bifidobacterium infantis + 6HMO) can promote immune benefits that last into adulthood.

[0101] Figure 18 - Early life nutritional intervention using synbiotics (Bifidobacterium infantis + 5HMO) can promote antiviral immune responses and reduce virus-induced lung inflammation during peak infection periods.

[0102] Figure 19- Schematic diagram of the experimental setup for a preclinical model used to test the efficacy of a transitional strain of Bifidobacterium longum in an infection model.

[0103] Figure 20 - The kinetics of body weight changes in mice following airway virus infection with pneumonia virus from 0 dpi to 10 dpi. Each point represents the mean, and error bars represent the standard error of the mean. N=8 / experimental group. Statistical differences between groups were calculated using two-way ANOVA. *，£ p-value < 0.05 **，££ p-value < 0.005 $$$,$$$$ p-value < 0.0001.

[0104] Figure 21 The effects of combinations of *Bifidobacterium adolescentis* (Bj) with *Bifidobacterium infantis* (Bi) and six HMOs on enhancing microbial-derived metabolites associated with immune benefits were tested. The effects of six HMOs combined with (i) *Bifidobacterium infantis* and *Bifidobacterium lactis* (BlBi), (ii) *Bifidobacterium adolescentis* (Bj), or (iii) combinations of *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium lactis* (BjBlBi) were tested, where N = 12 / experimental group. Statistical differences between groups were calculated using a nonparametric ANOVA (Friedman's test), and nominal p-values ​​were reported. Detailed Implementation

[0105] Unless otherwise specified, all percentages are by weight.

[0106] As used herein, the terms “about” or “approximately” when referring to measurable values ​​such as parameters, quantities, durations, etc., are intended to cover variations in a particular value and variations arising from that particular value, such as variations in a particular value and variations arising from that particular value being 1 / -10% or less, 1 / -5% or less, 1 / -1% or less, and + / 0.1% or less, provided that such variations are suitable for making in the disclosed invention. It should be understood that the values ​​referred to by the modifiers “about” or “approximately” are themselves specifically and preferably disclosed.

[0107] The terms “subject,” “individual,” and “patient” are used interchangeably to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets.

[0108] The term "infant" refers to a human individual under 12 months of age or a non-human animal of equivalent age.

[0109] As used in this article, the term "toddler" or "walking infant" may refer to an individual between 12 months and 5 years of age. Appropriately, "toddler" may refer to a non-human animal of equivalent age.

[0110] The term "child" refers to a human child between the ages of three and twelve. Preferably, the term "child" refers to a child between the ages of three and six.

[0111] "Premature infant" or "premature person" refers to an infant or toddler born before full term. Generally, it refers to an infant or toddler born before 36 weeks of gestation.

[0112] The terms "small for gestational age" or "SGA" refer to an infant or toddler whose size is smaller than the normal standard for birth at gestational age (most commonly defined as a weight below the 10th percentile for gestational age). In some implementations, SGA may be associated with intrauterine growth restriction (IUGR), a condition in which a fetus is unable to reach its potential size.

[0113] The term "low birth weight" should be understood as a birth weight of less than 2500g.

[0114] The terms "supplementary feeding period," "supplementary period," "transition period," "transitional feeding period," and "weaning period" are used interchangeably and refer to the period during which milk (breast milk or formula) is replaced by other foods in an infant's or toddler's diet. This typically involves a gradual transition from exclusive breastfeeding (breast milk or formula) to a mixed diet that includes milk and / or solid foods. The transition period varies depending on the infant or toddler, but is generally from about 4 months to about 18 months of age, such as from about 6 months to about 18 months, but in some cases it can extend to about 24 months or longer. For humans, weaning typically begins between 4 and 6 months of age and is considered complete once the infant and / or toddler is no longer breastfed or formula-fed, usually around 24 months of age. In some implementations, the weaning period is from 4 to 24 months.

[0115] The term "composition" or "nutritional composition" refers to any kind of composition or formulation that provides nutritional benefits to an individual and is safe for consumption by humans or animals. A nutritional composition may be in solid (e.g., powder), semi-solid, or liquid form and may contain one or more macronutrients, micronutrients, food additives, water, etc. For example, a nutritional composition may contain the following macronutrients: protein sources, lipid sources, carbohydrate sources, and any combination thereof. Furthermore, a nutritional composition may contain the following micronutrients: vitamins, minerals, fiber, phytochemicals, antioxidants, prebiotics, probiotics, bioactive agents, metabolites (e.g., butyrate, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA)), and any combination thereof. The composition may also contain food additives, such as stabilizers (when provided in liquid or solid form) or emulsifiers (when provided in liquid form). The amounts of various components (e.g., oligosaccharides) may be expressed in g / 100g of composition on a dry weight basis when the composition is in solid form (e.g., powder), or in g / L of composition concentration when the composition is in liquid form (the latter also covers liquid compositions that can be obtained by reconstituted powder in a liquid (such as milk, water), such as reconstituted infant formula or stage 2 infant formula or infant cereal products or any other formulation designed to provide nutrition for infants or young children). Typically, nutritional compositions are formulated for oral, enteral, parenteral, or intravenous administration, and generally include one or more nutrients selected from a variety of nutrients: lipid or fat sources, protein sources, and carbohydrate sources. Preferably, the nutritional composition is intended for oral administration.

[0116] In this specific implementation, the nutrient composition is a "synthetic nutrient composition." The expression "synthetic nutrient composition" means a mixture obtained by chemical and / or biological methods.

[0117] As used herein, the term "infant formula" refers to a food intended for specific nutritional purposes in the first few months after birth and which meets the nutritional needs of such infants (in accordance with Article 2(c) of European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 concerning infant formula and follow-up formula). It also refers to nutritional compositions intended for infants, as defined in the Codex Alimentarius Commission (STAN 72-1981) and in provisions concerning infant specialties (including foods for specific medical purposes). The term "infant formula" encompasses both "Stage 1 infant formula" and "Stage 2 infant formula" or "follow-up formula."

[0118] Stage 2 infant formula or follow-up formula is introduced starting from the 6th month. Infant formula constitutes the main liquid component of this group's gradually diversifying diet.

[0119] The term "infant food" refers to food designed for specific nutritional purposes for infants or young children during the first few years of their lives.

[0120] The term "infant cereal composition" refers to a food intended for specific nutritional purposes for infants or young children during the first few years of their lives.

[0121] The term "GUM" refers to a milk-based beverage that is typically fortified with vitamins and minerals and is intended for use by toddlers or children.

[0122] The term "fortifier" refers to a liquid or solid nutritional composition suitable for fortifying or mixing with human milk, infant formula, growing milk, or human breast milk fortified with other nutrients. Therefore, a fortifier may be administered after being dissolved in human breast milk, infant formula, growing milk, or human breast milk fortified with other nutrients, or it may be administered as a standalone composition. When administered as a standalone composition, a milk fortifier may also be identified as a "supplement".

[0123] The term "metabolism" is used here to refer to the ability of a substrate to be broken down, adsorbed, and / or utilized by microorganisms. For example, a substrate may promote and / or contribute to the growth and / or survival of microorganisms.

[0124] Suitablely, the term "capable of metabolizing glycan substrates" may mean that a Bifidobacterium longum transitional strain encodes at least one CAZyme capable of utilizing glycan substrates. For example, the CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within the glycan substrate. Suitablely, the Bifidobacterium longum transitional strain may encode at least one, at least two, at least three, at least four, or at least five CAZymes capable of utilizing glycan substrates. Suitablely, the term "capable of metabolizing glycan substrates" may mean that the glycan substrate (or fibers or components containing the glycan substrate) 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 can be determined, for example, by measuring the abundance of 16S rDNA using PCR methods. An exemplary assay for measuring the growth of the Bifidobacterium longum transitional strain in the presence of a glycan substrate (e.g., in fiber form) is provided in this example.

[0125] Suitablely, the glycan substrate can be metabolized by *Bifidobacterium longum* transitional microorganisms. Suitablely, the glycan substrate may be able to promote the growth and / or survival of *Bifidobacterium longum* transitional strains. Glycan substrates that can promote the growth and / or survival of *Bifidobacterium longum* transitional strains can be determined, for example, by an anaerobic culture of *Bifidobacterium longum* transitional strains with the glycan substrate to be tested. The growth and / or survival of *Bifidobacterium longum* transitional strains can be determined by measuring bacterial cell number, cell density (e.g., by optical density measurement), and / or 16S rDNA abundance, for example using PCR methods. Exemplary assays for measuring the growth of *Bifidobacterium longum* transitional strains in the presence of glycan substrates are provided in the examples. Compared to the number of *Bifidobacterium longum* transitional strains in control anaerobic cultures without HMOs, a polysaccharide substrate that promotes the growth and / or survival of *Bifidobacterium longum* transitional strains can increase the number of *Bifidobacterium longum* transitional bacteria in anaerobic cultures by at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 100%. Suitablely, a polysaccharide substrate that promotes the growth and / or survival of *Bifidobacterium longum* transitional strains can increase the number of *Bifidobacterium longum* transitional bacteria in anaerobic cultures by a statistically significant amount (e.g., p < 0.05 as determined by one-way ANOVA).

[0126] "Glycan substrate" refers to a glycan that can be metabolized by microorganisms. A glycan substrate can be, for example, a glycoconjugate, oligosaccharide, or polysaccharide. Glycan conjugates can include N-linked or O-linked glycans within glycoproteins and proteoglycans or glycolipids. For example, O-linked glycans can include proteins or peptides in which the oxygen atom of a serine or threonine residue is linked to a monosaccharide, oligosaccharide, or polysaccharide, as in the case of glycosaminoglycans (GAGs). Other examples of "glycan substrates" are cellulose, a glycan composed of β-1,4-linked D-glucose, and chitin, a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. As used herein, "glycan substrate" includes, for example, oligosaccharides and polysaccharides.

[0127] "Oligosaccharide" can refer to a carbohydrate having more than two but relatively few monosaccharide units (typically three, four, five, six, and up to ten). Exemplary oligosaccharides include, but are not limited to, fructooligosaccharides, galactooligosaccharides (raffinose, stachyose, verbascose), maltodextrin, gentiosaccharides, cellulose oligosaccharides, lactoosaccharides (e.g., those found in mammary gland secretions), isomaltooligosaccharides, lactulose oligosaccharides, mannan oligosaccharides, melibiose-derived oligosaccharides, pectin oligosaccharides, and xylooligosaccharides.

[0128] The term "polysaccharide" can refer to a carbohydrate having more than ten monosaccharide units. Exemplary polysaccharides include, but are not limited to, starch, arabinogalactan, laminarin, golden kelp laminarin, xylan, arabinoxylan, mannan, fucoidan, and galactomannan. It should be understood that there is no precise boundary or distinction between the terms oligosaccharide and polysaccharide, and such distinction is not necessary for carrying out this invention.

[0129] The term "glycosaminoglycan" (GAG), or mucopolysaccharide, refers to a long, linear polysaccharide composed of repeating disaccharide units (i.e., two sugar units). The repeating disaccharide units consist of aldoses and amino sugars, with galactose present at the aldose position, except for keratin. GAGs are classified into four groups based on their core disaccharide structure.

[0130] As used in this article, "mucins" can refer to a family of high-molecular-weight, highly glycosylated proteins (glycoconjugates). A key characteristic of mucins is their ability to form gels; therefore, they are key components in most gel-like secretions, playing roles ranging from lubrication to cell signaling to the formation of mechanical or chemical barriers.

[0131] The term "HMO" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, suggesting that their important functions may not be directly related to their calorific value. It has been specifically noted in the art that these carbohydrates play a crucial role in early infant and toddler development, such as the maturation of the immune system. Many different types of HMOs have been found in human milk. Each individual oligosaccharide is based on a variety of combinations of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine with these molecules, resulting in a large number of diverse oligosaccharides in human milk; over 130 such structures have been identified to date. Almost all oligosaccharides have a lactose molecule at the reducing end, and the non-reducing end is occupied by sialic acid and / or fucose (if present). Based on the presence of fucose and sialic acid in the oligosaccharide structure, HMOs can be classified as unfucosylated (neutral) or fucosylated (neutral) and sialylated (acidic) and non-sialylated molecules, respectively.

[0132] The term "fucosylated oligosaccharide" refers to oligosaccharides containing fucose residues. These oligosaccharides are neutral. Some examples are 2'-fucosylvose (2'-FL), 3-fucosylvose (3-FL), difucosylvose (DiFL), lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasaccharide, lactose-N-difucohexasaccharide I, fucosylvose-N-hexasaccharide, fucosylvose-N-neohexose, difucosylvose-N-hexasaccharide I, difucosylvose-N-neohexose II, and any combination thereof. Fucosylated oligosaccharides represent the largest portion of human milk, with 2'-FL comprising up to 30% of the total HMOs. Fucosylated oligosaccharides are believed to reduce the risk of infection and inflammation and promote the growth and metabolic activity of certain symbiotic microorganisms, thereby reducing inflammatory responses.

[0133] The term "N-acetylated oligosaccharide" encompasses both "N-acetyl-lactoside" and "oligosaccharides containing N-acetyl-lactoside." Such oligosaccharides are neutral oligosaccharides having N-acetyl-lactoside residues. Suitable examples are LNT (lactose-N-tetrasaccharide), para-lactose-N-neohexose (para-LNnH), LNnT (lactose-N-neohexose), DSLNT (disialyllactose-N-tetrasaccharide), and any combination thereof. Other examples are lactose-N-hexasaccharide, lactose-N-neohexose, para-lactose-N-hexasaccharide, para-lactose-N-neohexose, lactose-N-octasaccharide, lactose-N-neohexose, isol-lactose-N-octasaccharide, para-lactose-N-octasaccharide, and lactose-N-decansaccharide.

[0134] The expressions “at least one fucoidylated oligosaccharide” and “at least one N-acetylated oligosaccharide” should be understood as “at least one type of fucoidylated oligosaccharide” and “at least one type of N-acetylated oligosaccharide”.

[0135] The term "sialylated oligosaccharide" refers to an oligosaccharide having charged sialic acid residues. This oligosaccharide is acidic. Some examples are 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), and sialyllactose-N-tetrasaccharide (Lst, such as Lst-a, Lst-b, or Lst-c).

[0136] Appropriately, the term "capable of metabolizing HMOs" may mean that a Bifidobacterium longum transitional strain encodes at least one CAZyme capable of utilizing HMOs. For example, this CAZyme may be capable of catalyzing the hydrolysis of glycosidic bonds within the HMO. Appropriately, the Bifidobacterium longum transitional strain may encode at least one, at least two, at least three, at least four, or at least five CAZymes capable of utilizing HMOs. Appropriately, the term "capable of metabolizing HMOs" may mean that the HMO promotes 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 can be determined, for example, by measuring the abundance of 16S rDNA using PCR methods.

[0137] The term fiber is used herein to refer to carbohydrates that are indigestible in humans or animals. Such fibers in relation to carbohydrates are also discussed herein. Suitablely, fiber may be fermented by one or more Bifidobacterium longum transitional microorganisms provided in the uses or compositions of this invention and / or fermented in one or more regions of the gastrointestinal tract of an organism such as a human or non-human animal. In the context of this invention, as used herein, the expressions “fiber” or “multiple fibers” or “dietary fiber” or “various dietary fibers” refer to the portion of a plant-derived food that is indigestible in the small intestine, comprising two main components: soluble fiber, which dissolves in water; and insoluble fiber. Mixtures of fibers are included within the scope of the terms mentioned above. Soluble fiber readily ferments in the colon into gases and physiologically active byproducts and may be prebiotic and viscous. Insoluble fiber is insoluble in water, metabolically inert and bloated, or it may be prebiotic and fermented in the large intestine. Chemically, dietary fiber consists of carbohydrate polymers having three or more monomeric units that are not hydrolyzed by endogenous enzymes in the small intestine, such as arabinoxylan, cellulose, and many other plant components such as resistant starch, resistant dextrin, inulin, lignin, chitin, pectin, arabinogalactan, arabinogalactan, galactan, xylan, β-glucan, and oligosaccharides. Non-limiting examples of dietary fiber include: prebiotic fibers such as fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), fruit fiber, plant fiber, cereal fiber, and resistant starches such as high-amylose corn starch.

[0138] As used herein, “added fiber” or “added dietary fiber” means an ingredient that consists primarily or entirely of fiber added to a nutritional supplement composition, and the amount of fiber in which it constitutes the total fiber content of the composition. The total fiber content of a nutritional supplement composition is provided by the sum of the amount of fiber naturally present in the ingredients used in the formulation (e.g., from whole grain flour) and the amount of added fiber.

[0139] The term "prebiotic" refers to non-digestible carbohydrates that exert a beneficial effect on the host by selectively stimulating the growth and / or activity of beneficial healthy bacteria (such as Bifidobacteria in the human colon) (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995; 125:1401-12).

[0140] The term "probiotics" refers to a microbial cell preparation or microbial cell component that has a beneficial effect on the health or well-being of the host (Salminen S, Ouwehand A. Benno Y. et al., "Probiotics: how should they be defined" Trends Food Sci. Technol. 1999: 10 107-10). The microbial cells according to the present invention are typically bacteria.

[0141] The term "cfu" should be understood as colony-forming unit.

[0142] The gut microbiota is the composition of microorganisms (including bacteria, archaea, and fungi) that live in the digestive tract.

[0143] The term “gut microbiome” can include the “gut microbiota” and their “site of activity”, which can include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolites (signaling molecules, toxins, organic and inorganic molecules), and molecules produced by the coexisting host and structured by the surrounding environmental conditions (Berg, G. et al., 2020. Microbiome, 8(1), pp. 1-22).

[0144] The term "SCFA" refers to short-chain fatty acids.

[0145] The statement "increased SCFA production" means that individuals fed the nutritional composition according to the invention have higher levels of systemic and / or colonic SCFAs compared to those fed the standard composition. SCFA production can be measured using techniques known to those skilled in the art, such as gas-liquid chromatography.

[0146] In this article, the term "gastrointestinal tract" includes the mouth, pharynx, esophagus, stomach, small intestine, large intestine, rectum, and anus. The term "intestine" includes the small intestine, large intestine, and rectum.

[0147] In this article, the term "respiratory tract" refers to the passage formed by the nose, nasal cavity, pharynx, larynx, trachea, bronchi and lungs through which air passes during respiration.

[0148] Composition

[0149] The inventors have surprisingly discovered that the combination of Bifidobacterium longum transitional microorganisms and HMO mixtures consisting of 2'-fucosylated lactose (2FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL), as well as optional 3-fucosylated lactose (3FL) and / or lactose-N-neotetrasaccharide (LNnT) provides beneficial effects in early life for preventing and / or reducing the risk of infection.

[0150] The specific combination of Bifidobacterium longum transitional microorganisms and HMO mixtures consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT) is surprisingly effective in preventing and / or reducing the risk of infection. Therefore, in one aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0151] In another aspect, the present invention provides a nutritional composition comprising a combination of Bifidobacterium longum transitional microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0152] In another aspect, the present invention provides a combination comprising or consisting of: a transitional Bifidobacterium longum microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT). Preferably, the combination consists of a transitional Bifidobacterium longum microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0153] In one embodiment, the composition further comprises *Bifidobacterium longum* subsp. infantis and / or *Bifidobacterium lactis*. Suitably, the composition further comprises *Bifidobacterium longum* subsp. infantis. Suitably, the composition further comprises *Bifidobacterium lactis*. Preferably, the composition further comprises *Bifidobacterium longum* subsp. infantis and *Bifidobacterium lactis*.

[0154] The inventors also surprisingly discovered that symbiotic intervention with a combination of 6HMO and three probiotic strains (i.e., *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium lactis*) increased the level of the metabolite indole-3-propionic acid compared to 6HMO alone with *Bifidobacterium adolescentis* or 6HMO in combination with two strains, *Bifidobacterium lactis* and *Bifidobacterium infantis*.

[0155] Therefore, in one aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0156] In another aspect, the present invention provides a nutritional composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0157] In another aspect, the present invention provides a combination comprising or consisting of a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT). Preferably, the combination consists of a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0158] In one embodiment, the composition further comprises Bifidobacterium lactis.

[0159] In one embodiment, the combination comprises or consists of a transitional Bifidobacterium longum microorganism, an HMO mixture, Bifidobacterium longum subsp. infantis, and Bifidobacterium lactis, wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0160] Preferably, the combination consists of a transitional Bifidobacterium longum microorganism and an HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), as well as optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0161] Preferably, the combination comprises *Bifidobacterium longum* subsp. *infantii*. Suitably, the combination comprises *Bifidobacterium lactis*. Preferably, the combination comprises both *Bifidobacterium longum* subsp. *infantii* and *Bifidobacterium lactis*.

[0162] Bifidobacterium longum transitional microorganism

[0163] Previously, subspecies of *Bifidobacterium longum* have been identified within the gut microbiome of mammals, particularly humans, during the transitional feeding period. *Bifidobacterium longum* belonging to this clade are referred to herein as *Bifidobacterium longum transitional* (B. longum transitional) and are also known in the art as *Bifidobacterium longum* youth subspecies. Transitional strains of Bifidobacterium longum, NCC 5000, NCC 5001, NCC 5002, NCC 5003 and NCC 5004, were deposited on May 11, 2021, at the National Center for the Collection of Microbial Cultures (CNCM), Pasteur Institute (INSTITUTPASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) under the Budapest Treaty, with accession numbers CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687, respectively. U.S. Provisional Patent Application 63 / 216127 indicates that the relative abundance of *Bifidobacterium longum* transitional microbes is greater than that of either *Bifidobacterium infantis* or *Bifidobacterium longum* subsp. *longum* during the transition feeding period (e.g., weaning). Indeed, the relative abundance of *Bifidobacterium longum* subsp. *inf.* decreases at the beginning of the transition feeding period and continues until its end, coinciding with the increase in the abundance of *Bifidobacterium longum* subsp. *longum*. Vatanen et al. demonstrated that this unique *Bifidobacterium longum* clade expands 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).

[0164] Suitable, the Bifidobacterium longum transitional microorganism may encode one or more CAZymes selected from the groups listed in Table 1. Suitable, the Bifidobacterium longum transitional microorganism may encode one or two CAZymes selected from the groups listed in Table 1.

[0165] Appropriately, the Bifidobacterium longum transitional microorganism encodes at least one CAZyme selected from the groups listed in Table 1 and one or more CAZymes selected from the groups listed in Tables 2 and 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 Tables 2 and 3.

[0166] Appropriately, the transitional microbial code for Bifidobacterium longum is (i) at least one CAZyme selected from the groups listed in Table 1 and (ii) each of the CAZymes listed in Table 3 or except GH5_44 of the CAZymes listed in Table 3.

[0167] Appropriately, the transitional microbial code for Bifidobacterium longum is (i) at least one CAZyme selected from the groups listed in Table 1 and (ii) each of the CAZymes listed in Table 3 or except GH25 of the CAZymes listed in Table 3.

[0168] Suitablely, the Bifidobacterium longum transitional microorganism does not encode one or more of the CAZymes listed in Table 4. Suitablely, the Bifidobacterium longum transitional microorganism does not encode any of the CAZymes listed in Table 4.

[0169] The *Bifidobacterium longum* transitional microorganism of the present invention advantageously carries a gene encoding CAZyme, thereby allowing the cleavage of sialic acid residues from glycans such as sialylated oligosaccharides, glycoproteins, and glycolipids. This allows for the efficient utilization of sialylated oligosaccharides present in breast milk at weaning and can therefore participate in the proper development of the gut microbiome in infants and / or young children. It may also help prevent the presence of intestinal pathogens.

[0170] In some implementations, the transitional microorganism of Bifidobacterium longum includes a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene that has at least 60% identity with the BLON_2348 gene present in Bifidobacterium longum infant subspecies ATCC15697.

[0171] In some implementations, the transitional microorganism of Bifidobacterium longum includes a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene that has approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the BLON_2348 gene present in Bifidobacterium longum subsp. infantis ATCC15697.

[0172] In some embodiments, the transitional microorganism of Bifidobacterium longum includes a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene having 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 73%, at least 74%, 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%, or at least 99% identity with the BLON_2348 gene present in Bifidobacterium longum subsp. infantis ATCC15697.

[0173] In some embodiments, the transitional Bifidobacterium longum microorganism 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 has at least 60% identity with 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 has at least 60% identity with the BLON_2336 gene present in Bifidobacterium longum subsp. infantis ATCC 15697.

[0174] In some embodiments, the transitional microorganism of Bifidobacterium longum used according to the present invention includes a sialidase or neuraminidase family 33 (GH33, sialidase or neuraminidase) gene that has at least 60% identity with the BLON_2348 gene present in Bifidobacterium longum infant subspecies ATCC 15697.

[0175] In some embodiments, the Bifidobacterium longum transitional microorganism preferentially utilizes 3-fucosyllactose (3-FL) instead of 2'-fucosyllactose (2'-FL). Suitablely, the Bifidobacterium longum transitional microorganism may preferentially utilize 3-FL instead of 2'-FL at a ratio between 0.1:5, preferably between 0.1:4, and more preferably between 0.2:2.

[0176] In some embodiments, the Bifidobacterium longum transitional microorganism used in the present invention utilizes 3-FL more efficiently than 2'-FL, as demonstrated by better growth, for example as shown in this example.

[0177] In some embodiments, the *Bifidobacterium longum* transitional microorganism has at least 96% 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. In some embodiments, the *Bifidobacterium longum* transitional microorganism has at least 96% 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). Bifidobacterium longum strains comprising BAA-2753 and any combination thereof have an ANI of approximately 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 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%. In some embodiments, the transitional microorganism of *Bifidobacterium longum* and 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 have a content of at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, and at least 97.8%. ANI of at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, and at least 99.9%.

[0178] 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, and CNCM I-5687 and any combination thereof. Suitably, the Bifidobacterium longum transitional microorganism encodes one or more CAZymes selected from the groups listed in Table 1 and has at least 98% 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, and CNCM I-5687 and any combination thereof. Suitable, the Bifidobacterium longum transitional microorganism encodes one or more GH31 CAZymes and has at least 98% 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, and CNCM I-5687 and any combination thereof. In some embodiments, the Bifidobacterium longum transitional microorganism has about 98% to 100% 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, and CNCM I-5687 and any combination thereof. In some embodiments, the transitional microorganism of *Bifidobacterium longum* and at least one *Bifidobacterium longum* strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687 and any combination thereof have 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%. In some embodiments, the Bifidobacterium longum transitional microorganism and at least one Bifidobacterium longum strain selected from the group consisting of CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687 and any combination thereof have 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%.

[0179] Methods for microbial genome sequencing are known in the art (see, for example, Segerman; Front. Cell. Infect. Microbiol.; 2020; 10; Article 527102 & Donkor; Genes; 2013;4(4); 556-572). Metagenomic methods can be used by way of example. For example, shotgun sequencing data can be used for suitable metagenomic methods. Suitable metagenomic methods are known in the art and include, for example, MetaPhlAn 3.0 (see Beghini et al.; eLife 2021; 10: e65088; https: / / huttenhower.sph.harvard.edu / metaphlan) for metagenomic sequencing.

[0180] "Average Nucleotide Identity (ANI)" is a term in this field referring to a distance-based method of characterizing species based on pairwise comparisons of genome sequences, and is a computerized alternative to the traditional DNA-DNA hybridization (DDH) technique already used for the phylogenetic definition of species (Goris et al., 2007, "DNA-DNA hybridization values ​​and their relationship to whole-genome sequence similarities", Int. J. Syst. Evol. Microbiol. 57: 81-91). Based on DDH, strains with a correlation greater than 70% are considered to belong to the same species (see, for example, Wayne et al., 1987, Report of the Ad-Hoc-Committee on Reconciliation of Approaches to Bacterial Systematics. Int J Syst Bacteriol 37: 463-464). ANI is similar to the aforementioned 70% DDH cutoff value and can be used for species characterization. ANI has been evaluated in multiple laboratories and has become the gold standard for species demarcation (see, for example, Kim et al., 2014, “Towards ataxonomic coherence between average nucleotide identity and 16S rRNA genesequence similarity for species demarcation of prokaryotes”, Int. J.Syst.Evol.Micr.64: 346-351; Richter et al., 2009, “Shifting the genomic goldstandard for the prokaryotic species definition”, P Natl Acad Sci USA 106:19126-19131; and Chan et al., 2012, “Defining bacterial species in the genomic era:insights from the genus Acinetobacter”, Bmc.Microbiol.12)).

[0181] The ANI (Average Nucleus Indices) of shared genes between two strains is a powerful tool for comparing genetic relationships between strains, and approximately 95% of ANI values ​​correspond to the 70% DNA-DNA hybridization criterion used to define species. See, for example, Konstantinidis and Tiedje, Proc Natl Acad Sci USA, 102(7):2567-72 (2005); and Goris et al., Int Syst Evol Microbiol.57(Pt 1):81-91 (2007). The ANI between two bacterial genomes is calculated by pairwise comparisons of all sequences common to both strains and can be determined, for example, using any of a number of publicly available ANI tools, including but not limited to OrthoANI with usearch (Yoon et al., Antonie van Leeuwenhoek 110:1281-1286 (2017)); ANI calculator, JSpecies (Richter and Rossello-Mora, Proc Natl Acad Sci USA 106:19126-19131 (2009)); and JSpeciesWS (Richter et al., Bioinformatics 32:929-931 (2016)). Other methods for determining the ANI between two genomes are known in the art. See, for example, Konstantinidis, KT and Tiedje, JM, Proc. Natl. Acad. Sci. USA, 102: 2567-2572 (2005); and Varghese et al., Nucleic Acids Research, 43(14):6761-6771 (2015). In one specific implementation, the ANI between two bacterial genomes can be determined, for example, by averaging the nucleotide identity of homologous genes identified as bidirectional best hits (BBHs). Protein-coding genes in the first genome (genome A) and the second genome (genome B) are compared at the nucleotide level using a similar search tool, such as NSimScan (Novichkov et al., Bioinformatics 32(15): 2380-23811 (2016)). The results are then filtered to show at least 70% sequence identity for BBHs that retain only those shorter than 70% of the shorter sequence in each BBH pair. The ANI between genome A and genome B is defined as the percentage of identity multiplied by the sum of the alignment lengths of all BBH genes, then divided by the sum of the lengths of the BBH genes. These and ANI determination techniques are known in the art.

[0182] Appropriately, the following groups of Bifidobacterium longum transitional microorganisms were selected as reference genomes for comparison with the microbial genomes: CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686 and CNCM I-5687.

[0183] Appropriately, the following groups of Bifidobacterium longum microbial representatives were selected as reference genomes for comparison with microbial genomes: CNCM I-5683, CNCM I-5684, CNCM I-5685, CNCM I-5686, CNCM I-5687 and CMCC-P0001 (ATCC BAA-2753).

[0184] The genome sequences of the transitional strains of *Bifidobacterium longum* NCC 5000 (CNCM I-5683), NCC 5001 (CNCM I-5684), NCC5002 (CNCM I-5685), NCC 5003 (CNCM I-5686), and NCC 5004 (CNCM I-5687) can be obtained through the Joint Genome Project (JGI) research number Gs0156595 (… https: / / genome.jgi.doe.gov / portal / The analysis plan number and taxonomic unit number for each genome are as follows:

[0185] In some embodiments, the Bifidobacterium longum transitional microorganism used in this invention is isolated from humans.

[0186] In some other implementations, the transitional microorganism of Bifidobacterium longum is not a subspecies of Bifidobacterium longum or Bifidobacterium longum infantis.

[0187] Suitable, the Bifidobacterium longum transitional microorganism is provided in the form of a probiotic. Suitable, the Bifidobacterium longum transitional microorganism is provided in the composition.

[0188] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacterium longum transitional microorganism, preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 A transitional microorganism of *Bifidobacterium longum* between CFUs. Appropriately, the transitional microorganism of *Bifidobacterium longum* is at least about 10... 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8Administer CFU / day to individuals. Appropriately, Bifidobacterium longum transitional microorganisms are administered at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 CFU / day or less is administered to the individual.

[0189] In one implementation, the Bifidobacterium longum transitional microorganism is alive.

[0190] HMO mixture

[0191] The compositions or combinations of the present invention comprise HMO mixtures.

[0192] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, and 3SL.

[0193] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, and 3-FL.

[0194] In some implementations, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, and LNnT.

[0195] In some embodiments, the HMO mixture consists of 2'-FL, DFL, LNT, 6SL, 3SL, 3-FL, and LNnT.

[0196] In one embodiment, the HMO mixture contains 16 wt% to 85 wt% of 2'-FL. Suitably, the HMO mixture may contain 31 wt% to 82 wt%, preferably 41 wt% to 70 wt% of 2'-FL. Suitably, the HMO mixture may contain 16 wt% to 69 wt%, preferably 22 wt% to 59 wt% of 2'-FL. Suitably, the HMO mixture may contain 34 wt% to 85 wt%, preferably 40 wt% to 71 wt% of 2'-FL. Suitably, the HMO mixture may contain 20 wt% to 60 wt%, preferably 22 wt% to 55 wt% of 2'-FL.

[0197] In one embodiment, the HMO mixture contains 4% to 40% by weight of LNT. Suitably, the HMO mixture may contain 10% to 27% by weight, preferably 14% to 23% by weight of LNT. Suitably, the HMO mixture may contain 9% to 24% by weight, preferably 12% to 21% by weight of LNT. Suitably, the HMO mixture may contain 10% to 40% by weight, preferably 12% to 26% by weight of LNT. Suitably, the HMO mixture may contain 4% to 30% by weight, preferably 6% to 20% by weight of LNT.

[0198] In one embodiment, the HMO mixture contains 1% to 14% by weight of DFL. Suitably, the HMO mixture may contain 4% to 11% by weight, preferably 6% to 10% by weight of DFL. Suitably, the HMO mixture may contain 2% to 10% by weight, preferably 3% to 8% by weight of DFL. Suitably, the HMO mixture may contain 4% to 14% by weight, preferably 5% to 10% by weight of DFL. Suitably, the HMO mixture may contain 1% to 12% by weight, preferably 2% to 8% by weight of DFL.

[0199] In one embodiment, the HMO mixture comprises 6SL and 3SL in an amount combined from 7 wt% to 34 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 9 wt% to 34 wt%, preferably from 11 wt% to 29 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 8 wt% to 26 wt%, preferably from 11 wt% to 22 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 9 wt% to 31 wt%, preferably from 10 wt% to 28 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL in an amount combined from 7 wt% to 23 wt%, preferably from 8 wt% to 22 wt%.

[0200] In one embodiment, the HMO mixture contains 10% to 50% by weight of 3-FL. Suitably, the HMO mixture may contain 18% to 50% by weight, preferably 11% to 43% by weight of 3-FL. Suitably, the HMO mixture may contain 10% to 50% by weight, preferably 13% to 46% by weight of 3-FL.

[0201] In one embodiment, the HMO mixture contains 6% to 30% by weight of LNnT. Suitably, the HMO mixture may contain 6% to 30% by weight, preferably 7% to 22% by weight of LNnT. Suitably, the HMO mixture may contain 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0202] In some implementations, the HMO mixture is essentially composed of the following substances:

[0203] i. 31% to 82% by weight of 2'-FL;

[0204] ii. 10% to 27% by weight of LNT;

[0205] iii. 4% to 11% by weight of DFL; and

[0206] iv. Combinations of 6SL and 3SL ranging from 9% to 34% by weight.

[0207] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0208] i. 41% to 70% by weight of 2'-FL;

[0209] ii. 14% to 23% by weight of LNT;

[0210] iii. 6% to 10% by weight of DFL; and

[0211] iv. Combinations of 6SL and 3SL ranging from 11% to 29% by weight.

[0212] In some implementations, the HMO mixture is essentially composed of the following substances:

[0213] i. 16% to 69% by weight of 2'-FL;

[0214] ii. 9% to 24% LNT by weight;

[0215] iii. 2% to 10% by weight of DFL;

[0216] iv. Combinations of 6SL and 3SL ranging from 8% to 26% by weight; and

[0217] v. 18% to 50% by weight of 3-FL.

[0218] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0219] i. 22% to 59% by weight of 2'-FL;

[0220] ii. 12% to 21% by weight of LNT;

[0221] iii. 3% to 8% by weight of DFL;

[0222] iv. Combinations of 6SL and 3SL ranging from 11% to 22% by weight; and

[0223] v. 11 wt% to 43 wt% 3-FL.

[0224] In some implementations, the HMO mixture is essentially composed of the following substances:

[0225] i. 34% to 85% by weight of 2'-FL;

[0226] ii. 10% to 40% by weight of LNT;

[0227] iii. 4% to 14% by weight of DFL;

[0228] iv. Combinations of 6SL and 3SL ranging from 9% to 31% by weight; and

[0229] v. 6% to 30% by weight of LNnT.

[0230] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0231] i. 40% to 71% by weight of 2'-FL;

[0232] ii. 12% to 26% by weight of LNT;

[0233] iii. 5% to 10% by weight of DFL; and

[0234] iv. Combinations of 6SL and 3SL ranging from 10% to 28% by weight; and

[0235] v.7% to 22% LNnT by weight.

[0236] In some implementations, the HMO mixture is essentially composed of the following substances:

[0237] i. 20% to 60% by weight of 2'-FL;

[0238] ii. 4% to 30% by weight of LNT;

[0239] iii. 1% to 12% by weight of DFL;

[0240] iv. Combinations of 6SL and 3SL ranging from 7% to 23% by weight;

[0241] v. 10 wt% to 50 wt% 3-FL; and

[0242] vi. 3% to 25% by weight of LNnT.

[0243] In some preferred embodiments, the HMO mixture is essentially composed of the following substances:

[0244] i. 22% to 55% by weight of 2'-FL;

[0245] ii. 6% to 20% by weight of LNT;

[0246] iii. 2% to 8% by weight of DFL;

[0247] iv. Combinations of 6SL and 3SL ranging from 8% to 22% by weight;

[0248] v. 13 wt% to 46 wt% 3-FL; and

[0249] vi. 5% to 20% by weight of LNnT.

[0250] When the composition or combination is in liquid form, the total HMO concentration is typically in the range of 0.5 g / L to 10 g / L, preferably in the range of 1 g / L to 7.5 g / L. Specific examples of total HMO concentration levels when the composition or combination is in liquid form include 1 g / L to 5 g / L, 1 g / L to 4 g / L, 2 g / L to 5 g / L, 1 g / L to 3 g / L, or 2 g / L to 4 g / L.

[0251] When the composition or combination is in solid form, the total HMO concentration is typically in the range of 0.35 wt% to 7 wt% (g total HMO / 100g dry composition), preferably in the range of 0.35 wt% to 5 wt%. Specific examples of total HMO concentration levels when the composition or combination is in dry form include 0.5 wt% to 3.5 wt% (g total HMO / 100g dry composition), 0.5 wt% to 2.5 wt%, 1 wt% to 3.5 wt%, 0.5 wt% to 2 wt%, or 1 wt% to 2.5 wt%.

[0252] Suitablely, the Bifidobacterium longum transitional microorganism is capable of metabolizing one or more HMOs in the HMO mixture. Suitablely, the Bifidobacterium longum transitional microorganism is capable of metabolizing the HMOs contained in the HMO mixture. Suitablely, the HMO mixture may be able to promote the growth and / or survival of the Bifidobacterium longum transitional strain as described herein.

[0253] Bifidobacterium longum infant subspecies

[0254] Bifidobacterium longum is a type of Bifidobacterium found in the human gastrointestinal tract. In 2002, three previously distinct species of Bifidobacterium (Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis) were merged with biotype Bifidobacterium infantis, Bifidobacterium longum, and Bifidobacterium suis into a single species named Bifidobacterium longum (Sakata, S. et al., 2002. International journal of systematic and evolutionary microbiology, 52(6), pp. 1945-1951).

[0255] Any suitable strain of *Bifidobacterium longum* subsp. infantis can be used in this invention. Such strains are well known to those skilled in the art. Suitable strains include *Bifidobacterium longum* subsp. infantis LMG 11588 (also known as *Bifidobacterium longum* subsp. infantis NCC3039 or *Bifidobacterium longum* subsp. infantis ATCC 17930) and *Bifidobacterium longum* subsp. infantis ATCC 15697 (also known as *Bifidobacterium longum* subsp. infantis NCC 3078).

[0256] Bifidobacterium longum subsp. infantis can be a strain known to those skilled in the art that has at least 99% (appropriately, at least 99.9%) of the ANI of Bifidobacterium longum subsp. infantis.

[0257] Suitably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 (also known as *Bifidobacterium longum* subsp. *infantitidis* NCC3039 or *Bifidobacterium longum* subsp. *infantitidis* ATCC 17930) have an ANI of at least 99% (suitably, 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%). Preferably, *Bifidobacterium longum* subsp. *infantitidis* and *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 have an ANI of at least 99.9%.

[0258] Examples of microbial genomes with at least 99.9% ANI of *Bifidobacterium longum* subsp. *infantii* 11588 can be found on PATRIC (https: / / www.patricbrc.org), genome ID 1678.111. Therefore, appropriately, *Bifidobacterium longum* subsp. *infantii* with PATRIC genome ID 1678.111 can be used in this invention.

[0259] Bifidobacterium longum infantis subspecies LMG 11588 was sold by the Belgian Microbial Collection Center (BCCM) under the LMG accession number LMG 11588.

[0260] Bifidobacterium longum infantis ATCC 15697 was sold by the American Type Culture Collection (ATCC) under accession number ATCC 15697.

[0261] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacterium longum infantis subsp., preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 Bifidobacterium longum infantis subsp. *CFU*. Appropriately, Bifidobacterium longum infantis subsp. *CFU* is at least about 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 Administer CFU / day to the individual. Appropriately, *Bifidobacterium longum* subsp. infantis is administered at approximately 10... 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 CFU / day or less is administered to the individual.

[0262] In one implementation, *Bifidobacterium longum* subsp. infantis is live.

[0263] Bifidobacterium lactis

[0264] Any suitable strain of *Bifidobacterium animalis* subsp. *lactobacter* (Bifidobacterium lactis) can be used in this invention. Such strains are well known to those skilled in the art. Suitable strains include *Bifidobacterium lactis* CNCM 1-3446.

[0265] Bifidobacterium lactis can be a strain known to those skilled in the art that has at least 99% (appropriately, at least 99.9%) of the ANI of Bifidobacterium lactis.

[0266] Suitably, Bifidobacterium lactis and Bifidobacterium lactis CNCM 1-3446 have an ANI of at least 99% (suitably, 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%). Preferably, Bifidobacterium lactis and Bifidobacterium lactis CNCM 1-3446 have an ANI of at least 99.9%.

[0267] Bifidobacterium lactis CNCM 1-3446 was deposited by NESTEC SA (NESTEC SA, AVENUE NESTLE 55, CH-1800 VEVEY) on June 7, 2005, at the National Center for Culture Collection of Microorganisms (CNCM) of the Pasteur Institute in France (INSTITUTPASTEUR, 25 RUE DU DOCTEUR ROUX, F-75724 PARIS CEDEX 15, FRANCE) under the Budapest Treaty, with accession number CNCM 1-3446.

[0268] Based on dry weight, the composition or combination according to the present invention may contain 10 g of the composition or combination per g of the composition or combination. 3 cfu to 10 12 CFU of Bifidobacterium lactis, preferably 10 7 cfu with 10 12 Between CFU, such as 10 8 cfu with 10 10 Bifidobacterium lactis between CFU. Appropriately, Bifidobacterium lactis in a quantity of at least approximately 10 6 CFU / day, at least approximately 10 7 CFU / day or at least approximately 10 8 Administer Bifidobacterium lactis to the individual at a dose of approximately 10 CFU / day. Appropriately, administer Bifidobacterium lactis at approximately 10 CFU / day. 12 CFU / day or less, approximately 10 11 CFU / day or less or about 10 10 CFU / day or less is administered to the individual.

[0269] In one implementation, the Bifidobacterium lactis is live.

[0270] Therapeutic uses

[0271] Preventing infection, reducing the risk of infection, and / or treating infection.

[0272] In another aspect, the present invention provides a composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0273] In another aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0274] In another aspect, the present invention provides a composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0275] In another aspect, the present invention provides a nutritional composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the nutritional composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0276] In another aspect, the present invention provides a nutritional composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the nutritional composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0277] In another aspect, the present invention provides a nutritional composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a mixture of HMOs, the HMO mixture being composed of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT), the nutritional composition being used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

[0278] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual.

[0279] In another aspect, the present invention provides the use of the nutritional composition according to the invention in the preparation of a medicament for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual.

[0280] In another aspect, the present invention provides methods for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual, wherein the method comprises administering a composition according to the present invention to the individual.

[0281] In another aspect, the present invention provides methods for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual, wherein the method comprises administering a nutritional composition according to the present invention to the individual.

[0282] Preferably, the composition or nutritional composition is used to prevent infection in an individual and / or reduce the individual's risk of infection.

[0283] The present invention also provides a combination of a transitional microorganism of Bifidobacterium longum and an HMO mixture for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual; wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0284] The present invention also provides a combination of a transitional microorganism of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, and an HMO mixture for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual; wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0285] In another aspect, the present invention provides a combination of a transitional microorganism of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and an HMO mixture for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual; wherein the HMO mixture comprises 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0286] In another aspect, the present invention provides the use of the combination according to the invention for preparing a medicament for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual.

[0287] In another aspect, the present invention provides methods for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in an individual, wherein the method comprises administering to the individual a combination according to the present invention.

[0288] Preferably, the composition or nutritional composition is used to prevent infection in an individual and / or reduce the individual's risk of infection.

[0289] Transitional Bifidobacterium longum can be a transitional Bifidobacterium longum as described in this article.

[0290] HMO mixtures can be HMO mixtures as described herein.

[0291] Bifidobacterium longum infantis subspecies can be as described in this article.

[0292] Bifidobacterium lactis can be the Bifidobacterium lactis described in this article.

[0293] Combinations (e.g., a combination of Bifidobacterium longum transitional microorganisms and HMO mixtures) may be provided in any form as described herein. For example, the combination may be provided in a composition as described herein.

[0294] The mixture of Bifidobacterium longum transitional microorganism and HMO can be applied alone, simultaneously, or sequentially.

[0295] Appropriately, a mixture of Bifidobacterium longum transitional microorganisms and HMOs can be applied as a combined composition.

[0296] Bifidobacterium longum transitional microorganisms, HMO mixtures, Bifidobacterium longum infantis subsp. and / or Bifidobacterium lactis can be applied alone, simultaneously, or sequentially.

[0297] Appropriately, Bifidobacterium longum transitional microorganisms, HMO mixtures of Bifidobacterium longum infantis subsp. and Bifidobacterium lactis can be applied in combination as a combined formulation.

[0298] Appropriately, a combination of Bifidobacterium longum transitional microorganisms (and optionally Bifidobacterium longum infantis subsp. and / or Bifidobacterium lactis) and HMO mixtures may be referred to as "synbiotics".

[0299] As used in this article, “infection” can refer to a disease or disorder (including its symptoms) caused by an infectious agent or pathogen.

[0300] As used herein, “prevention” can mean the application of the compositions and / or combinations of the present invention and / or prebiotics to an individual who is not yet infected and / or does not exhibit any symptoms of such infection, in order 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. An individual may be predisposed to developing the disease, or is considered to be at risk of developing the disease.

[0301] "Reducing the risk of infection" can refer to the application of the compositions and / or combinations and / or prebiotics of the present invention to individuals who are not yet infected and / or do not exhibit any symptoms of such infection, thereby reducing the likelihood of an infant or young child developing a disease caused by an infectious agent or pathogen. Application may prevent or weaken the cause of the disease, or reduce or prevent the development of at least one symptom associated with the disease. The individual may have a predisposition to develop the disease, or be considered to be at risk of developing the disease.

[0302] The term "treating" refers to reducing the duration and / or severity of a physical condition, ailment, or its consequences (e.g., the reduction or elimination of symptoms of the condition). Treatment also encompasses reducing, alleviating, or eliminating one or more symptoms associated with the treated disease, disorder, or ailment, and / or slowing, reducing, or halting the progression of the treated disease, disorder, or ailment.

[0303] Prevention and / or treatment of physical conditions, illnesses, or their consequences may occur during treatment (i.e., during the administration of the compositions of the present invention, either immediately after the start of administration or some time after the start of administration, such as days or weeks after the start). However, it may also cover subsequent prevention and / or treatment. The term "subsequent" covers effects after the intervention or treatment has ended. "Subsequent" effects may last from one week to several months or even several years, such as two to four weeks, two to six weeks, two to eight weeks, one to six months, or two to twelve months. Suitablely, after the termination of the intervention or treatment, "subsequent" effects may last from twelve months to twelve years, such as two to ten years or four to five years. Suitablely, "subsequent" effects may persist until the individual is at least five years old, such as at least ten years old, at least twenty years old, or at least thirty years old.

[0304] The use of this invention for preventing infection and / or reducing the risk of infection may be referred to as a preventive use for delaying or preventing the onset of infection symptoms and / or reducing the number or severity of infection symptoms.

[0305] Appropriately, administering the compositions and / or combinations of the present invention and / or prebiotics to an individual may reduce the magnitude and / or amount of infection symptoms caused by infectious agents or pathogens.

[0306] Suitable, the compositions and / or combinations and / or prebiotics of the present invention may be applied to infants, toddlers or children.

[0307] Suitable, the compositions and / or combinations of prebiotics of the present invention can prevent infection in an individual and / or reduce the individual's risk of infection.

[0308] Suitable, the compositions and / or combinations and / or prebiotics of the present invention can be administered to an individual to prevent infection and / or reduce the individual's risk of infection.

[0309] Appropriately, the composition and / or combination and / or prebiotics are not intended to reduce or prevent the presence of intestinal pathogens. Appropriately, the composition and / or combination and / or prebiotics are not intended to reduce or prevent the presence of intestinal pathogens in an individual's gut.

[0310] Appropriately, the composition and / or combination and / or prebiotics may increase the level of IL-6 in an individual.

[0311] IL-6 is secreted by macrophages in response to pathogen-associated molecular patterns (PAMPs). Therefore, IL-6 is an important component of fever and the acute phase response. Furthermore, IL-6 is responsible for stimulating acute-phase protein synthesis and the production of neutrophils in the bone marrow. It supports B cell growth and antagonizes regulatory T cells. IL-6 has been shown to play an important role in the prevention and / or control of many infections, including, for example, vaccinia virus and Listeria monocytogenes (Kopf et al.; 1994; Nature; 368; 339-342); herpes simplex virus (LeBlanc et al.; 1999; J Virol; 73(10)); influenza virus (Pyle et al.; 2017; PLoS Pathogens; 13(9), Dienz et al.; 2012; Mucosal Immunol; 5(3); 258-266, Gou et al.; 2019; Front Immunol; 10:3102); enteric bacterial pathogens (Dann et al.; 2008; J Immunol; 180(10); 6816-6826); and Escherichia coli (Dalrymple et al.; 1996; Infect Immun; 64(8): 3231-3235); pulmonary aspergillosis (Cenci et al.; 2001; J Infect Dis; 184(5); 610-617) and Candida albicans (van Enckevort et al.; 1999; Med Mycol; 37(6): 419-426).

[0312] Appropriately, the composition and / or combination and / or prebiotics can increase the level of short-chain fatty acids (SCFAs) in an individual.

[0313] Appropriately, SCFA may be selected from acetate (C1:0), butyrate (C4:0) and / or propionate (C3:0).

[0314] Dietary fiber produces SCFAs when it ferments in the colon. SCFAs have various physiological roles in bodily functions; they can affect the production of lipids, energy, and vitamins; influence appetite and cardiovascular metabolic health; and have a blood pressure-lowering effect in experimental models.

[0315] SCFAs have been shown to play an important role in the prevention and / or control of many infections and immune responses (Kim et al.; Cell Host & Microbe; 2016; 20(2); 202-214). For example, SCFAs have been shown to provide protection against RSV (Antunes et al.; Nat Comm; 2019; 10; 3273); influenza virus (Trompette; Immunity; 2018; 48(5); 992-1005 and Moriyama and Ichinobe; PNAS; 2018; 16(8); 3118-3125); viral bronchiolitis (Lynch et al.; J Exp Med; 2018; 215(2); 537-557) and common microbial infections (Schulthess et al.; Immunity; 2019; 50(2); 432-445). It is noteworthy that SCFAs produced in the gut affect systemic levels and local SCFA levels in other local organs (e.g., the lungs).

[0316] The cytokine and SCFA effects mediated by the compositions and / or combinations and / or prebiotics of the present invention can be systemic. Therefore, cytokine effects (e.g., increased levels of IL-6 and / or SCFA) can systemically prevent or reduce the risk of infections as described herein. Cytokine effects can occur locally in an individual's gut, lungs, and / or skin. Suitablely, SCFA effects can systemically prevent or reduce the risk of infections as described herein. SCFA effects can occur locally in an individual's gut, lungs, and / or skin. Suitablely, cytokine or SCFA effects can occur in an individual's gut. Suitablely, cytokine or SCFA effects can occur in an individual's lungs. Therefore, cytokine or SCFA effects can prevent or reduce the risk of infection in a specific organ or system.

[0317] Appropriately, the composition and / or combination and / or prebiotics can increase the level of indole-3-propionic acid in an individual. Indole-3-propionic acid has been shown to play an important role in the immune response (Li et al., Front. Pharmacol., 2021, 12:769501).

[0318] Suitablely, the composition and / or combination and / or prebiotics can modulate the permeability of an individual's intestinal epithelial barrier. Suitablely, the composition and / or combination and / or prebiotics can reduce the permeability of the intestinal epithelial barrier. Increased permeability of the intestinal epithelial barrier may be associated with, for example, increased passage of pathogens across the intestinal epithelium. Therefore, decreased permeability of the intestinal epithelial barrier may be associated with, for example, reduced passage of pathogens across the intestinal epithelium.

[0319] The composition and / or combination and / or prebiotics may reduce and / or prevent the exacerbation of symptoms of infection. For example, the composition and / or combination and / or prebiotics may reduce and / or prevent the exacerbation of symptoms caused by inflammation. Inflammation can be, for example, a pro-inflammatory response to an existing infection. An existing infection can be a current infection or a separate infection caused by a different infectious agent or pathogen. For example, current examples show that a transitional Bifidobacterium longum reduces the level of increased permeability in a model of intestinal epithelial barrier function following pro-inflammatory injury. It is not desired to be bound by theory that reduced intestinal epithelial barrier permeability following inflammatory injury may reduce the number / level of pathogens crossing the intestinal epithelial barrier during an inflammatory episode, and thus prevent infection and / or reduce the risk of infection; and / or prevent the exacerbation of symptoms of existing infection and / or reduce the risk of exacerbation of symptoms of existing infection.

[0320] The combinations or prebiotics used in this invention can be provided in the form of compositions.

[0321] The compositions of the present invention can be suitably administered to an individual, such as an infant or toddler, in any suitable form, such as dosage units (e.g., tablets, capsules, powder sachets, etc.). The compositions can be in powder, semi-liquid, or liquid form. The compositions can be added to nutritional compositions, infant formula, food compositions, supplements, baby food, stage 2 infant formula, growing-up milk, infant cereals, or fortifiers. In some embodiments, the compositions of the present invention are infant formula, baby food, infant cereals, growing-up milk, supplements, or fortifiers intended for use in infants, toddlers, or children.

[0322] By way of example, the composition may include other components that may be beneficial in preventing infection and / or reducing the risk of infection. Additionally or alternatively, the composition may include other components that may be beneficial during the weaning period.

[0323] For example, the composition may contain additional probiotics, such as probiotics known to have an effect on preventing infection and / or reducing the risk of infection (e.g., Bifidobacterium lactis, Lactobacillus rhamnosus, Bifidobacterium infantis), formula foods (e.g., partially hydrolyzed formula foods, extensively hydrolyzed formula foods, amino acid-based formula foods, or complete formula foods), infant foods (with or without milk fat), milk fat, cereals, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), butyrate, and / or gamma-linolenic acid (GLA).

[0324] Appropriately, additional probiotics may be Bifidobacterium infantis microorganisms.

[0325] The infection can be a viral infection, a bacterial infection, or a fungal infection.

[0326] Viral infection

[0327] Viral respiratory infections, such as respiratory syncytial virus (RSV), affect nearly 90% of children under two years of age (Karpinnen et al., Clin Microbiol Infect, 2016; 22; 208.e1-e6). These viral respiratory infections in infants and young children often result in bronchiolitis, an inflammatory bronchial response in infants and young children (Pickles et al., J Pathol, 2015; 235; 266-276). Severe RSV-induced bronchiolitis is a leading cause of infant morbidity and mortality worldwide (Nair et al., Lancet, 2010;375;9725;2545-1555).

[0328] Viral infections can be viral gastrointestinal infections or viral respiratory infections. Viral gastrointestinal infections can be viral intestinal infections or viral gastric infections.

[0329] In a preferred embodiment, the viral infection is a viral respiratory infection. The viral respiratory infection can be a viral infection in the upper or lower respiratory tract.

[0330] Illnesses associated with viral infections are typically the common cold, influenza (flu), bronchitis, bronchiolitis, pneumonia, sore throat (pharyngitis), sinusitis, non-allergic rhinitis, severe acute respiratory syndrome (SARS), viral croup, otitis media, meningitis, or diarrhea. Generally, when the viral infection is located in the respiratory tract, illnesses associated with respiratory infections include the common cold, influenza (flu), bronchitis, bronchiolitis, pneumonia, sore throat (pharyngitis), sinusitis, non-allergic rhinitis, severe acute respiratory syndrome (SARS), viral croup, or otitis media. Most commonly, illnesses associated with viral respiratory infections are the common cold, influenza (flu), bronchitis, bronchiolitis, or pneumonia.

[0331] Therefore, in a preferred embodiment of the invention, the compositions, combinations, and / or prebiotics of the invention are used to treat and / or prevent diseases associated with viral respiratory infections, selected from the common cold, influenza (flu), bronchitis, bronchiolitis, and pneumonia. In a more preferred embodiment, the respiratory infection-associated disease is selected from bronchiolitis and pneumonia, particularly RSV-induced bronchiolitis and / or pneumonia, i.e., bronchiolitis and / or pneumonia caused by RSV. In an even more preferred embodiment, the respiratory infection-associated disease is bronchiolitis, particularly RSV-induced bronchiolitis.

[0332] The symptoms most commonly associated with viral infections and that can be alleviated by the compositions of the present invention are lung irritation, lung congestion, excessive mucus production, fever, cough, wheezing, shortness of breath, abdominal cramps, diarrhea, or vomiting.

[0333] The above-mentioned infections can be caused by a variety of different viruses, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza viruses such as influenza A (IVA) and / or influenza B (IVB), rhinovirus (RV), adenovirus (ADV), metapneumovirus (MPV), bocavirus (BoV), coronavirus (CoV), myxovirus, herpesvirus, enterovirus (EV), parachovirus (PeV), or combinations thereof.

[0334] For example, infections can be caused by influenza virus infection, respiratory syncytial virus infection, rhinovirus infection, parainfluenza virus infection, metapneumovirus infection, coronavirus infection, adenovirus infection, and bocavirus infection.

[0335] Appropriately, the infection can be an influenza virus infection, a respiratory syncytial virus infection, or a rhinovirus infection. In a typical embodiment of the invention, the viral respiratory infection is caused by respiratory syncytial virus (RSV).

[0336] Influenza viruses are the infectious agents that cause influenza (the flu). Symptoms range from mild to severe and typically include fever, runny nose, sore throat, muscle aches, headache, cough, and fatigue. These symptoms begin one to four days (usually two days) after exposure to the virus and last for about two to eight days. Diarrhea and vomiting may occur, especially in children. There are four types of influenza viruses, called influenza A, influenza B, influenza C, and influenza D. Waterfowl are the primary source of influenza A virus (IAV), which is also widespread in various mammals, including humans and pigs. Influenza B virus (IBV) and influenza C virus (ICV) primarily infect humans, and influenza D virus (IDV) has been found in cattle and pigs. IAV and IBV circulate in humans and cause seasonal epidemics, while ICV causes mild infections, primarily in children. IDV can infect humans but has not been found to cause disease. In humans, influenza viruses are primarily spread through respiratory droplets produced by coughing and sneezing. Transmission also occurs through aerosols, intermediate objects, and surfaces contaminated with the virus.

[0337] Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus. It is the single most common cause of respiratory hospitalization in infants, with infection rates typically higher during the cold winter months, causing bronchiolitis. RSV spreads through contaminated airborne droplets and can cause outbreaks in both community and hospital settings. Following initial infection via the eyes or nose, the virus infects the epithelial cells of the upper and lower airways, causing inflammation, cell damage, and airway obstruction.

[0338] Rhinoviruses are the most common viral pathogens in humans and a major cause of the common cold. The three rhinovirus classes (A, B, and C) comprise approximately 160 recognized types of human rhinoviruses, distinguished by their surface proteins (serotypes). They are inherently lytic and belong to the smallest viruses, approximately 30 nanometers in diameter. Symptoms of rhinovirus infection can include sore throat, runny nose, nasal congestion, sneezing, and cough; sometimes accompanied by muscle pain, fatigue, malaise, headache, muscle weakness, or loss of appetite.

[0339] Using a mouse pneumonia virus (PVM) model infected with human RSV, the inventors surprisingly discovered that synbiotic intervention (i.e., a combination of Bifidobacterium longum transitional microbes, Bifidobacterium longum infantis subsp., Bifidobacterium lactis and HMO, and a combination of Bifidobacterium longum infantis subsp. and HMO) provided protection against virus-induced bronchiolitis in early life. These findings confirm the use of synbiotics in the protection and treatment of viral infections, particularly viral bronchiolitis, in early life and reveal the functional benefit of synbiotics in establishing an effective antiviral immune response associated with faster disease resolution.

[0340] Therefore, the compositions and / or combinations of prebiotics of the present invention are particularly effective for use in the treatment and / or prevention of viral infections in individuals.

[0341] The compositions and / or combinations of prebiotics of the present invention are particularly effective in treating RSV-induced viral infections, preventing RSV-induced viral infections, reducing the risk of RSV-induced viral infections, and / or alleviating the symptoms of RSV-induced viral infections. Therefore, the compositions of the present invention are particularly preferably used in treating RSV-induced bronchiolitis or RSV-induced pneumonia, preventing RSV-induced bronchiolitis or RSV-induced pneumonia, reducing the risk of RSV-induced bronchiolitis or RSV-induced pneumonia, and / or alleviating the symptoms of RSV-induced bronchiolitis or RSV-induced pneumonia.

[0342] The compositions and / or combinations of prebiotics of the present invention can be used to treat and / or prevent viral infections, particularly respiratory infections in people of any age. Therefore, individuals to be treated with the compositions of the present invention may be selected from those aged 0 to <1 year (infants), 1 to <3 years (toddlers), and 3 to <6 years (children), including those aged 3 to <5 years (preschool children).

[0343] Sustained immune benefits

[0344] Viral infections can also disrupt normal host function and lead to more serious infection-related disorders, including immunopathological manifestations following respiratory viral infections (Newton et al., Semin Immunopathol, 2016;38;471-482), such as long-term changes in the immune system (e.g., inflammatory responses) and subsequent allergic or inflammatory diseases. For example, uncontrolled inflammatory responses following respiratory viral infections can lead to pathological airway smooth muscle remodeling, a hallmark feature of asthma reported to begin early in life (O'Reilly et al., JACI, 2013;131;1024-1032), and in chronic obstructive pulmonary disease (COPD; Yan F et al., J Transl Med, 2018;16;262-270). Therefore, severe viral airway infection early in life represents a major independent risk factor for subsequent respiratory diseases such as allergic airway diseases (e.g., asthma; Feldman et al., Am J Respir Crit Care Med, 2015;191;34-44) and later chronic obstructive pulmonary disease (Savran O et al., Int J Chron Obstruct Pulmon Dis. 2018; 13: 683–693).

[0345] Therefore, the compositions and / or combinations of the present invention are particularly effective in promoting sustained immune benefits in individuals.

[0346] Therefore, in another aspect, the present invention provides a composition according to the invention for promoting long-term immune benefits in an individual.

[0347] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for promoting long-term immune benefits in an individual.

[0348] In another aspect, the present invention provides a method for promoting long-term immune benefits in an individual, the method comprising administering to the individual a composition according to the present invention.

[0349] In another aspect, the present invention provides a composition according to the invention for promoting long-term immune benefits in an individual.

[0350] In another aspect, the present invention provides the use of the combination according to the invention for preparing a medicament for promoting long-term immune benefits in an individual.

[0351] In another aspect, the present invention provides a method for promoting long-term immune benefits in an individual, the method comprising administering to the individual a combination according to the present invention.

[0352] The benefits of promoting long-term immunity include:

[0353] i. Promote long-term respiratory health;

[0354] ii. To prevent allergen sensitization and / or reduce the risk of allergen sensitization; and / or

[0355] iii. To prevent and / or reduce the risk of developing respiratory illnesses in the future.

[0356] Where appropriate, promoting long-term immune benefits means promoting long-term respiratory health. Where appropriate, promoting long-term immune benefits means preventing allergen sensitization and / or reducing the risk of allergen sensitization. Where appropriate, promoting long-term immune benefits means preventing future respiratory illnesses and / or reducing the risk of future respiratory illnesses.

[0357] As used herein, the phrase “long-term” encompasses effects following the termination of intervention or treatment. “Long-term” effects can range from one week to several years after the termination of intervention or treatment, for example, 2 to 4 weeks, 2 to 6 weeks, 2 to 8 weeks, 1 to 6 months, 2 to 12 months, 12 months to 12 years, such as 2 to 10 years, or 4 to 5 years. Therefore, “long-term” effects can exist when an individual reaches 3 years of age or older, preferably 3 to 12 years, more preferably 3 to 10 years, even more preferably 3 to 8 years, most preferably 3 to 6 years, and particularly 3 to 5 years or 3 to 4 years. Appropriately, long-term benefits persist until the individual is at least 5 years old, such as at least 10 years, at least 20 years, or at least 30 years old.

[0358] Suitablely, the compositions and / or combinations of the present invention can prevent complications associated with respiratory viral infections. Suitablely, this effect can be long-term prevention of complications associated with respiratory viral infections. Such complications can be those related to the immune system, such as inflammatory responses, and include immunopathological manifestations following respiratory viral infection, such as long-term alterations to the immune system (e.g., long-term alterations to inflammatory responses) and pathological airway smooth muscle remodeling. These complications may further predispose an individual to subsequent allergic or inflammatory diseases, such as allergic respiratory diseases or chronic inflammatory diseases of the respiratory tract.

[0359] Allergic reactions and respiratory symptoms

[0360] Given that early-life viral respiratory infections represent a major independent risk factor for subsequent asthma, recurrent wheezing, and chronic obstructive pulmonary disease (Savran et al., Int J Chron Obstruct, 2015; 191; 34-44; and Feldman et al., 2015 Am J Respir Crit Care Med, 191; 34-44), the compositions and / or combinations of the present invention are also effective in preventing and / or reducing the risk of respiratory conditions, such as chronic inflammatory diseases of the airways and allergic airway diseases.

[0361] Since viral infections, particularly RSV infections, are associated with the subsequent development of allergic airway diseases, such as later-onset asthma (Feldman et al. 2015 Am J Respir Crit Care Med, 191; 34-44), the compositions and / or combinations of the present invention are also effective in preventing individual allergen sensitization and / or the development of allergic respiratory diseases and / or reducing the risk of individual allergen sensitization and / or the development of allergic respiratory diseases.

[0362] Therefore, in another aspect, the present invention provides a composition according to the invention for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0363] In another aspect, the present invention provides a composition according to the invention for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma.

[0364] In another aspect, the present invention provides the use of the composition according to the invention in the preparation of a medicament for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0365] In another aspect, the present invention provides the use of the compositions according to the invention in the preparation of a medicament for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma.

[0366] In another aspect, the present invention provides i) methods for preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) methods for preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms, the method comprising administering a composition according to the present invention to the individual.

[0367] In another aspect, the present invention provides a method for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma, the method comprising administering a composition according to the present invention to the individual.

[0368] Therefore, in another aspect, the present invention provides a combination according to the invention for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0369] In another aspect, the present invention provides a combination according to the invention for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma.

[0370] In another aspect, the present invention provides the use of the combination according to the invention for preparing a medicament for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0371] In another aspect, the present invention provides the use of the combination according to the invention for preparing a medicament for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma.

[0372] In another aspect, the present invention provides i) methods for preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) methods for preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms, the method comprising administering to an individual a combination according to the present invention.

[0373] In another aspect, the present invention provides a method for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma, the method comprising administering to an individual a combination according to the present invention.

[0374] Respiratory disorders include chronic inflammatory diseases of the respiratory tract and allergic respiratory diseases.

[0375] Chronic inflammatory diseases of the respiratory tract include chronic obstructive pulmonary disease (COPD) and asthma, including allergic asthma and non-allergic asthma.

[0376] COPD is a term used for a group of lung diseases, including chronic bronchitis, emphysema, and chronic obstructive airway disease. People with COPD experience difficulty breathing, primarily due to narrowing of their airways.

[0377] Asthma is a chronic respiratory condition characterized by inflammation and bronchospasm, leading to difficulty breathing. It is often associated with allergic reactions or other forms of hypersensitivity. Inflammation and narrowing of the small airways in the lungs can cause asthma symptoms, which can be any combination of cough, wheezing, shortness of breath, and chest tightness. Asthma typically develops in childhood, particularly during the preschool years (3 to 5 years old).

[0378] Allergic respiratory diseases include recurrent wheezing and asthma, including allergic asthma.

[0379] To i) prevent individual allergen sensitization and / or reduce the risk of individual allergen sensitization and / or ii) prevent individual respiratory illness and / or reduce the risk of individual respiratory illness, the compositions of the present invention are preferably applied to individuals aged 0 to <3 years, preferably 0 to 2 years, more preferably 0 to <1 year, such as 0 to 6 months. When an individual is 3 years or older, preferably 3 to 12 years, more preferably 3 to 10 years, even more preferably 3 to 8 years, most preferably 3 to 6 years, and particularly 3 to 5 years or 3 to 4 years, this further prevents the occurrence of respiratory illness or reduces the risk of developing respiratory illness.

[0380] Since viral infections, particularly RSV infections, are often associated with bacterial co-infections (Thorburn et al., Thorax, 2006; 61(7); 611-615) or secondary infections (Sande et al., Nature Communications, 2019;10;2218), including antibiotic use, the compositions and / or combinations of the present invention are also effective in preventing or reducing the risk of bacterial co-infections and / or secondary bacterial infections associated with respiratory viral infections in mammals, particularly humans. Pathogens commonly involved in co-infections or secondary infections include Staphylococcus aureus, Streptococcus pneumoniae, and / or Haemophilus influenzae.

[0381] prebiotics

[0382] The present invention also provides a prebiotic for preventing, reducing the risk of infection and / or treating infection in an individual by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the individual's gut, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0383] In another aspect, the present invention provides the use of a prebiotic in the preparation of a medicament for preventing, reducing the risk of infection and / or treating an individual’s infection by promoting the growth of Bifidobacterium longum transitional microorganisms in the gut of an infant or young child, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0384] In another aspect, the present invention provides a method for preventing, reducing the risk of infection and / or treating infection in an individual by promoting the growth of Bifidobacterium longum transitional microorganisms in the individual's gut, wherein the method comprises administering a prebiotic to an infant or young child, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0385] HMO mixtures may be as defined herein.

[0386] In some implementations, prebiotics are used to prevent infection in an individual and / or reduce the individual's risk of infection.

[0387] Preferably, the individual is an infant or toddler.

[0388] In some embodiments, the compositions and / or combinations of the present invention may also contain prebiotics (i.e., in addition to the HMO mixtures described herein). Suitablely, the prebiotics are polysaccharide substrates.

[0389] Polysaccharide substrate / carbohydrate-active enzyme (CAZyme)

[0390] Bifidobacterium longum transitional microbes encode a carbohydrate-active enzyme (CAZyme) profile. Without being bound by theory, it is believed that targeting these CAZymes, for example, by providing a mixture of HMOs as described herein and / or suitable glycan substrates in prebiotic form, could promote the growth and / or survival of Bifidobacterium longum transitional microbes in the gut microbiota of infants or young children.

[0391] Promoting the growth and / or survival of Bifidobacterium longum transitional microbes can be defined as increasing the number and / or concentration of Bifidobacterium longum transitional microbes in the gut microbiota.

[0392] In particular, CAZyme encoded by each of the transitional strains of Bifidobacterium longum NCC 5000, NCC 5001, NCC 5002, NCC 5003 and NCC 5004, which were deposited at the Pasteur Institute on May 11, 2021, in accordance with the Budapest Treaty, with accession numbers CNCM I-5683, CNCM I-5684, CNCM I.5685, CNCM I-5686 and CNCM I-5687, respectively.

[0393] Carbohydrate active enzymes (CAZymes) are responsible for the synthesis and breakdown of glycoconjugates, oligosaccharides, and polysaccharides. They typically correspond to 1%–5% of the genes in living organisms. Glycoconjugates, oligosaccharides, and polysaccharides play important roles in many biological functions, such as serving as structural and energy storage components, and in many intracellular and intercellular events. The CAZyme classification is a sequence-based family classification system associated with the structure and molecular mechanisms of CAZymes (www.cazy.org).

[0394] CAZyme includes glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE), and the carbohydrate binding module family (CBM).

[0395] Appropriately, CAZyme can be a glycosidic 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 the glycosidic bond is catalyzed by two amino acid residues of the enzyme: the common acid (proton donor) and the nucleophile / base. Depending on the spatial position of these catalytic residues, the hydrolysis occurs via overall retention or overall inversion of the terminal isomer conformation.

[0396] The GH classification system is provided by the CAZy classification. In this paper, GH are divided into families (e.g., GH1, GH2, GH3, GH4, etc.) based on molecular function. These families are then further divided into subfamilies based on subgroups found within each family, which share a more recent ancestor and are generally more consistent in molecular function (e.g., GH13_1, GH13_2, GH13_3, GH13_4, etc.).

[0397] Table 1 provides detailed information on CAZymes specific to transitional strains of Bifidobacterium longum (i.e., those not coded by Bifidobacterium longum suis / suillum, Bifidobacterium longum longum, or Bifidobacterium longum infantis). Table 1 also provides a summary of the glycan substrates metabolized by each CAZyme and exemplary dietary fiber sources / components.

[0398] Table 1

[0399] Table 2 lists strains present in at least one transitional strain of *Bifidobacterium longum* but not in strains selected from... Figure 7 Detailed information on CAZyme in at least one of the groups of *Bifidobacterium longum* subsp. suis, *Bifidobacterium longum* subsp. spp., or *Bifidobacterium longum* subsp. spp. infantis strains is shown. Table 2 also provides a summary of the polysaccharide substrates metabolized by each CAZyme and exemplary dietary fiber sources / components.

[0400] Table 2

[0401] Table 3 provides detailed information on CAZymes present in all analyzed Bifidobacterium longum strains (i.e., transitional Bifidobacterium longum, Bifidobacterium longum suis subsp., Bifidobacterium longum subsp., and Bifidobacterium longum infantis subsp.). Table 3 also provides a summary of the glycan substrates metabolized by each CAZyme and exemplary dietary fiber sources / components.

[0402] Table 3

[0403] Table 4 provides detailed information on CAZymes that are not encoded by transitional strains of Bifidobacterium longum but are encoded by one or more of the following: Bifidobacterium longum suis subsp., Bifidobacterium longum subsp. longum, and Bifidobacterium longum infantis subsp.

[0404] Table 4

[0405] The representative sequences of CAZyme listed in Tables 1 to 4 are shown in... Figure 7 In appropriate context, the CAZyme referred to in any of Tables 1 through 4 may include... Figure 7 The corresponding sequence shown or composed of it is included. Appropriately, CAZyme may include... Figure 7 The variants of the corresponding sequences shown, or those constituting them, retain at least one function of the corresponding CAZyme listed in Tables 1 to 4. Suitablely, the variants may provide each functional activity of the corresponding CAZyme listed in Tables 1 to 4. Suitablely, the variants may include... Figure 7 The sequences listed herein have at least 70% sequence identity with amino acid sequences or are composed of such sequences, and retain at least one functional activity of the corresponding CAZyme listed in Tables 1 to 4, preferably each functional activity. Suitable variants may include amino acid sequences with... Figure 7 The corresponding sequences listed herein have or consist of 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. Variants retain at least one functional activity of the corresponding CAZyme listed in Tables 1 to 4, preferably each functional activity.

[0406] Suitable prebiotics used in this invention may include polysaccharide substrates selected from any of the groups listed in Tables 1 to 3.

[0407] Suitable prebiotics used in this invention may include combinations of glycan substrates selected from any of the groups listed in Tables 1 to 3.

[0408] 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.

[0409] Appropriately, prebiotics may include one or more polysaccharide substrates selected from the groups listed in Table 1 or Table 2.

[0410] Prebiotics may include at least 2, at least 4, at least 10, at least 20, or at least 30 glycan substrates listed in Tables 1 and 2. Prebiotics may include each of the glycan substrates listed in Tables 1 and 2.

[0411] Appropriately, the polysaccharide substrate may include or consist of pectin, arabinogalactan, and / or starch.

[0412] Appropriately, the polysaccharide substrate may include or consist of pectin.

[0413] Appropriately, the polysaccharide substrate may include or consist of arabinogalactan.

[0414] Appropriately, the polysaccharide substrate may include or consist of starch.

[0415] Appropriately, the polysaccharide substrate is provided in the form of dietary fiber. For example, dietary fiber can be prebiotic fiber.

[0416] Where appropriate, the polysaccharide substrate may be included in the ingredients, such as dietary ingredients.

[0417] This ingredient contains one or more polysaccharide substrates, optionally from the group consisting of: purified polysaccharides or purified oligosaccharides, dietary fiber components, semi-purified food components, raw food components, food additives, HMOs, and semi-purified or purified peptidoglycans.

[0418] Semi-purified food ingredients can be fruit, vegetable, or grain extracts.

[0419] Raw food ingredients can be fruits, vegetables, grains, seaweed, or microalgae.

[0420] Food additives can be guar gum or gum arabic.

[0421] Appropriately, peptidoglycan can be GAG.

[0422] Appropriately, the polysaccharide substrate may be included in the purified cellulose.

[0423] Tables 1 through 3 provide exemplary components and / or purified fibers that include suitable glycan substrates. In particular, dietary fibers and / or components that include a given glycan substrate are identified in the same row as the glycan substrate.

[0424] Pectin can be included in fruit or vegetable pectin. Therefore, suitable ingredients that include pectin include, but are not limited to: fruits (e.g., apples, pears), vegetables, legumes (peas), and roots (e.g., sugar beets). Suitable purified fibers that include arabinogalactan include peach pectin. Pectin extracted from sugar beets suitably contains arabinogalactan, galactan, and arabinogalactan, and can be provided as an ingredient.

[0425] Arabinogalactan can be included in fruit or vegetable pectin. Typical suitable ingredients that include arabinogalactan include, but are not limited to, dietary fiber from fruits, vegetables, whole grains, and seaweed. Suitable purified fibers that include arabinogalactan include peach pectin, larch wood arabinogalactan, and gum arabic. Suitablely, arabinogalactan can be provided in larch wood arabinogalactan.

[0426] Starch can be included in resistant starch from grains (whole grains), legumes, vegetables (e.g., corn), and roots (e.g., potatoes). Suitable, illustrative, starch-containing ingredients include, but are not limited to, corn. Suitable purified fibers including starch include high amylose and resistant dextrin. Starch may suitably be provided in potatoes, corn, or other ingredients. Starch may suitably be included in potato ingredients.

[0427] Human milk oligosaccharides (HMOs)

[0428] Appropriately, the prebiotic contains one or more additional HMOs. Appropriately, the additional HMOs are different from those provided in the HMO mixtures described herein.

[0429] Suitablely, the added HMO can be metabolized by *Bifidobacterium longum* transitional microorganisms. Suitablely, the added 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, for example, by an anaerobic culture of the *Bifidobacterium longum* transitional strain and the HMO to be tested. The growth and / or survival of the *Bifidobacterium longum* transitional strain can be determined by measuring the number of bacterial cells, cell density (e.g., by optical density measurement), and / or the abundance of 16S rDNA, for example using PCR methods. An exemplary assay for measuring the growth of *Bifidobacterium longum* transitional strains in the presence of HMOs is provided in Example 6. Compared to the number of *Bifidobacterium longum* transitional bacteria in a control anaerobic culture without HMOs, HMOs that can promote the growth and / or survival of *Bifidobacterium longum* transitional strains can increase the number of *Bifidobacterium longum* transitional bacteria in an anaerobic culture by at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 100%. Appropriately, HMOs that promote the growth and / or survival of Bifidobacterium longum transitional bacteria can increase the number of Bifidobacterium longum transitional bacteria in anaerobic cultures by a statistically significant amount (e.g., p < 0.05 as determined by one-way ANOVA) compared to the number of Bifidobacterium longum transitional bacteria in control anaerobic cultures that do not contain HMOs.

[0430] HMOs can be fucosylated oligosaccharides (i.e., oligosaccharides with fucose residues; for example, 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasose, lactose-N-difucohexasose I, fucosyllacto-N-hexasose, fucosyllacto-N-neohexose, difucosyllacto-N-hexasose I, difucosyllacto-N-neohexose II, and any combination thereof). (e.g., sialized oligosaccharides, N-acetylated oligosaccharides, such as p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neohexose), DSLNT (disialyllactose-N-tetrasaccharide), lactose-N-hexasaccharide, lactose-N-neohexose, p-lactose-N-hexasaccharide, p-lactose-N-neohexose, lactose-N-octasaccharide, lactose-N-neohexose, isolatose-N-octasaccharide, p-lactose-N-octasaccharide and lactose-N-decanose and any combination thereof) and / or sialylated oligosaccharides (e.g., Lst (sialyllactose-N-tetrasaccharide), Lst-a, Lst-b or Lst-c)).

[0431] individual

[0432] In one implementation, the individual is an infant. In another implementation, the individual is a toddler. In yet another implementation, the individual is a child.

[0433] The compositions or combinations according to the invention are for use in infants, toddlers, or children. This nutritional composition is particularly suitable for infants under 6 months of age.

[0434] Generally, formula-fed infants have an underdeveloped immune system compared to adults and are more susceptible to viral infections than breastfed infants, with the immune system becoming less developed the younger the infant. Therefore, the composition or combination is particularly useful for preterm infants and / or low or very low birth weight infants, as these infants are even more vulnerable to injury and viral infections. In another embodiment of particular interest, the composition or combination is used for infants delivered by cesarean section. Cesarean-born infants are born in a hospital environment with more pathogens, and antibodies against these pathogens transferred from the mother to the infant are not effective against them. Furthermore, antibiotic administration is a recommended medical practice for cesarean delivery to prevent infection. Such interventions can severely disrupt the (mother's or child's) microbiome, and early-life antibiotic treatment is associated with an increased risk of developing immune-mediated disorders later in life. Cesarean-born infants have delayed and less desirable colonization of the large intestine and are therefore also more susceptible to infection.

[0435] The infant, toddler, or child may be full-term or premature. In one specific embodiment, the composition or combination of the present invention is used for premature infants, toddlers, or children. Premature infants may have an increased risk of developing poor nutrient utilization, lean body mass syndrome, visceral fat accumulation, and metabolic diseases later in life.

[0436] In one implementation, the individual is for infants, toddlers, or children who are born small for gestational age or have low birth weight.

[0437] Low birth weight infants, toddlers, or children may or may not be preterm infants, and similarly, small for gestational age infants, toddlers, or children may or may not be preterm infants.

[0438] The compositions or combinations of the present invention can also be used for infants, toddlers or children born by cesarean section or vaginal delivery.

[0439] All infants, toddlers, and children can benefit from this invention because they are all prone to, or may be prone to, an unbalanced gut microbiota at some age.

[0440] In some advantageous embodiments of the invention, the composition or combination is used for infants, toddlers or children with a fragile or unbalanced microbiome or microbiome dysbiosis, such as premature infants, infants born by cesarean section, infants born small for gestational age or with low birth weight, hospitalized infants / toddlers / children, infants / toddlers / children who have received or have received antibiotic treatment, and / or infants / toddlers / children who have or have had intestinal infections and / or intestinal inflammation.

[0441] Indeed, it is foreseeable that the compositions or combinations of the present invention may be even more beneficial to infants whose gut microbiota may be impaired at birth or to vulnerable infants / toddlers / children (such as premature infants and / or infants born by cesarean section). Furthermore, it is foreseeable that the compositions or combinations of the present invention may be even more beneficial, particularly to infants / toddlers / children who exhibit intestinal disturbances (such as diarrhea, infection, or colic), especially after birth, for example, during the first four weeks after birth.

[0442] In embodiments of the invention, the compositions or combinations thereof are intended for infants who are premature, born by cesarean section, born small for gestational age, or have low birth weight, or exhibit an unbalanced or abnormal gut microbiota, or have had or have had intestinal infections and / or intestinal inflammation, particularly when the infant is 0 to 6 months of age. Without being bound by theory, it is believed that younger infants benefit even more from the compositions or combinations thereof of the invention, particularly when the infant has an unbalanced gut microbiota (or is at risk of having an unbalanced gut microbiota) and / or has a weakened health condition (as illustrated in the conditions cited above).

[0443] The age and duration of administration (or administration or feeding) of a composition (e.g., a nutritional composition) or combination can be determined as needed.

[0444] In one embodiment, the infant or toddler is 0-36 months old, such as 0-12 months or 0-6 months. It is foreseeable that the compositions or combinations of the present invention may be more advantageous for newborns (0 to 4 weeks or 0 to 8 weeks) because their intestines may be more vulnerable.

[0445] In some embodiments, the compositions (e.g., nutritional compositions) or combinations according to the invention may be used before and / or during the weaning period.

[0446] In some embodiments, the compositions (e.g., nutritional compositions) or combinations according to the invention are used for individuals at risk and / or in need.

[0447] Individuals at risk and / or in need may be bottle-fed and / or formula-fed.

[0448] In one embodiment, the composition or combination of the present invention is given to an individual as a supplemental composition to breast milk. In some embodiments, the individual is breastfed for at least the first 2 weeks, the first 1 month, 2 months, 4 months, or 6 months. In one embodiment, the composition or combination of the present invention (e.g., a nutritional composition) is given to the individual after this period of nutrition provided by breast milk, or given to the individual along with breast milk during this period of nutrition provided by breast milk. In another embodiment, the composition or combination is given to the individual as the sole or primary nutritional composition for at least a period of time (e.g., after the first, second, or fourth month of life), for at least 1 month, 2 months, 4 months, or 6 months. In one embodiment, the nutritional composition of the present invention is a complete nutritional composition (meeting all or most of the individual's nutritional needs). In another embodiment, the nutritional composition of the present invention is a supplement or fortifier intended for use, for example, supplementing human milk or supplementing infant formula or follow-up formula.

[0449] Nutritional composition

[0450] In some embodiments, the compositions of the present invention are in the form of nutritional compositions.

[0451] The nutritional compositions according to the invention may be, for example, infant formula, stage 1 infant formula, follow-up or stage 2 infant formula, growing milk, baby food, infant cereal compositions, fortifiers (such as human milk fortifiers), or supplements. In some specific embodiments, the compositions of the invention are infant formula, fortifiers, or supplements intended for infants aged 4 months or 6 months. In a preferred embodiment, the nutritional compositions of the invention are infant formula.

[0452] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier may be a breast milk fortifier (e.g., human milk fortifier) ​​or a formula food fortifier (such as an infant formula fortifier or a follow-up formula / stage 2 infant formula fortifier).

[0453] When a nutritional composition is a supplement, it can be provided in unit doses. In such cases, it is particularly useful to limit the amount of oligosaccharides and probiotics according to the daily dose administered to infants or young children.

[0454] When the nutritional composition is a supplement, it may contain an HMO mixture and Bifidobacterium longum subsp. microorganisms as described herein, and may contain no additional nutrients other than the excipients necessary to obtain a stable nutritional composition.

[0455] The nutritional composition of the present invention may be in solid (e.g., powder), liquid, or gel form. In one particular embodiment, the nutritional composition is a supplement, wherein the supplement is in powder form and provided in sachets, preferably with 0.1 g / sachet to 20 g / sachet, for example, 1 g / sachet to 10 g / sachet, or provided in syrup form, preferably with a total solids concentration of 5 g / 100 mL to 75 g / 100 mL (5% to 75% (w / v)). When the supplement is in powder form, it may contain a carrier. However, it is preferred that the supplement does not contain a carrier. When the supplement is in syrup form, the components are preferably dissolved or suspended in water acidified with citrate.

[0456] In one embodiment, the nutritional composition according to the invention is a hypoallergenic composition. In another embodiment, the composition according to the invention is a hypoallergenic nutritional composition.

[0457] Other ingredients

[0458] The compositions or combinations according to the invention may also contain other types of oligosaccharides, polysaccharides, and / or fibers and / or their precursors. Other oligosaccharides and / or fibers and / or their precursors may be selected from the list including: human milk oligosaccharides (HMOs), galactooligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), cellulose oligosaccharides (COS), arabinoxylan, arabinogalactan, xylan, inulin, polydextrose, β-glucan, pectin, and any combination thereof and any derivative thereof (e.g., partially hydrolyzed). Their amounts may be between 0% by weight and 10% by weight of the composition. In one specific embodiment, the nutritional composition may also contain at least one BMO (a bovine milk-derived oligosaccharide).

[0459] Additional HMOs that may be included in the nutritional compositions according to the invention may be selected from lactose-N-fucopentose (e.g., lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V), lactose-N-fucohexasose, lactose-N-difucohexasose I, fucosyllacto-N-hexasose, fucosyllacto-N-neohexose, and difucosyllacto-N-hexasose. Fucose-N-hexasaccharide I, fucose-N-neohexaccharide II, p-lactose-N-neohexaccharide (p-LNnH), lactose-N-hexasaccharide, lactose-N-neohexaccharide, p-lactose-N-hexasaccharide, p-lactose-N-neohexaccharide, lactose-N-octasaccharide, lactose-N-neohexaccharide, isolose-N-octasaccharide, p-lactose-N-octasaccharide, lactose-N-decasaccharide, and any combination thereof.

[0460] In some embodiments, the composition or combination according to the invention comprises at least one additional HMO.

[0461] In other embodiments, the compositions or combinations according to the invention do not contain any additional HMOs. Therefore, the HMO mixtures as described herein may be the only HMOs in the compositions or combinations of the invention.

[0462] The compositions or combinations of the present invention may further comprise at least one additional probiotic (or probiotic strain), such as at least one additional probiotic strain.

[0463] The most commonly used probiotics are mainly bacteria and yeasts from the following genera: Lactobacillus spp., Lacticaseibacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp., and Saccharomyces spp.

[0464] In some specific embodiments, the probiotics are probiotic bacterial strains. In some specific embodiments, they are specifically Bifidobacterium and / or Lactobacillus.

[0465] Suitable probiotic strains include Lactobacillus rhamnosus ATCC 53103, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM I-2116, Lactobacillus johnsonii CNCMI-1225 (produced by Valio Oy, Finland), Streptococcus salivarius DSM 13084 (produced by BLIS Technologies Limited, New Zealand under the name KI2), Bifidobacterium longum CNCM I-2618 (Bifidobacterium longum NCC2705), Bifidobacterium breve (produced by Danisco under the trademark Bb-03), Bifidobacterium breve (produced by Morinaga under the trademark M-16V), and Bifidobacterium infantis (produced by Procter & GambIe Co. under the trademark Bifantis), as well as Institut... Rosell (Lallemand) sells Bifidobacterium breve under the trademark R0070.

[0466] On a dry weight basis, the composition or combination according to the invention may contain at least one (additional) probiotic strain of 10e3 cfu to 10e12 cfu per g of composition or combination, more preferably a probiotic strain of between 10e7 cfu and 10e12 cfu (such as between 10e8 cfu and 10e10 cfu).

[0467] In one embodiment, the probiotics are live. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, both live and inactivated probiotics may be present simultaneously. Probiotic components and metabolites may also be added.

[0468] The nutritional compositions according to the invention typically contain a protein source. The amount of protein can be from 1.6 g / 100 kcal to 3 g / 100 kcal. In some embodiments, particularly when the composition is intended for preterm infants, the amount of protein can be between 2.4 g / 100 kcal and 4 g / 100 kcal or more than 3.6 g / 100 kcal. In some other embodiments, the amount of protein can be less than 2.0 g / 100 kcal, for example, from 1.8 g / 100 kcal to 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0469] Protein sources based on whey, casein, or mixtures thereof, or soy-based protein sources, may be used. Regarding the whey protein of interest, the protein source may be based on acidic whey or sweet whey, or mixtures thereof, and may contain any desired proportions of α-lactalbumin and β-lactoglobulin.

[0470] In some advantageous implementations, the protein source is whey-based (i.e., more than 50% of the protein comes from whey protein, such as 60% or 70%).

[0471] The protein can be whole or hydrolyzed, or a mixture of whole and hydrolyzed proteins. The term "whole" means that the major components of the protein are intact, i.e., the molecular structure is not altered, for example, at least 80% of the protein is unchanged, such as at least 85% of the protein is unchanged, preferably at least 90% of the protein is unchanged, even more preferably at least 95% of the protein is unchanged, such as at least 98% of the protein is unchanged. In one specific embodiment, 100% of the protein is unchanged.

[0472] The term "hydrolyzed" means, in the context of this invention, that a protein has been hydrolyzed or broken down into its constituent amino acids. The protein may be completely or partially hydrolyzed. For example, providing partially hydrolyzed protein (with a degree of hydrolysis between 2% and 20%) may be desirable for infants or young children considered at risk of bovine milk allergies. If a hydrolyzed protein is required, the hydrolysis process can be carried out as needed and as is known in the art. For example, whey protein hydrolysates can be prepared by enzymatic hydrolysis of whey fractions in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it has been found that the protein undergoes far less lysine blockage during the hydrolysis process. This allows the degree of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which significantly improves the nutritional quality of the protein source.

[0473] In one embodiment of the invention, at least 70% of the protein is hydrolyzed, preferably at least 80%, such as at least 85%, even more preferably at least 90%, such as at least 95%, and particularly at least 98%. In one specific embodiment, 100% of the protein is hydrolyzed.

[0474] In one specific embodiment, the protein in the nutritional composition is hydrolyzed, fully hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be between 8 and 40, or between 20 and 60, or between 20 and 80, or greater than 10, 20, 40, 60, 80, or 90.

[0475] Alternatively, the protein component may be replaced with a mixture or synthetic amino acids, for example, for premature or low birth weight infants.

[0476] In one embodiment, the nutritional composition or growing milk according to the invention is a hypoallergenic composition. In another embodiment, the composition according to the invention is a hypoallergenic nutritional composition or growing milk.

[0477] The nutritional compositions according to the invention typically contain a carbohydrate source. This is particularly preferred when the nutritional compositions of the invention are for infant formula. In this case, any carbohydrate source commonly found in infant formula can be used, such as lactose, sucrose, saccharin, maltodextrin, starch, and mixtures thereof, but one of the preferred carbohydrate sources is lactose.

[0478] The nutritional compositions according to the invention typically contain a lipid source. This is particularly relevant when the nutritional compositions of the invention are for infant formula. In this case, the lipid source can be any lipid or fat suitable for use in infant formula. Some suitable fat sources include palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids linoleic acid and α-linolenic acid, as well as small amounts of oils containing large amounts of pre-formed arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oil, may also be added. The ratio of n-6 fatty acids to n-3 fatty acids in the fat source can be from about 5:1 to about 15:1, for example from about 8:1 to about 10:1.

[0479] The nutritional compositions of the present invention may also contain all vitamins and minerals considered essential for a daily diet and required in significant amounts. Minimum requirements for certain vitamins and minerals have been determined. Examples of minerals, vitamins, and other nutrients optionally present in the compositions of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are typically added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the target population.

[0480] If necessary, the nutritional compositions of the present invention may contain emulsifiers and stabilizers, such as soybean, lecithin, mono- and di-citrate citrate, etc.

[0481] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, nucleosides, etc.

[0482] The nutritional composition of the present invention may further contain carotenoids. In some specific embodiments of the present invention, the nutritional composition of the present invention does not contain any carotenoids.

[0483] Manufacturing of nutritional compositions

[0484] The nutritional compositions according to the invention can be prepared in any suitable manner. The compositions will now be described by way of example.

[0485] For example, formulated foods (such as infant formula) can be prepared by blending protein sources, carbohydrate sources, and fat sources together in appropriate proportions. If used, an emulsifier may be added at this stage. Vitamins and minerals may be added at this stage, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc., can be dissolved in the fat source. Water (preferably water subjected to reverse osmosis) can then be mixed in to form a liquid mixture. The water temperature is suitably between about 50°C and about 80°C to aid in the dispersion of the components. Commercially available liquefying agents can be used to form the liquid mixture.

[0486] Oligosaccharides can be added at this stage, especially when the final product is in liquid form. If the final product is in powder form, these components can also be added at this stage as needed.

[0487] Then, the liquid mixture is homogenized in, for example, in two stages.

[0488] The liquid mixture can then be heat-treated to reduce the bacterial load, for example by rapidly heating the liquid mixture to a temperature between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes. This can be done by steam injection, autoclaving, or a heat exchanger (e.g., a plate heat exchanger).

[0489] The liquid mixture is then cooled, for example, to between approximately 60°C and approximately 85°C by rapid cooling. It is then homogenized again, for example, in two stages, between approximately 10 MPa and approximately 30 MPa in the first stage and between approximately 2 MPa and approximately 10 MPa in the second stage. The homogenized mixture can then be further cooled to add any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture can then be conveniently adjusted.

[0490] If the final product is a powder, the homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted into a powder. The moisture content of the powder should be less than about 5% by weight. Oligosaccharides may also be added at this stage, or alternatively, by dry mixing them with probiotic strains in the form of crystalline syrup, or by blending them with probiotic strains, followed by spray drying or freeze drying of the mixture.

[0491] If a liquid composition is preferred, the homogenized mixture can be sterilized and then filled into a suitable container under aseptic conditions, or it can be filled into a container first and then distilled.

[0492] In another embodiment, the composition of the present invention may be a supplement. The supplement may be in the form of, for example, tablets, capsules, lozenges, or liquids. The supplement may also contain protective hydrophilic colloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulation agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, flavor masking agents, weighting agents, gelling agents, and gelling agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients, and diluents, including but not limited to: water, gelatin of any source, plant gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, fillers, etc.

[0493] In addition, supplements may contain organic or inorganic carrier materials suitable for oral or parenteral administration, as well as vitamins, trace minerals, and other micronutrients recommended by government agencies such as the USRDA.

[0494] Implementation Plan

[0495] This invention provides embodiments according to the following numbered clauses:

[0496] 1. A composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture comprising 2'-fucosyllactose (2'-FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0497] 2. A composition comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a mixture of HMOs, said HMO mixture comprising 2'-fucosyllactose (2'-FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0498] 3. An assembly comprising a transitional form of Bifidobacterium longum and an HMO mixture, the HMO mixture comprising 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL) and 3'-sialylated lactose (3SL) and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0499] 4. An assembly comprising a transitional form of Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium lactis, and a mixture of HMOs, wherein the HMO mixture comprises 2'-fucosyllactose (2'-FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0500] 5. The composition or combination according to any one of the preceding clauses, wherein the composition or combination further comprises Bifidobacterium longum subsp. infantis.

[0501] 6. The composition or combination according to any one of the preceding clauses, wherein the composition or combination further comprises Bifidobacterium lactis.

[0502] 7. The composition or combination according to Clause 5 or Clause 6, wherein the Bifidobacterium longum infant subsp. is Bifidobacterium longum infant subsp. LMG 11588 or has at least 99.9% average nucleotide identity (ANI) with Bifidobacterium longum infant subsp. LMG 11588.

[0503] 8. The composition or combination according to Clause 6 or Clause 7, wherein the Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446 or an ANI having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium lactis CNCM 1-3446.

[0504] 9. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is:

[0505] (a) Capable of metabolizing one or more of the HMOs, preferably all of the HMOs; and / or

[0506] (b) 3-FL is preferred over 2'-FL.

[0507] 10. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism is capable of metabolizing a glycan substrate selected from any of the groups listed in Tables 1 to 3.

[0508] 11. The composition or combination according to any one of the preceding clauses, wherein the Bifidobacterium longum transitional microorganism encodes one or more CAZymes selected from the groups listed in Table 1, preferably wherein the Bifidobacterium longum transitional microorganism also encodes one or more CAZymes selected from Tables 2 and 3.

[0509] 12. The composition or combination according to any one of the preceding clauses, wherein 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, CMCC-P0001 (ATCC BAA-2753) and any combination thereof.

[0510] 13. The composition or combination according to any one of clauses 1 to 11, wherein 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 and CNCM I-5687 and any combination thereof.

[0511] 14. The composition or combination according to any one of the preceding clauses, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL and 3SL.

[0512] 15. The composition or combination according to any one of clauses 1 to 13, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL and 3-FL.

[0513] 16. The composition or combination according to any one of clauses 1 to 13, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL and LNnT.

[0514] 17. The composition or combination according to any one of clauses 1 to 13, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL, 3-FL and LNnT.

[0515] 18. The composition or combination according to any one of clauses 1 to 14, wherein the HMO mixture is substantially composed of the following substances:

[0516] i. 31% to 82% by weight, preferably 41% to 70% by weight of 2FL;

[0517] ii. 10% to 27% by weight, preferably 14% to 23% by weight of LNT;

[0518] iii. 4% to 11% by weight, preferably 6% to 10% by weight of DFL; and

[0519] iv. 9% to 34% by weight, preferably 11% to 29% by weight of a combination of 6SL and 3SL.

[0520] 19. The composition or combination according to any one of clauses 1 to 13 or 15, wherein the HMO mixture is substantially composed of the following substances:

[0521] i. 16% to 69% by weight, preferably 22% to 59% by weight of 2'-FL;

[0522] ii. 9% to 24% by weight, preferably 12% to 21% by weight of LNT;

[0523] iii. 2% to 10% by weight, preferably 3% to 8% by weight of DFL;

[0524] iv. 8% to 26% by weight, preferably 11% to 22% by weight of a combination of 6SL and 3SL; and

[0525] v. 18% to 50% by weight, preferably 11% to 43% by weight of 3-FL.

[0526] 20. The composition or combination according to any one of clauses 1 to 13 or 16, wherein the HMO mixture is substantially composed of the following substances:

[0527] i. 34% to 85% by weight, preferably 40% to 71% by weight of 2'-FL;

[0528] ii. 10% to 40% by weight, preferably 12% to 26% by weight of LNT;

[0529] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL;

[0530] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL; and

[0531] v. 6% to 30% by weight, preferably 7% to 22% by weight of LNnT.

[0532] 21. The composition or combination according to any one of clauses 1 to 13 or 17, wherein the HMO mixture is substantially composed of the following substances:

[0533] i. 20% to 60% by weight, preferably 22% to 55% by weight of 2'-FL;

[0534] ii. 4% to 30% by weight, preferably 6% to 20% by weight of LNT;

[0535] iii. 1% to 12% by weight, preferably 2% to 8% by weight of DFL;

[0536] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL;

[0537] v. 10% to 50% by weight, preferably 13% to 46% by weight of 3-FL; and

[0538] vi. 3% to 25% by weight, preferably 5% to 20% by weight of LNnT.

[0539] 22. The composition according to any one of clauses 1 to 3 or 6 to 21, wherein the composition further comprises a polysaccharide substrate selected from any group listed in Tables 1 to 3.

[0540] 23. The composition according to any one of clauses 1 to 3 or 6 to 22, wherein the composition is a nutritional composition selected from infant formula, stage 1 infant formula, follow-up formula or stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers, or supplements.

[0541] 24. The composition according to any one of clauses 1 to 3 or 6 to 23, wherein the composition is used to prevent an individual from infection, reduce the risk of an individual from infection, and / or treat an individual from infection.

[0542] 25. The combination according to any one of Clauses 4 to 23, wherein the combination is used to prevent an individual from becoming infected, to reduce the individual’s risk of becoming infected, and / or to treat the individual’s infection.

[0543] 26. Use of the composition according to any one of Clauses 1 to 3 or 6 to 23 in the preparation of a medicament for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in said individual.

[0544] 27. Use of the combination of any one of Clauses 4 to 23 in the preparation of a medicament for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in said individual.

[0545] 28. A method for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in said individual, said method comprising administering to said individual a composition according to any one of clauses 1 to 3 or 6 to 23.

[0546] 29. A method for preventing infection in an individual, reducing the risk of infection in an individual, and / or treating infection in said individual, said method comprising administering to said individual a combination according to any one of clauses 4 to 23.

[0547] 30. A prebiotic, wherein the prebiotic is used to prevent, reduce the risk of infection in an individual, and / or treat an individual’s infection by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the individual’s gut, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0548] 31. The use of prebiotics in the preparation of medicaments for preventing, reducing the risk of infection in, and / or treating infection in an individual by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the individual's gut, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0549] 32. A method for preventing, reducing the risk of infection in an individual, and / or treating an individual’s infection by promoting the growth and / or survival of a transitional Bifidobacterium longum microorganism in the individual’s gut, the method comprising administering a prebiotic to the individual, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

[0550] 33. A composition, combination, use, method, or prebiotic for the purpose of any one of Clauses 24 to 32, wherein the composition, combination, or prebiotic is used to prevent infection in the individual and / or reduce the individual's risk of infection.

[0551] 34. A composition, combination, use, method, or prebiotic used for the purpose according to any one of clauses 24 to 33, wherein the infection is a viral, bacterial, or fungal infection.

[0552] 35. A composition, combination, use, method, or prebiotic used for the purpose according to any one of clauses 24 to 34, wherein the infection is an airway infection.

[0553] 36. A composition, combination, use, method, or prebiotic used for the purpose according to any one of clauses 24 to 35, wherein the infection is a viral airway infection.

[0554] 37. A composition for the stated purpose, a combination for the stated purpose, the stated purpose, the method thereof, or a prebiotic for the stated purpose, wherein the viral airway infection is selected from influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza virus, metapneumovirus, coronavirus, adenovirus, and bocavirus.

[0555] 38. A composition, combination, use, method, or prebiotic used for the stated purpose in accordance with Clause 36 or Clause 37, wherein the viral airway infection is influenza virus, respiratory syncytial virus, or rhinovirus.

[0556] 39. A composition, combination, use, method, or prebiotic for the purpose according to any one of clauses 34 to 38, wherein the viral infection causes a disease selected from the group consisting of: the common cold, influenza, bronchitis, bronchiolitis, and pneumonia, preferably bronchiolitis or pneumonia, more preferably bronchiolitis.

[0557] 40. The composition according to any one of clauses 1 to 3 or 6 to 23, wherein the composition is used to promote long-term immune benefits in an individual.

[0558] 41. The combination according to any one of clauses 4 to 23, wherein the combination is used to promote long-term immune benefits in an individual.

[0559] 42. Use of the composition according to any one of Clauses 1 to 3 or 6 to 23 for the preparation of a medicament for promoting long-term immune benefits in an individual.

[0560] 43. Use of the combination of any one of clauses 4 to 23 in the preparation of a medicament for promoting long-term immune benefits in an individual.

[0561] 44. A method for promoting long-term immune benefits in an individual, the method comprising administering to the individual a composition according to any one of clauses 1 to 3 or 6 to 23.

[0562] 45. A method for promoting long-term immune benefits in an individual, the method comprising administering to the individual a combination according to any one of clauses 4 to 23.

[0563] 46. ​​A composition, combination, use, or method for the stated purpose according to any one of clauses 40 to 45, wherein the promotion of long-term immune benefits in an individual includes:

[0564] i. Promote long-term respiratory health;

[0565] ii. To prevent allergen sensitization and / or reduce the risk of allergen sensitization; and / or

[0566] iii. To prevent and / or reduce the risk of developing respiratory illnesses.

[0567] 47. The composition according to any one of clauses 1 to 3 or 6 to 23, wherein the composition is used for i) preventing an individual from becoming sensitized to an allergen and / or reducing the individual’s risk of becoming sensitized to an allergen and / or ii) preventing the individual from developing respiratory symptoms and / or reducing the individual’s risk of developing respiratory symptoms.

[0568] 48. The combination of any one of the clauses or 4 to 23 is used for i) preventing an individual from becoming sensitized to an allergen and / or reducing the individual’s risk of becoming sensitized to an allergen and / or ii) preventing the individual from developing respiratory symptoms and / or reducing the individual’s risk of developing respiratory symptoms.

[0569] 49. The use of the composition according to any one of Clauses 1 to 3 or 6 to 23 for the preparation of a medicament for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0570] 50. Use of the combination of any one of the clauses or 4 to 23 for the preparation of a medicine for i) preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms.

[0571] 51. i) a method for preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) a method for preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms, said method comprising administering to said individual a composition according to any one of clauses 1 to 3 or 6 to 23.

[0572] 52. i) methods for preventing an individual from becoming sensitized to an allergen and / or reducing the risk of an individual becoming sensitized to an allergen and / or ii) methods for preventing an individual from developing respiratory symptoms and / or reducing the risk of an individual developing respiratory symptoms, said methods comprising administering to said individual a combination according to any one of clauses 4 to 23.

[0573] 53. A composition, combination, use, or method thereof for the stated purpose, according to any one of clauses 46 to 53, wherein the respiratory condition is a chronic inflammatory disease of the respiratory tract or an allergic respiratory disease.

[0574] 54. The composition used for the stated purpose, the combination used for the stated purpose, the stated purpose, or the method thereof, wherein the chronic inflammatory disease of the respiratory tract is asthma or chronic obstructive pulmonary disease (COPD).

[0575] 55. The composition, combination, use, or method thereof for the stated purpose, as described in Clause 53, wherein the allergic respiratory disease is recurrent wheezing and asthma, preferably allergic asthma.

[0576] 56. A composition, combination, use, or method thereof for the stated purpose according to any one of Clauses 47 to 55, wherein the composition and / or combination is used to i) prevent the individual from becoming sensitized to an allergen in the future and / or reduce the risk of the individual becoming sensitized to an allergen in the future and / or ii) prevent the individual from developing respiratory symptoms in the future and / or reduce the risk of the individual developing respiratory symptoms in the future.

[0577] 57. The composition according to any one of clauses 1 to 3 or 6 to 23, wherein the composition is used to prevent an individual from developing asthma and / or to reduce the individual's risk of developing asthma.

[0578] 58. The combination according to any one of Clauses 4 to 23, wherein the combination is used to prevent an individual from developing asthma and / or reduce the individual's risk of developing asthma.

[0579] 59. Use of the composition according to any one of Clauses 1 to 3 or 6 to 23 for the preparation of a medicament for the prevention of asthma in an individual and / or the reduction of the risk of developing asthma in an individual.

[0580] 60. Use of the combination of any one of Clauses 4 to 23 in the preparation of a medicament for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma.

[0581] 61. A method for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma, said method comprising administering to said individual a composition according to any one of clauses 1 to 3 or 6 to 23.

[0582] 62. A method for preventing an individual from developing asthma and / or reducing the risk of an individual developing asthma, said method comprising administering to said individual a combination according to any one of clauses 4 to 23.

[0583] 63. A composition, combination, use, or method thereof for the stated purpose, according to any one of clauses 57 to 62, wherein the asthma is allergic asthma.

[0584] 64. A composition, combination, use, or method thereof for the purpose according to any one of clauses 47 to 63, wherein the composition is used to prevent the individual from developing asthma later in life and / or to reduce the individual's risk of developing asthma later in life.

[0585] 65. The composition, combination, use, or method used for the stated purpose under Clause 56 or Clause 64, wherein the subsequent period is from 12 months to 12 years after the termination of the treatment.

[0586] 66. A composition, combination, use, method, or prebiotic used for the purpose according to any one of clauses 24 to 65, wherein the individual is an infant, toddler, or child.

[0587] 67. A composition, combination, use, method, or prebiotic used for the purpose pursuant to Clause 66, wherein the individual is an infant or young child.

[0588] 68. A composition, combination, use, method, or prebiotic used for the purpose according to any one of clauses 24 to 67, wherein the composition, combination, and / or prebiotic increases the level of IL-6 in the individual.

[0589] 69. A composition, combination, use, method, or prebiotic for the purpose according to any one of clauses 24 to 68, wherein the composition, combination, and / or prebiotic increases the level of short-chain fatty acids (SCFA) in the individual.

[0590] 70. A composition, combination, use, method, or prebiotic used for the purpose pursuant to Clause 69, wherein the SCFA is selected from acetate, butyrate, and / or propionate.

[0591] 71. A composition, combination, use, method, or prebiotic for the purpose according to any one of clauses 24 to 68, wherein the composition, combination, and / or prebiotic increases the level of indole-3-propionic acid in the individual.

[0592] 72. A composition, combination, use, method, or prebiotic for the purpose according to any one of clauses 24 to 71, wherein the composition, combination, and / or prebiotic modulates the permeability of the intestinal epithelial barrier; preferably wherein the composition, combination, and / or prebiotic reduces the permeability of the intestinal epithelial barrier.

[0593] Those skilled in the art will understand that they are free to combine all the features of the invention disclosed herein. In particular, features described for the products of the invention can be combined with the methods of the invention, and vice versa. Furthermore, features described for different embodiments of the invention can be combined. Where known equivalents exist for a particular feature, such equivalents are incorporated as expressly mentioned in this specification.

[0594] Further advantages and features of the invention will become apparent upon reference to the accompanying drawings and non-limiting embodiments.

[0595] Example

[0596] Example 1: Transitional Bifidobacterium longum increases the production of short-chain fatty acids

[0597] 3-Fucose-based lactose (3-FL), short-chain fatty acids (SCFA), tricarboxylic acid (TCA) intermediates, and SCFA intermediates were measured using 1H-NMR. Results are shown in... Figure 2 and Figure 3 The heatmap highlights the dynamics of consumption and production of key metabolites in the SCFA pathway by displaying the Z-scores of the abundance of each metabolite at T0, T24, and T48.

[0598] Total SCFA corresponds to the sum of peak integrals for acetate, butyrate, and propionate. Significant differences in metabolite Z-scores between transitional *Bifidobacterium longum* or *Bifidobacterium longum* species and those without supplementation were calculated using ANOVA and highlighted with an asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001). Significant differences in metabolite Z-scores between transitional *Bifidobacterium longum* and *Bifidobacterium longum* species were calculated using ANOVA and highlighted with a circle (° p < 0.05, °° p < 0.01, ° ° ° p < 0.001). Box plots indicate the abundance of transitional *Bifidobacterium longum* or *Bifidobacterium longum* species at 48 hours or during fermentation (i.e., strain-specific gene copies measured by qPCR). Figure 2 and Figure 3 This demonstrates that the transitional Bifidobacterium longum is well integrated into the microbial community, exhibits metabolic activity towards 3-FL or pea fiber, and produces more SCFA than Bifidobacterium longum infantis subsp.

[0599] Figure 2 The experiment showed that SCFA (acetate, butyrate and propionate) was produced by batch fermentation of 3-fucosyl lactose (3-FL) over 48 hours.

[0600] Figure 3 The experiment showed that SCFA (acetate, butyrate, and propionate) was produced by batch fermentation of pea fiber (rich in arabinogalactan) over 48 hours.

[0601] Example 2: Transitional Bifidobacterium longum increases the anti-infective cytokine IL-6

[0602] Monocytes were isolated from the yellow layer of blood clots from healthy donors. One hundred thousand monocytes were seeded into each well of a 96-well plate and incubated with 1e6 CFU of transitional Bifidobacterium longum for 24 hours for immunotraining. Cells were washed by centrifugation and allowed to stand for 6 days. Monocytes were stimulated with LPS for 24 hours. IL-6 in the cell culture supernatant was then measured to assess immunotraining (see [link to relevant documentation]). Figure 4 The bars indicate the median IL-6 production from the three donors, and the dashed lines indicate the IL-6 levels in untrained monocytes.

[0603] method

[0604] Immunospectral analysis using PBMC cells

[0605] Peripheral blood mononuclear cells (PBMCs) were isolated from the yellow layer of blood clots obtained from healthy adults using a density gradient. The PBMCs were then separated at a density of 1.5 × 10⁻⁶. 6 PBMCs were seeded at 100 cells / ml in 48-well Isocove modified Dulbecco medium (cIMDM) containing 10% fetal bovine serum, 1% glutamine, 1% penicillin / streptomycin, and 0.1% gentamicin. PBMCs were stimulated for 36 hours in the presence of different bacterial strains, including 10... 7 All transitional Bifidobacterium longum isolates and probiotic strains were collected at CFU / ml. Cell culture supernatant was collected to assess cytokine expression of IL-10 and IL-12p40 by ELISA. Standard curves for each cytokine were used to calculate the absolute amount (picograms / ml) from optical density readings.

[0606] Example 3: Transitional Bifidobacterium longum increases intestinal epithelial barrier resistance

[0607] In vitro experiments using the human colorectal adenocarcinoma cell line (Caco-2) have shown that transitional Bifidobacterium longum strains can increase transmembrane resistance (TEER) when incubated with epithelial cells.

[0608] Caco-2 cells were seeded on Transwell plates and grown for 3 weeks. A Caco-2 monolayer was then inoculated with transitional Bifidobacterium longum NCC5002 (black line) (4.10). 6 CFU / well), Bifidobacterium lactis NCC2818 (gray line) (4.10) 6CFU / well or vector (dashed line) were pre-incubated for 24 hours (0–24) in the presence of 10 ng / mL IFNγ. Following this period, cells were restimulated with 50 ng / mL TNFα (a pro-inflammatory cytokine) for 24 hours (24–48), followed by a 24-hour recovery period (48–72). Transmembrane resistance was measured at 0, 24, 48, and 72 hours. Data are presented as mean ± SD. For each time point, statistical differences were assessed using a two-way ANOVA with Dunnett's test for multiple comparisons, and are indicated by asterisks or hash marks for NCC5002 and NCC2818, respectively. * / # = p<0.05;** / ## =P<0.01 (compared to the control group) Figure 5 ).

[0609] Caco-2 cells were seeded on Transwell plates and grown for 3 weeks. A Caco-2 monolayer was then inoculated with transitional Bifidobacterium longum NCC5002 (black line) (4.10). 6 CFU / well), Bifidobacterium lactis NCC2818 (gray line) (4.10) 6 CFU / well or vector (dashed line) were pre-incubated for 24 hours (0–24) in the presence of 10 ng / mL IFNγ. Following this period, cells were restimulated with 50 ng / mL TNFα pro-inflammatory cytokine for 24 hours (24–48), followed by a 24-hour recovery period (48–72). At the 72-hour time point, the permeability of the Caco-2 monolayer was assessed by measuring the flux of fluorescein sulfonic acid (478 Daltons) across the epithelium over 180 minutes. Data are presented as mean ± SD. For each time point, statistical differences were assessed using a two-way ANOVA with Dunnett's test for multiple comparisons and indicated by an asterisk. * = p < 0.05 (compared to control group) Figure 6 ).

[0610] method

[0611] CACO-2 cell culture and transmembrane resistance measurement

[0612] Caco-2 cells (HTB-37; American Type Culture Collection) were seeded into 24-well semi-permeable inserts. Caco-2 monolayers were cultured for 14 days, with the medium changed three times weekly, until a functional cell monolayer with transmembrane resistance (TEER) was obtained. Cells were maintained in Dulbecco modified Eagle medium (DMEM) containing glucose and glutamine, supplemented with HEPES and 20% (v / v) heat-inactivated fetal bovine serum. The TEER of the Caco-2 monolayer (=0-hour time point) was measured before adding bacteria to the top compartment. The TEER of the empty insert was subtracted from all readings to calculate the residual resistance of the insert. A probiotic strain (directly derived from the glycerol stock) was then diluted in Caco-2 complete medium and added to the top of the Caco-2-containing insert at a colony-forming unit (CFU) of 2 × 10⁶. Cells were also exposed to Caco-2 complete medium (CM) in two control compartments and 0.75% glycerol in the top compartment as a vector 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 values ​​(to account for differences in initial TEER between different inserts) and expressed as a percentage of the initial values.

[0613] Example 4: Analysis of carbohydrate-active enzyme (CAZyme) genes in a transitional microorganism of Bifidobacterium longum

[0614] Combined with the dbCAN2 tool (Zhang et al., Nucleic Acids Res.46(W1):W95-W101 (2018)) and the databases HMMdb (v9) and Diamond (v2.0.8). Figure 7 The genomes of the subspecies of *Bifidobacterium longum* listed in the documentation were annotated as CAZyme. Query sequences with a coverage >0.50 and an e-value <1e-15 were annotated using 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., NucleicAcids Res.50(D1):D571-D577 (2022)) (http: / / www.cazy.org / ), with an identity >0.90 and an e-value <1e-102. In cases where CAZyme annotations for query sequences did not match between the HMMER and DIAMOND tools, HMMER annotation was preferred. Only the CAZyme family and subfamilies encoding glycoside hydrolases (GH) and polysaccharide lyases (PL) were used for comparative analysis of *Bifidobacterium longum* subspecies (see [link to documentation]). Figure 7 ).

[0615] Example 5: Utilization of polysaccharide substrates

[0616] The pulverized or homogenized fecal sample was mixed 10-fold with PBS / glycerol (1 / 10) (w / v) and then centrifuged at 2000g for 2 minutes. The slurry and pellets were then stored at -80°C. Frozen fecal samples were thawed from -80°C storage before centrifugation at 2000g for 2 minutes. The resulting supernatant was inoculated using a medium based on the medium disclosed in the following literature: Daguet et al. (Journal of Functional Foods; 2016; 20; 369-379). This medium was supplemented with 5 g / L of the specific fiber to be tested and 5 E07 CFU / ml of Bifidobacterium supplement.

[0617] Cultures were established at 37°C under a nitrogen gas flow to ensure anaerobic conditions and gentle stirring. Aliquots were taken and analyzed at specified time points.

[0618] The growth of the Bifidobacterium longum transitional strain NCC5001 was promoted by pectin (sugar beet) and arabinogalactan (larch wood). Figure 8 ).

[0619] The growth of the Bifidobacterium longum transitional strain NCC5002 was promoted by arabinogalactan (larch wood) and starch (potato). Figure 9 ).

[0620] Example 6: Characterization of the transitional microorganism Bifidobacterium longum

[0621] Transitional strains of *Bifidobacterium longum* were isolated from feces of breastfed infants using Eugon tomato agar (ETA). The obtained isolates were sequenced using PacBio to obtain the fully closed-assembled genome of each strain. Each strain, along with its genome sequence data, was deposited at the Nestlé In-House Culture Collection (NCC, Lausanne, Switzerland) and the National Collection of Microorganisms (CNCM) at the Pasteur Institute (Paris, France). The genomes of the strains were compared using OrthoAni (https: / / www.ezbiocloud.net / tools / orthoani) by average nucleotide identity (ANI) to other publicly available genomes representing the overall diversity of *Bifidobacterium longum* species (Table 5), and to metagenomically assembled genomes (MAGs) obtained from metagenomic sequences published from feces of infants in the same cohort.

[0622] Table 5 - List of genomes used for ANI analysis and their publicly available references. (T) represents the type strain.

[0623] Analysis showed that the newly described strains, grouped together with MAG from the same cohort, defined a well-defined clade belonging to the *Bifidobacterium longum* species. Two previously isolated strains, BSM11-5 and 3_mod, were found to be grouped within this newly described clade. This clade is genetically distinct from the *Bifidobacterium longum* subspecies (96.40% ANI). This clade is associated with *Bifidobacterium longum* subspecies (98.207%) and with the previously identified group of strains (JDM301, CMCC_P0001, and BXY01) (O'Callaghan et al., 2015), but still exhibits significant differences, sharing 98.260% identity with that group. Figure 1 A phylogenetic tree based on ANI UPGMA is shown. The scale indicates the percentage (%) of identity at each branch point.

[0624] The selection of the above-mentioned genomes, representing the diversity of *Bifidobacterium longum* subspecies, was annotated with carbohydrate-active enzymes (CAZY) using the dbCAN annotation pipeline (http: / / bcb.unl.edu / dbCAN / ). The results showed that *Bifidobacterium longum* subsp. *suis*, *Bifidobacterium longum* subsp. *suillum*, and *Bifidobacterium longum* subsp. *suillum* strains contain the GH20 (lactose-N-glucosidase) enzyme involved in the degradation and metabolism of lactose-N-tetrasaccharides (LNT). Similar to *Bifidobacterium longum* subsp. *infant* strains, *Bifidobacterium longum* transitional strains also possess similar enzymes and additionally carry the GH29 (fucosidase) gene encoding fucosylated human milk oligosaccharides (such as 2'-FL, 3-FL, or diFL). In addition, three strains (CNCM I-5684, BSM1-15 and 3_mod) also carry the GH 33 (sialidase) encoding gene involved in the degradation and metabolism of sialylated HMOs (such as 3'SL or 6'SL) (Table 6).

[0625] Table 6 - GH20 (lacto-N-glucosidase) and GH29 (α-fucosidase) are encoded in the genomes of each representative. The genes for GH95 (α-fucosidase / α-galactosidase) and GH33 (sialidase) glucosyl hydroxylase family enzymes. quantity.

[0626] All newly obtained genomes were compared and aligned with the genomes of two strains (Bifidobacterium longum infantis ATCC15697 and Bifidobacterium kanamycin DSM 21854), which belong to species in which genes responsible for the utilization of fucosylated HMOs have been elucidated (James et al., 2019).

[0627] result

[0628] like Figure 11 As shown, all newly described strains contain genes responsible for utilizing fucosylated HMOs. The NCC5001 tissue reflects one of the *Bifidobacterium longum* subsp. *infant* ATCC 15697, while all other strains (NCC 5000, NCC5002, NCC 5003, NCC 5004) carry gene tissues more closely related to *Bifidobacterium kanamycin* DSM 21854. Overall, the similarity of fucosidases to the fully described *Bifidobacterium longum* subsp. *infant* ATCC 15697 was higher than 77% (for BLON_2334) and 88% (for BLON_2335) among all newly described strains.

[0629] Example 7: Utilization of Fucosylated HMOs

[0630] All strains obtained from the Nestlé Culture Collection were reactivated from freeze-dried stock solutions using two consecutive culture steps (16 h, 37 °C, anaerobic) in MRS supplemented with 0.05% cysteine ​​(MRSc). (Table 7) The reactivated cultures were then centrifuged, washed, and resuspended in 1 volume of PBS. The washed cells were then inoculated into carbon-free MRS-based medium (MRSc-C) (10 g l⁻¹ bactoproteose peptone n°3, 5 g l⁻¹ bacterial yeast extract, 1 g l⁻¹ Tween 80, 2 g l⁻¹ diammonium citrate, 5 g l⁻¹ sodium acetate, 0.1 g l⁻¹ magnesium sulfate, 0.05 g l⁻¹ manganese sulfate, 2 g l⁻¹ disodium phosphate, 0.5 g l⁻¹ cysteine), with glucose, 2'-FL, or 3'-FL added at a concentration of 0.5% as the sole carbon source. The microplates were then grown in 96-well microplates, with each well containing 200 µl. Incubation was performed anaerobically for 48 h, and the optical density was measured at 600 nm using a spectrophotometer. Figure 9 As shown, all Bifidobacterium longum transitional strains were grown on fucosylated HMOS.

[0631] result

[0632] All Bifidobacterium longum transitional strains grew better on 3'-FL than on 2'-FL, and achieved higher cell densities on this carbohydrate. This behavior indicates that 3'-FL is superior to 2'-FL (ratio 1.8 to 2.8 – see [link]). Figure 12 This was not observed in Bifidobacterium longum infant subspecies LMG 11588.

[0633] Table 7 - List of strains (and corresponding numbers) used for studies on the growth of single fucosylated HMOs

[0634] Example 8: Efficacy testing of synbiotics in a PVM infection model and a pollution-enhanced allergic airway inflammation model

[0635] Detailed experimental design

[0636] Figure 14 A schematic diagram of a model of early-life viral airway infection and allergic airway inflammation exacerbated by pollution is shown. The experimental design is described in more detail below.

[0637] Starting three weeks before gestation, WT C57BL / 6 mice were fed a control fiber or low-fiber diet. Throughout the experiment, from day 0 (PND) to day 66 (PND66), the offspring of these mice were fed a diet corresponding to that of their mothers.

[0638] At PND5, young mice fed a low-fiber diet were randomly assigned to different experimental groups (n=14-16 / group). Mice were fed once daily with different combinations of nutrients (6HMO; Bifidobacterium infantis LGM11588; Bifidobacterium infantis LGM11588+6HMO) via intragastric gavage (ig) at a concentration of 50 μl in saline solution. This continued until PND20. The control group was fed saline solution only.

[0639] In PND10, all animals (n=13–16 / group) were intranasally infected with 10 PFU of PVM (PVM, J3666) in a total volume of 40 μl saline solution (20 μl / nostril) to induce bronchiolitis. The virus-containing solution was delivered as droplets into the nostrils using a P20 pipette. Mice anesthetized with isoflurane were kept in a supine position to ensure proper aspiration of the solution.

[0640] Animals (n=7-8 / group) were sacrificed at PND20 (the peak of the immunopathological period of bronchiolitis) to measure virus-induced lung inflammation and related pathology.

[0641] Starting from PND42, animals recovering from PVM infection (n=7-8 / group) were intranasally sensitized once a week (i.e., at PND42, PND49, PND56, PND63) with a combination of cockroach allergen extract (CRE, 1 mg / application) and particulate PM2.5 (10 mg), ending at PND63, to induce allergic airway inflammation exacerbated by pollution.

[0642] Animals were sacrificed at PND20 (N=7-8 / group, peak of immunopathological bronchiolitis) to test the effect of nutritional intervention on promoting protective immunity, and at PND66 (N=7-8 / group) to assess susceptibility to contamination-induced allergic respiratory inflammation following viral airway infection.

[0643] Probiotic dosage

[0644] 10 6 CFU / day.

[0645] HMO dosage

[0646] Based on the latest recommendations for HMOs in infant formula, a concentration of 4 mg / day and a mixture of 6 HMOs (2FL / DFL, LNT, 3SL, 6SL, 3FL) were selected (Phase 1: 1.8-2 g / L / day for the first 6 months).

[0647] The calculations are based on the following: 1-week-old infants consume 500 mL of breast milk per day and 10-day-old mouse pups consume up to 2 mL of milk per day (Source: contemporary topics in laboratory animal science / American Association for Laboratory Animal Science 43(3):50-3).

[0648] The ratios of HMO components are inspired by the composition of breast milk: 2FL / DFL component 53%; LNT component 19%; 6SL component 8%; 3SL component 6.2%; 3FL component 13.8%.

[0649] In some experiments, based on the latest recommendations for HMOs in infant formula, a concentration of 4 mg / day and a blend of 5 HMOs (2FL, DFL, LNT, 3SL, 6SL) were selected (Phase 1: 1.8-2 g / L / day for the first 6 months). The proportions of the 5 HMO blends used were: 2FL / DFL 65.70%; LNT 23.40%; 6SL 9%; and 3SL 2%.

[0650] low fiber diet

[0651] WT C57BL / 6 mice effectively cleared PVM virus. This mild infection was not associated with changes in lung tissue remodeling, which predispose infants to allergic airway inflammation later in life. To increase susceptibility to PVM infection and associated risk of allergic airway inflammation in adulthood, mother mice and their corresponding offspring were fed a low-fiber diet throughout the experiment (susceptible group; Trompette, Nat. Med, 2014). Animals fed a normal-fiber diet served as protected controls (protected group).

[0652] Statistical analysis

[0653] Nonparametric two-way ANOVA was used, followed by Bonferroni post-hoc tests to determine statistical significance. A result was considered significant if p ≤ 0.05. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. All statistical analyses were performed using Prism GraphPad software.

[0654] result

[0655] PVM infection model

[0656] Mouse pneumonia virus (PVM) is associated with the human respiratory syncytial virus (RSV) pathogen, affecting >90% of children under two years of age. PVM has been used to study respiratory viral replication and subsequent inflammatory responses as part of the natural host-pathogen relationship. Therefore, PVM infection in mice reproduces many of the clinical and pathological features of a more severe form of RSV infection in infants—which, if not properly controlled, has lasting consequences for lung function and predisposes the individual to anaphylactic airway reactions later in life (Dyer KD, Viruses, 2012). The highly pathogenic PVM strain J3666 was chosen for these studies to align with the human pathophysiology of RSV infection in infants.

[0657] Severe bronchiolitis early in life is associated with excessive mucus secretion and airway epithelial cell shedding. Dead epithelial cells, along with viscous exudate, can form dense plugs in the bronchoalveolar spaces, obstructing respiration. Controlled mucus secretion and epithelial cell death are beneficial defense mechanisms that limit viral transmission. These responses need to be strictly regulated after viral clearance to avoid chronic and pathological tissue remodeling.

[0658] The pathological features of severe viral lower respiratory tract infections include airway epithelial cell (AEC) shedding, excessive mucus secretion, and airway smooth muscle (ASM) remodeling. AEC shedding is characteristic of viral bronchiolitis and is associated with disease severity and viral load.

[0659] AEC shedding was quantified by measuring the length of shed airway epithelium and expressing it as a percentage of the basement membrane length of at least five airways in each mouse. Quantitative assessment of mucus secretion and pathological lung tissue remodeling (as another marker of severe bronchiolitis) was performed by quantifying Muc5ac-positive AECs, and ASM remodeling was assessed by area / airway circumference (Lynch JP, JEM 2018).

[0660] Compared with placebo, early life nutritional intervention using synbiotics (Bifidobacterium infantis + 6HMO) significantly reduced infection-mediated airway epithelial cell (AEC) shedding. Figure 15A) Mucus secretion ( Figure 15 B) and pathological tissue remodeling ( Figure 15 C). Notably, synbiotic intervention showed superior efficacy compared to prebiotic (6HMO) or probiotic (Bifidobacterium infantis) intervention alone in reducing infection-mediated AEC shedding, mucus secretion, and pathological airway remodeling.

[0661] To evaluate whether early life nutritional supplementation with synbiotics, in addition to evoking an effective antiviral immune response, also leads to controlled resolution of lung inflammation after viral clearance, the number of neutrophils and / or eosinophils in the airways of mice infected with PVM was assessed by flow cytometry. Importantly, early life supplementation with synbiotics resulted in controlled resolution of lung inflammation after viral clearance, as assessed by a significant reduction in the number of granulocytes (neutrophils and eosinophils) in the airways in response to PVM infection.

[0662] Early life nutritional intervention using a second synbiotic (Bifidobacterium infantis + 5HMO) resulted in a significant upregulation of the antiviral cytokine IFN-λ 5 days post-infection (peak of viral infection). Figure 18 A), which is related to factors such as the number of lung neutrophils ( Figure 18 B) and the number of eosinophils in the lungs ( Figure 18 C) The significantly reduced quantified granulocyte infiltration into the inflamed lung is associated with intervention with synbiotics (Bifidobacterium infantis + 5HMO) in early life, indicating that intervention can reduce virus-induced severe lung inflammation. These data suggest that early life supplementation with synbiotics can control an effective antiviral immune response and promote rapid resolution of lung inflammation, thus facilitating recovery from respiratory infections.

[0663] A model of allergic airway inflammation exacerbated by pollution

[0664] Severe RSV infection early in life represents a significant risk factor for the later development of allergic airway diseases. Outdoor air pollution is a major health problem worldwide. Exposure to particulate matter (PM) is associated with the exacerbation of several respiratory diseases, including allergic asthma. To simulate this globally relevant condition, animals recovering from severe PVM infection were sensitized with cockroach allergens in the presence of pollutant PM2.5 to induce allergic airway inflammation. This experimental approach allows testing whether early-life (window of opportunity) nutritional interventions not only alter susceptibility to viral bronchiolitis but may also have lasting immune benefits into adulthood.

[0665] Early-life respiratory viral infections represent a potential tipping point in the balance between long-term respiratory health and chronic airway diseases such as airway hypersensitivity. This study shows that early-life synbiotic intervention not only reduces the severity of airway viral infections but also promotes immune benefits that persist into adulthood, as assessed by reducing susceptibility to pollution-exacerbated allergic airway inflammation.

[0666] This is manifested by lung ILC2 as assessed by flow cytometry ( Figure 16 A) and eosinophil count ( Figure 16 Both (B) decreased. The reduction in lung inflammation was also associated with a decrease in mucus score ( Figure 17 A) and organizational restructuring and improvement ( Figure 17 (B) is associated with this. Notably, it is consistent with reduced susceptibility to viral airway infections (see [link]). Figure 15 Synbiotic intervention (Bifidobacterium infantis + 6HMO) showed superior efficacy to prebiotic (6HMO) or probiotic (Bifidobacterium infantis) intervention alone in reducing allergic airway inflammation exacerbated by pollution in adulthood.

[0667] in conclusion

[0668] Using a PVM-induced bronchiolitis and early-life nutritional intervention model in newborn mice, we demonstrated the effectiveness of synbiotic intervention (a combination of Bifidobacterium infantis and human milk oligosaccharides (5HMO or 6HMO)). It can provide fine-grained antiviral inducement Prevention of bronchitis. Importantly, we demonstrated that synbiotic intervention (a combination of Bifidobacterium infantis and human milk oligosaccharides (6HMOs)) provides defense against virus-induced bronchiolitis. Provides excellent efficacy It is superior to 6HMO alone or Bifidobacterium infantis alone. Specifically, synbiotic intervention leads to rapid resolution of virus-induced lung inflammation and appropriate lung tissue remodeling after virus clearance.

[0669] Since the initial host response to respiratory viruses represents a potential tipping point in the balance between long-term respiratory health and chronic respiratory disease, these findings suggest that early-life nutritional interventions using these synbiotics (Bifidobacterium infantis + 6 HMO or Bifidobacterium infantis + 5 HMO) may represent a preventative strategy against severe bronchiolitis in infants and the associated risk of developing allergic airway disease in school age. Indeed, early-life nutritional synbiotic interventions not only reduced the severity of airway viral infections but also promoted immune benefits that persist into adulthood, as assessed by reducing susceptibility to pollution-exacerbated allergic airway inflammation. In particular, these results provide strong evidence that early-life nutritional synbiotic interventions attenuate pathological airway remodeling, which is known to increase the risk of developing chronic inflammatory diseases later in life, such as asthma or COPD.

[0670] Example 9: A preclinical model for efficacy testing of Bifidobacterium adolescentis in an infection model.

[0671] Detailed experimental design

[0672] In vivo preclinical models of infection have been developed, such as Figure 19 As shown.

[0673] Starting three weeks before gestation, WT C57BL / 6 mice were fed either a control fiber or low-fiber diet. Throughout the experiment, the offspring of these mice were fed the same diet as their mothers.

[0674] The control group was fed by mothers who were fed a low-fiber diet only (susceptible group) or a high-fiber diet only (protected group) before weaning and maintained the same diet after weaning.

[0675] Five days after birth (PND), pups fed a low-fiber diet were randomly assigned to different experimental groups (n=8 / group). C57BL / 6 WT pups received different combinations of nutrients via oral gavage (6 HMOs + 2 Bifidobacterium strains) (Bifidobacterium infantis LGM11588 and Bifidobacterium lactis CNCM 1-3446); 6 HMOs + 3 Bifidobacterium strains (Bifidobacterium infantis LGM11588, Bifidobacterium lactis CNCM 1-3446, and Bifidobacterium adolescentis) while being fed by their low-fiber-dieted mothers. A wide range of antibiotics were administered via drinking water from PND16 to PND26. After weaning at PND21, a selective fiber mixture (adapted to a transitional strain of Bifidobacterium longum) was introduced into the diet of these mice, while they were orally gavaged with the same nutrients (reduced doses of HMOs + Bifidobacterium strains). Table 8 shows the HMO mixtures used in the pre- and post-weaning phases.

[0676] PND35 mice were infected with pneumonia virus.

[0677] Table 8 - HMO mixtures used in the study

[0678] result

[0679] like Figure 20 As shown, when compared with a low-fiber diet (Group 2) or a low-fiber diet mixture without Bifidobacterium longum transition strain (Group 3), the nutritional composition containing a mixture of Bifidobacterium longum transition strains (Group 4) provides better protection against post-weaning airway viral infections, as shown by weight gain (%PND35) over the days following infection.

[0680] Example 10: Efficacy test of the combination of Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium lactis. Cryptobiotix Research

[0681] The cryptobiotix study was conducted to test the effects of combinations of Bifidobacterium adolescentis with Bifidobacterium infantis and Bifidobacterium lactis and six HMOs (2'-FL, DFL, 3-FL, LNT, 3SL, 6SL) on enhancing microbial-derived metabolites associated with immune benefits.

[0682] In particular, indole-3-propionic acid has been shown to play an important role in the immune response (Li et al., Front. Pharmacol., 2021, 12: 769501).

[0683] For example, Van den Abbeele et al. (Van den Abbeele et al. Front. Front. Microbiol. (2023)).

[0684] In summary, batch fermentation experiments were used to simulate colonic fermentation of different test products (Table 5) by gut microbiota derived from 12 weaned infants (11 to 14 months).

[0685] Each test product was added to the fecal inoculum and appropriate culture medium in the container and fermented for 24 h. After fermentation, the supernatant from each container was collected and subjected to metabolomics analysis.

[0686] Table 9 below shows the research conditions used.

[0687] Table 9

[0688] To assess the therapeutic effect of microbial metabolites, a nonparametric ANOVA analysis (Friedman test) was performed, which demonstrates the fact that values ​​are compared between samples from a given donor.

[0689] Specifically, Friedman's test (non-parametric ANOVA) was performed to test whether the combination of 6HMO with Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium lactis increased the level of the metabolite indole-3-propionic acid compared to 6HMO alone with Bifidobacterium adolescentis or the combination of 6HMO with Bifidobacterium infantis and Bifidobacterium lactis alone (i.e., without Bifidobacterium adolescentis).

[0690] The results showed that compared with 6HMO + Bifidobacterium adolescentis and 6HMO + Bifidobacterium lactis + Bifidobacterium infantis, the combination of 6HMO + Bifidobacterium adolescentis + Bifidobacterium lactis + Bifidobacterium infantis significantly increased (nominal p-value) (see [link to relevant documentation]). Figure 21 ).

[0691] Linear regression models have also been used to test for Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium lactis.

[0692] The gut microbiota emerges and diversifies from infancy into early childhood (Laursen, Ann Nutr Metab, 2021, 77(Supplement 3): 21–34). Clostridium microbes are known to produce indole-3-propionic acid (Zhang et al., FrontEndocrinol, 2022, 13: 841703) and typically colonize the infant gut in the later stages of weaning and microbiome diversification. Therefore, a mature microbiome that has diversified to include Clostridium microbes favors the production of indole-3-propionic acid.

[0693] It was assumed that not all infants included in the study had a mature microbiome at this stage of infancy, as samples were collected at a single time point between 11 and 14 months of age. Therefore, the microbiome maturity of the samples was tested.

[0694] One indicator of microbiome maturity is butyrate level, which increases with microbiome maturation. Therefore, the butyrate level of samples was tested, and donors with samples containing high levels of butyrate (from 2.5 mM or higher) were considered to have mature microbiomes. Five donors were classified as having mature microbiomes. This classification was validated by microbiome analysis, which showed that donors classified as having mature microbiomes had more complex and diverse microbiomes, including members of the Lachnospiracae family and Clostridium species.

[0695] Linear regression models were used to examine interactions among all donors (n=12) and among the donor subset (n=5) with more mature microbiomes: *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium lactis*. The results are shown in Table 10.

[0696] Table 10 shows the p-values ​​for the production of indole-3-propionic acid obtained through linear regression analysis.

[0697] The results showed that compared to 6HMO alone with *Bifidobacterium adolescentis* or 6HMO alone with *Bifidobacterium infantis* and *Bifidobacterium lactis* (i.e., without *Bifidobacterium adolescentis*), specific interactions existed among the three probiotics *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium lactis* when used with 6HMO in mature donors. In particular, significant p-values ​​were observed when the three probiotics were used in combination in mature donors. This indicates an unexpected interaction between *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium lactis*.

[0698] The lack of statistically significant effects observed across the entire donor group can be explained by the absence of key species in some donors that do not possess a mature microbiome. Since the microbiome diversifies over time, it is hypothesized that the effects observed in the mature donor group will correlate with the entire population over time.

[0699] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention protected by the claims should not be unduly limited to such specific embodiments. Indeed, various modifications to the described modes of carrying out the invention that will be apparent to those skilled in molecular biology or related fields are intended to fall within the scope of the following claims.

Claims

1. A composition comprising a Bifidobacterium longum transitional microorganism and an HMO mixture, the HMO mixture comprising 2'-fucosyllactose (2'-FL), difucosyllactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

2. The composition according to claim 1, wherein the Bifidobacterium longum transitional microorganism: (i) It is capable of metabolizing the HMO; (ii) Prefer 3-FL over 2'-FL; (iii) Capable of metabolizing glycan substrates selected from any of the groups listed in Tables 1 to 3; and / or (iv) Encoding one or more CAZyme selected from the groups listed in Table 1, preferably wherein the Bifidobacterium longum transitional microorganism also encodes one or more CAZyme selected from Tables 2 and 3.

3. The composition according to claim 1 or claim 2, wherein 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, CMCC-P0001 (ATCC BAA-2753) and any combination thereof.

4. The composition according to any one of the preceding claims, wherein the composition further comprises Bifidobacterium longum subsp. infantis.

5. The composition according to any one of the preceding claims, wherein the composition further comprises Bifidobacterium lactis.

6. The composition according to claim 4 or claim 5, wherein: (i) The *Bifidobacterium longum* subsp. *infantitidis* is *Bifidobacterium longum* subsp. *infantitidis* LMG 11588 or has at least 99.9% average nucleotide identity (ANI) with *Bifidobacterium longum* subsp. *infantitidis* LMG 11588; and (ii) The Bifidobacterium lactis is Bifidobacterium lactis CNCM 1-3446 or an ANI having at least 99.9% average nucleotide identity (ANI) with Bifidobacterium lactis CNCM 1-3446.

7. The composition according to any one of the preceding claims, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL and 3SL.

8. The composition according to any one of claims 1 to 6, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL and 3-FL.

9. The composition according to any one of claims 1 to 6, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL and LNnT.

10. The composition according to any one of claims 1 to 6, wherein the HMO mixture comprises 2'-FL, DFL, LNT, 6SL, 3SL, 3-FL and LNnT.

11. The composition according to any one of the preceding claims, wherein the composition is a nutritional composition selected from infant formula, stage 1 infant formula, follow-up formula or stage 2 infant formula, infant food, infant cereal composition, growing milk, fortifiers such as human milk fortifiers, or supplements.

12. The composition according to any one of claims 1 to 11, wherein the composition is used to prevent infection in an individual, reduce the risk of infection in an individual, and / or treat infection in an individual.

13. The composition according to any one of claims 1 to 11, wherein the composition is used to promote long-term immune benefits in an individual.

14. The composition of claim 13 for the said use, wherein the long-term immune benefits to an individual include: i. Promote long-term respiratory health; ii. Prevent allergen sensitization and / or reduce the risk of allergen sensitization; And / or iii. To prevent the occurrence of respiratory illnesses and / or reduce the risk of developing respiratory illnesses, preferably said respiratory illnesses being chronic inflammatory diseases of the airways, such as asthma or chronic obstructive pulmonary disease (COPD), or allergic respiratory diseases, such as recurrent wheezing or asthma.

15. The composition according to any one of claims 1 to 11, wherein the composition is used to prevent an individual from developing asthma and / or to reduce the risk of an individual developing asthma.

16. A prebiotic, wherein the prebiotic is used to prevent, reduce the risk of infection in an individual, and / or treat an individual’s infection by promoting the growth and / or survival of Bifidobacterium longum transitional microorganisms in the individual’s gut, wherein the prebiotic is an HMO mixture consisting of 2'-fucosylated lactose (2'-FL), difucosylated lactose (DFL), lactose-N-tetrasaccharide (LNT), 6'-sialylated lactose (6SL), and 3'-sialylated lactose (3SL), and optionally 3-fucosylated lactose (3-FL) and / or lactose-N-neotetrasaccharide (LNnT).

17. The composition of claim 12 for the stated purpose or the prebiotic of claim 16 for the stated purpose, wherein the infection is a viral, bacterial, or fungal infection, preferably wherein the infection is an airway infection.

18. The composition or prebiotic for the purpose according to any one of claims 12, 16 or 17, wherein the infection is a viral airway infection, preferably wherein the viral airway infection is selected from influenza virus, respiratory syncytial virus, rhinovirus, parainfluenza virus, metapneumovirus, coronavirus, adenovirus and bocavirus.

19. The composition or prebiotic for the said use according to any one of claims 12 to 18, wherein the individual is an infant, toddler, or child.

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