The composition comprises a bifidobacterium animalis subsp. Lactis strain and 2apos; composition of fucosyllactose

Abstract

In particular, the present disclosure relates to a composition comprising a Bifidobacterium animalis subsp. Lactis strain (Bifidobacterium animalis subsp. Lactis), which is preserved as DSM 15954 by the Deutsche Sammneck von Microorganism strain preservation center (Deutsche Sammneck von Microorganism strain GmbH, DSMZ), and 2 '-fucosyllactose (2' FL), and to a method for preparing the same. The present disclosure also relates to a composition that also comprises a strain of Bifidobacterium longum subsp. Infantis, which is preserved as DSM 15953, and to a method for producing the same. The present disclosure also relates to a composition that also comprises a strain of Bifidobacterium longum subsp. Infantis, which is preserved as DSM 15953. The use of the compositions in supporting gastrointestinal health, including promoting intestinal barrier integrity and growth of beneficial microbiota in the gastrointestinal tract, while also having beneficial effects on immune function is also part of the present disclosure.

Description

Technical Field

[0001] This disclosure relates to compositions comprising a strain of Bifidobacterium animalis subsp. lactis deposited as DSM 15954 at the German Collection of Microbial Cultures (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, DSMZ). Bifidobacterium animalum subsp. milk The disclosure also relates to a strain of *Bifidobacterium longum* subsp. *infanthi* deposited as DSM 15953, containing 2'-fucosylated lactose (2'FL). Bifidobacterium long subsp. child The composition of strain. The use of said composition in supporting gastrointestinal health, including promoting intestinal barrier integrity and the growth of beneficial microbiota in the gastrointestinal tract, while also having a beneficial effect on immune function, is also part of this disclosure. Background Technology

[0002] Bifidobacteria are natural inhabitants of the gastrointestinal tract, possessing genetic adaptations that enable them to colonize this harsh and complex environment. Bifidobacteria are believed to interact with key elements of intestinal function and contribute to maintaining homeostasis. It is believed that strain-dependent interactions with the host can reduce mucosal antigen load, improve the intestinal barrier, and induce regulation of local and systemic immune responses.

[0003] For these and other reasons, Bifidobacterium can be used as a probiotic. Probiotics are cultures of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms, and extracts or supernatants of microorganisms that, when applied to humans or animals, have a beneficial effect on the host (Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic).

[0004] Digestive health issues affect the daily lives of millions. Antibiotics, diet, and travel can all disrupt the balance of the microbiome. Furthermore, mounting evidence suggests that a healthy microbiome benefits more than just an individual's gastrointestinal and digestive health. Normal immune system function is linked to a healthy microbiome, and there is growing interest in establishing a beneficial microbiome immediately after birth to ensure healthy development and overall health for both mother and child.

[0005] Probiotics can offer solutions to digestive health problems and help establish an early healthy microbiome. There are approximately 12 clinically proven Bifidobacteria species (…). Bifidobacterium The strain is commercially available.

[0006] Specifically, the use of probiotics for premature infants and other infants with birth problems (such as extremely low birth weight) has been evaluated.

[0007] Therefore, there remains a need for probiotic compositions that are useful in supporting gastrointestinal health, including, for example, protecting against intestinal tissue damage. Furthermore, probiotic compositions that may have beneficial effects on the immune system will be of interest. Summary of the Invention

[0008] This invention relates to a strain of *Bifidobacterium animalis* subsp. *lactamase* deposited by the German Society for the Preservation of Microbial Cultures (DSMZ) as DSM 15954 and optionally a strain of *Bifidobacterium longum* subsp. *infant* deposited as DSM 15953. Both strains were found to be sensitive to all relevant antibiotics in MIC testing according to EFSA 2018 guidelines. Consistent with this, no antibiotic resistance genes were identified. Furthermore, cytotoxicity of the strains was tested by Vero cell assays and found to be non-cytotoxic.

[0009] Based on the in vitro results provided in Example 1, the *Bifidobacterium animalis* subsp. *lactamase* strain deposited under DSM 15954 and the *Bifidobacterium longum* subsp. *infant* strain deposited under DSM 15953 are considered safe.

[0010] Example 2 provides information on the ability of the *Bifidobacterium animalis* subsp. *lactamase* strain deposited as DSM 15954 and the *Bifidobacterium longum* subsp. *infant* strain deposited as DSM 15953 to lyse human bile salts.

[0011] Example 3 demonstrates that inoculation of both the infant and the mother can potentially increase the amount of Bifidobacteria in the infant's gut microbiome. This example also shows that inoculation of infants with Bifidobacteria can have a positive effect on bowel movement frequency, stool consistency, and / or the percentage of breastfed infants exhibiting uncomfortable crying and irritability.

[0012] Example 4 provides information on *Bifidobacterium longum* subsp. *infantitidis* preserved as DSM 15953. B. long subsp. child Information on the ability to use HMOs as a carbon source for growth.

[0013] Example 5 provides results of transepithelial resistance (TEER) measurements, which indicate intestinal barrier integrity.

[0014] When grown on transfer plates, mature cell lines of Caco-2, originally derived from human colon adenocarcinoma, form a polarized monolayer, which is commonly used as a model of intestinal permeability.

[0015] Example 5 demonstrated that the *Bifidobacterium animalis* subsp. *lactamase* strain deposited in DSM 15954 and the *Bifidobacterium longum* subsp. *infant* strain deposited in DSM 15953 increased the electrical resistance across the Caco-2 cell monolayer, as measured by TEER. These strains are believed to have potential roles in supporting gastrointestinal health, including resisting intestinal tissue damage.

[0016] Bacteria that activate immune cells to secrete cytokines through dose administration may modulate the inflammatory state of the gastrointestinal tract. For example, Foligne et al. (Foligne, B., Nutten, S., Grangette, C., Dennin, V., Goudercourt, D., Poirot, S., Dewulf, J., Brassart, D., Mercenier, A. & Pot, B. (2007) World J Gastroenterol, 13(2), 236-243) found a significant relationship between bacterial-mediated in vivo protection against experimental colitis and the in vitro bacterial-induced cytokine profile (particularly the ratio of IL-10 to interleukin-12 (IL-10 / IL-12)). The authors showed how in vivo protection against trinitrobenzenesulfonate (TNBS)-induced colitis in mice was mediated by bacterial strains capable of in vitro inducing high levels of the “anti” inflammatory cytokine IL-10 and low levels of the “pro” inflammatory cytokine IL-12. Conversely, strains induced with a low IL-10 / IL-12 ratio did not alleviate inflammatory colitis.

[0017] Therefore, in vitro regulation of human inflammatory cells can be a useful tool for predicting bacterial-induced pro-inflammatory or anti-inflammatory gut environments.

[0018] Example 6 provides information on cytokines derived from Bifidobacterium longum subsp. infantis (Bifidobacterium longum infantis subsp. infantis). B. long subsp. child Information on the secretion of human peripheral blood mononuclear cells (PBMCs) stimulated by 2'-O-fucosylated lactose (2'FL) (DSM 15953).

[0019] Example 7 provides cytokines derived from Bifidobacterium longum subsp. infantis (DSM 15953) or Bifidobacterium animalis subsp. lactis (DSM 15953). B. animal subsp. milk ), BB-12 ®(DSM 15954) information on the secretion of peripheral blood mononuclear cells (PBMCs) stimulated by human stimulation.

[0020] To further investigate whether BB-12 may exist and provide Bifidobacterium animalis subsp. lactis, ® The combination of both (DSM15954) and Bifidobacterium longum infantis subsp. (DSM 15953) is effective in driving specific T helper cells (T cells). H The study investigated the benefits related to adaptive immune responses, including their effects on dendritic cells and the ratio of IL-10 to interleukin-12 (IL-10 / IL-12).

[0021] Example 8 provides the following information: Bifidobacterium longum infantis subsp. (DSM 15953) and Bifidobacterium animalis lactis subsp. BB-12 are being used. ® In DCs stimulated with a combination of DSM 15954 and Bifidobacterium longum subsp. infantis (DSM 15953), the IL-10 / IL-12 ratio was significantly increased. This effect appears to be driven by the Bifidobacterium longum subsp. infantis (DSM 15953) strain, suggesting anti-inflammatory properties and potentially stimulating adaptive T cells. H Regulatory response.

[0022] Therefore, this probiotic composition can be useful in supporting gastrointestinal health, including promoting, for example, intestinal barrier integrity and the growth of beneficial microbiota in the gastrointestinal tract. The compositions of the present invention may also have beneficial effects on immune function. Attached Figure Description

[0023] Figure 1 Figure 1 The activity of bile salt hydrolases was shown. Quantification of glycine and taurine debinding activities of (A and B) *Bifidobacterium animalis* subsp. *lactamase* (DSM 15954) and (C and D) *Bifidobacterium longum* subsp. *infant* (DSM 15953) was also presented. (E and F) [The text abruptly ends here, likely due to an incomplete translation or missing information.] L. rhamnosus To compare binding activity (standard error bars as SD±).

[0024] Figure 2 Figure 2 The recoveries of the strains at three dosages are shown, expressed as the percentage of infants with detectable strains and the number of strains per μl per DNA material. Data for *Bifidobacterium longum* subsp. infantis (DSM 15953) are provided in Figures (a) and (b); LGG data are provided in Figures (c) and (d). ® And data for *Bifidobacterium animalis* subsp. *lactamase* (DSM 15954) in Figures (e) and (f). IS = infant vaccination, MS = maternal vaccination. Data are presented as mean (SSEM).

[0025] Figure 3 Figure 3A This paper presents a comparison of the microbial composition in fecal samples from infants at birth, day 14, day 28, and day 42 in studies of infant vaccination versus maternal vaccination. Figure 3B ) shows the basis with Figure 3A More detailed insights into the same data.

[0026] Figure 4 Figure 4 The differences in defecation frequency (a), stool consistency (b), and percentage of infants exhibiting uncomfortable crying and irritability (c) between the two treatment groups (infant vaccination versus maternal vaccination) are shown. Figure (d) illustrates the differences in aromatic lactate, indole-3-lactic acid, p-hydroxyphenyllactic acid, and 3-phenyllactic acid, (e) N-acetyl-lysine, and (f) methionine at birth, day 14, day 28, and day 42 in the infant vaccination versus maternal vaccination study.

[0027] Figure 5 Figure 5 The results show the OD values ​​after incubation for 96 hours in broth without carbohydrates (white column) and in broth containing 2% 2'-fucosylated lactose, 2'FL (grey column). 600 Growth of *Bifidobacterium longum* subsp. *infantitidis* (DSM 15953) was measured spectrophotometrically. Growth on MRS was used as a positive control (black column).

[0028] Figure 6 Figure 6 This illustrates the use of Bifidobacterium animalis subsp. lactis, BB-12 ® The ability of DSM 15954, Bifidobacterium longum subsp. infantis (DSM 15953), and 2'FL, alone or in combination, to induce higher transepithelial resistance (TEER) across the Caco-2 monolayer. A) represents real-time TEER measurements, while B) represents the area under the curve (AUC) after 10 hours of measurement.

[0029] Figure 7 Figure 7The secretion of cytokines from human PBMCs stimulated with 2'FL, *Bifidobacterium longum* subsp. infantis (DSM 15953), or a combination of 2'FL and *Bifidobacterium longum* subsp. infantis (DSM 15953) is shown. The mean cytokine concentration (pg / ml) of A. IL-10 or B. IFN-γ was measured in the supernatant of human PBMCs co-incubated with 2'FL, *Bifidobacterium longum* subsp. infantis (DSM 15953), or a combination of 2'FL and *Bifidobacterium longum* subsp. infantis (DSM 15953) for 20 hours under steady-state (hollow column) or virus-like challenge (grid column) conditions. Data columns represent the mean concentration of immune cells isolated from four healthy donors, and error bars represent the standard error (SEM) of the mean.

[0030] Figure 8 Figure 8 The study showed that cytokines were derived from Bifidobacterium animalis subsp. lactis, BB-12. ® The secretion of human PBMCs stimulated by either (DSM 15954) or *Bifidobacterium longum* subsp. infantis (DSM 15953) is shown. The secretion is also shown in the presence of *Bifidobacterium animalis* subsp. lactis, BB-12. ® Mean cytokine concentrations (pg / ml) were measured in the supernatant of human PBMCs co-incubated with or kept unstimulated for 20 hours with *Bifidobacterium longum* subsp. *infantitidis* (DSM 15954) and *Bifidobacterium longum* subsp. *infantitidis* (DSM 15953). Data columns represent the mean cytokine concentrations of immune cells isolated from four healthy donors, and error bars represent the standard error (SEM) of the mean.

[0031] Figure 9 Figure 9 The study showed that cytokines were derived from Bifidobacterium longum subsp. infantis (DSM 15953), Bifidobacterium animalis subsp. lactis, and BB-12. ® (DSM 15954) or a combination of stimuli to the secretion of human dendritic cells (DCs). In contrast to *Bifidobacterium longum* subsp. infantis (DSM 15953) (1:10), *Bifidobacterium animalis* subsp. lactis, BB-12 ® (DSM 15954) (1:10), Bifidobacterium animalis subsp. lactis, BB-12 ® Mean cytokine concentrations (pg / ml) of A. IL-10 and B. IL-12 were measured in the supernatant of human DCs co-incubated with a combination of DSM 15954 and Bifidobacterium longum subsp. infantis (DSM 15953) (1:20) or kept unstimulated for 20 hours. C. IL-10 / IL-12 cytokine ratio. Data bars represent the mean concentration of immune cells isolated from 4 healthy donors, and error bars represent the standard error (SEM) of the mean. Detailed Implementation

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While any methods and materials equivalent to or similar to those described herein may be used in practice with respect to this disclosure, typical methods and materials are described. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order.

[0033] Unless otherwise stated herein or obviously contradicted by the context, the use of the terms “a,” “an,” and “the,” and similar indicators, in the context of describing the invention (particularly in the context of the following claims), shall be considered to cover both singular and plural forms. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be considered open-ended terms (i.e., meaning “including (but not limited to)”). Unless otherwise stated herein, the enumeration of numerical ranges herein is intended only as a way of abbreviating individual values ​​belonging to a range including both endpoints, and each individual value is introduced into this specification as it is individually enumerated herein. Unless otherwise asserted, the use of any and all embodiments or exemplary language (e.g., “as”) provided herein is intended only to better describe the invention and does not limit the scope of the invention.

[0034] As used herein, the term “and / or” is intended to indicate both combined (“and”) and exclusive (“or”) uses, i.e., “A and / or B” is intended to mean “A alone, or B alone, or A and B together”.

[0035] As used herein, the term "composition" refers to a composition comprising a carrier and at least one bacterial strain described herein.

[0036] As used herein, the terms “effective amount,” “effective concentration,” or “effective dose” are defined as the amount, concentration, or dose of bacterial strains sufficient to improve the overall health of an animal and to impart benefits similar to those shown in the examples.

[0037] The actual effective dose, expressed in absolute values, depends on factors including the health status of the subject under discussion and other components present. The “effective amount,” “effective concentration,” or “effective dose” of a bacterial strain can be determined by routine assays known to those skilled in the art.

[0038] As used herein, the term "isolated" means that the bacterial strain described herein is in a form or environment not present in nature, i.e., the strain is at least partially removed from one or more of the naturally occurring components associated with it in nature.

[0039] As used herein, a bacterial "strain" refers to bacteria that are genetically unchanged during growth or proliferation and originate from a single isolate or pure culture. Probiotics are categorized by their genus (e.g., Bifidobacterium). Bifidobacterium Probiotics are classified by species and subspecies (e.g., *Bifidobacterium longum* subsp. *infantii*) and strain (e.g., DSM 15953). The FAO / WHO states that the effects of probiotics are strain-specific, and therefore, most probiotic characteristics of a particular strain cannot be extrapolated to other strains of the same species.

[0040] As used herein, the term "improved immune health" is intended to refer to providing an increased IL-10 / IL-12 ratio and / or promoting Th1 responses, as evaluated in Examples 6-8.

[0041] The language used in this specification should not be construed as indicating any unclaimed element necessary for the practice of this invention.

[0042] The present invention has been described with reference to various embodiments, aspects, examples, etc. These elements are not intended to be understood in isolation. Therefore, this disclosure provides for two or more combinations of embodiments, aspects, examples, etc.

[0043] All embodiments described herein are intended to be within the scope of the disclosed invention. These and other embodiments of the invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments, which are not limited to any of the particularly preferred embodiments disclosed.

[0044] This disclosure provides isolates of *Bifidobacterium animalis* subsp. *lactamase*, deposited as DSM 15954 by the German Society for the Preservation of Microbial Cultures (DSMZ), and *Bifidobacterium longum* subsp. *infant*, deposited as DSM 15953. Both strains were sensitive to gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin when tested as described in ISO 10932:2010. Neither strain possessed antibiotic resistance genes when analyzed as described in Example 1, and neither strain exhibited cytotoxicity when tested by Vero cell assay as described in Example 1.

[0045] For the effects of probiotics that depend on viability and physiological activity in the gut, the survival of probiotics in the presence of gastric acid and bile in the upper gastrointestinal tract may be important.

[0046] The *Bifidobacterium animalis* subsp. *lactamase* strain preserved under DSM 15954 exhibits a degree of tolerance to gastric acid and bile. This strain also contains enzymes encoding bile salt hydrolases (…). bsh ) genes, bsh It is an enzyme that can break down human bile salts. Figure 1 The presence of this enzyme can provide an advantage to the strain because it allows for a response to high bile salt concentrations by cleaving toxic bile salts into less toxic compounds. While not wishing to be bound by theory, it is believed that ingestion of substances containing... bsh Genetically modified probiotic strains may reduce daily vomiting in pregnant women.

[0047] As described in Example 2, it is believed that the *Bifidobacterium animalis* subsp. *lactamase* strain deposited under DSM 15954 and the *Bifidobacterium longum* subsp. *infant* strain deposited under DSM 15953 have the ability to debind human bile salts. When bile salts are bound to *Bifidobacterium animalis* subsp. *lactamase* strain (BB-12) deposited under DSM 15954... ® Alternatively, incubation with *Bifidobacterium longum* subsp. infantis* strain preserved under DSM 15953 revealed the unbinding of bile salts bound to glycine and taurine, resulting in the release of free glycine and taurine. Therefore, this example demonstrates that both *Bifidobacterium animalis* subsp. lactis strain preserved under DSM 15954 and *Bifidobacterium longum* subsp. infantis strain preserved under DSM 15953 exhibit statistically significant BSH activity compared to the control, which could be beneficial to subjects ingesting these strains. *Bifidobacterium animalis* subsp. lactis strain preserved under DSM 15954 has been shown to be superior to other probiotics in lysing human bile salts.

[0048] Adhesion of probiotic microorganisms to the intestinal mucosa can be a selection criterion for probiotic microorganisms. The *Bifidobacterium animalis* subsp. *lactamase* strain deposited in DSM 15954 has demonstrated adhesiveness in various adhesion models used in the field to evaluate in vitro adhesion. This supports the theory that the strain has the ability to transiently colonize and persist on the mucosal surface of the intestine, thereby increasing the likelihood of delivering beneficial effects to subjects who ingest the strain.

[0049] Pathogens are microorganisms that can cause disease in their host. The ability to inhibit pathogens is one of the three main mechanisms of probiotics, the other two being enhanced barrier function and immune interactions. The *Bifidobacterium animalis* subsp. *lactamase* strain preserved in DSM 15954 exhibits antagonistic activity against several relevant human gastrointestinal pathogens by producing antimicrobial substances and by competing for mucosal adhesion.

[0050] Epithelial and endothelial cells form barriers in the body. The strength and integrity of these barriers can be obtained by measuring transcellular resistance (TEER) in vitro.

[0051] TEER is a recognized method for evaluating and monitoring epithelial tissue in a non-destructive assay. Specifically, it rapidly identifies the confluence of monolayers. The confluence can be tracked and monitored in real time because TEER measurements rise as gaps in the monolayer close.

[0052] TEER is often used as a strong indicator of cell barrier integrity and permeability for epithelial and endothelial cells in monolayers.

[0053] Measuring transepithelial electrical resistance (TEER) after the addition of different substances is a method to evaluate whether a given composition may be beneficial to intestinal barrier integrity. Therefore, Example 5 of this disclosure uses Bifidobacterium animalis subsp. lactis BB-12... ® The efficacy was evaluated in TEER assays using (DSM 15954), *Bifidobacterium longum* subsp. infantis (DSM 15953), and 2'FL. Both bacteria, alone or in combination, induced moderate TEER readings, indicating that administration of *Bifidobacterium animalis* subsp. lactis BB-12 may be effective. ® A composition containing both DSM 15954 and Bifidobacterium longum subsp. infantis (DSM 15953) can achieve some effects. If a composition containing all three components (Bifidobacterium animalis subsp. lactis BB-12) is provided, some effects can be achieved. ® (DSM 15954), Bifidobacterium longum infantis subsp. ( B. long subsp. child The combination of (DSM 15953) and 2'FL) resulted in significantly higher TEER readings compared to all other groups. Figure 6 B). While not wishing to be bound by theory, it is considered that compositions containing more than one probiotic strain and at least one HMO may improve cell barrier integrity and permeability more than compositions containing only one probiotic strain or none of the probiotic strain.

[0054] The compositions disclosed herein may contain at least one strain of Bifidobacterium longum subsp. infantis, such as the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953.

[0055] An infant's gut microbiome develops after birth to ensure healthy functioning of the gastrointestinal system and optimal maturation of the immune system. Four different types of Bifidobacteria are considered particularly important for infant gut health. These are *Bifidobacterium longum* subsp. infantis, *Bifidobacterium breve* (…), and others. B. short Bifidobacterium bifidum ( B. bifidum ) and Bifidobacterium longum subsp. ( B. long subsp. longThese Bifidobacteria digest human milk oligosaccharides (HMOs) present in breast milk. By breaking down these HMOs, they form a variety of metabolites that are believed to be beneficial for infant intestinal maturation and immune system development, such as acetates and various aromatic lactic acids.

[0056] Bifidobacterium longum subsp. infantis is effective at utilizing HMOs. Bifidobacterium longum subsp. infantis strains, such as those preserved as DSM 15953, have the ability to utilize HMOs as a growth carbon source, thus growing well on HMOs present in the infant colon where other carbon sources are lacking.

[0057] HMOs present in breast milk feed Bifidobacterium longum infantis strains, thus improving the chances of beneficial Bifidobacterium longum infantis colonizing in the infant's intestine.

[0058] For example, by inoculating the infant with a combination of probiotics, including *Bifidobacterium longum* subsp. infantis and / or the mother, it is possible to increase the amount of Bifidobacteria in the infant's gut microbiome (Example 3). Inoculation of infants with probiotic combinations has been found to have effects on bowel movement frequency, stool consistency, and / or crying and irritability.

[0059] Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine, and tyrosine) into their respective aromatic lactates (indole-3-lactic acid, phenyllactic acid, and p-hydroxyphenyllactic acid) via previously unidentified aromatic lactate dehydrogenases. This triangular relationship between the gut microbiome, metabolites, and the developing immune system supports the possibility that *Bifidobacterium longum* subspecies *Infant* DSM15953 may influence immune maturation through the production of indole-3-lactic acid (Example 3).

[0060] While not wishing to be bound by theory, it is believed that providing a composition containing more than one probiotic component would provide benefits to subjects receiving such a composition, as each added probiotic is believed to provide different positive effects and functions, and these can work together and result in a synergistic effect.

[0061] This disclosure relates to Bifidobacterium animalis subsp. lactis (BB-12), deposited under DSM 15954. ® Use as a probiotic in a composition comprising the strain and at least one HMO.

[0062] In one embodiment, this disclosure provides a supplement containing a strain of Bifidobacterium animalis subsp. lactis (BB-12) deposited as DSM 15954. ®The supplement comprises a composition of at least one HMO. The supplement can be used to provide beneficial effects to subjects in need. For example, the compositions of the present invention can promote intestinal barrier integrity in subjects in need, promote the growth of beneficial microbiota in the gastrointestinal tract, etc. The supplement can also be used to modulate the immune health of subjects in need. For example, the compositions of the present invention can increase the IL10 / IL12 ratio and / or promote Th1 responses, etc.

[0063] In the context of this invention, the term "intestinal barrier integrity" refers to the natural integrity of the intestinal wall, which acts as a barrier between the intestinal interior and surrounding tissues and lumens. One method for measuring it is the TEER method as described in Example 5, which measures intestinal barrier tightness.

[0064] This disclosure relates to a method for promoting intestinal barrier integrity, improving immune health, and promoting the growth of beneficial microbiota in the gastrointestinal tract in adults and / or infants and / or children in need thereof, the method comprising administering to the adults and / or infants and / or children a composition comprising Bifidobacterium animalis subsp. lactis and at least one human milk oligosaccharide and optionally at least one Bifidobacterium longum subsp. infantis.

[0065] The compositions disclosed herein can be used to support intestinal barrier integrity and the growth of beneficial microbiota in the gastrointestinal tract in subjects, such as adult subjects with digestive health problems and / or infants with immature gastrointestinal tracts, with the aim of providing a healthy gut microbiome by promoting the growth of beneficial microbiota in the gastrointestinal tract.

[0066] The subjects may be infants (aged 0-12 months) or pregnant women, such as pregnant women aged 18-45 years, with the aim of directly inoculating the infants by applying the composition to them or by inoculating the expectant mothers, who then pass the beneficial bifidobacteria to their children (see Example 3), thereby creating a bifidobacteria-inoculated environment for the infants.

[0067] The compositions disclosed herein can be used to help enhance the immunity of children aged 1-6 years, for example. As shown in Examples 6-8, the compositions of the present invention can promote a Th1 response, which is particularly beneficial for infants and children because they have an immature immune system, and therefore a Th1 response can help them fight off infections more quickly and effectively. An elevated IL10 / IL-12 ratio can indicate anti-inflammatory properties, which can provide benefits to subjects who ingest compositions containing this strain through immunomodulation.

[0068] Compositions comprising Bifidobacterium animalis subsp. lactis, deposited as DSM 15954, and at least one human milk oligosaccharide and optionally at least one Bifidobacterium longum subsp. infantis, deposited as DSM 15953, may be useful for intestinal barrier protection and protection of the properties of the gastrointestinal microbiota in subjects with a normal gastrointestinal tract and in subjects with an immature or dysfunctional gastrointestinal tract, during and after pregnancy and lactation.

[0069] This composition can be used to reduce intestinal tissue damage, that is, to support intestinal barrier function by preventing, reducing or treating intestinal mucosal ruptures or lesions.

[0070] This composition, comprising Bifidobacterium animalis subsp. lactis as deposited in DSM 15954, at least one human milk oligosaccharide, and optionally at least one Bifidobacterium longum subsp. infantis, can be useful for administration to adult subjects, preferably female subjects, such as adult women aged 18 to 45 years. In one embodiment, the female subject is pregnant or lactating.

[0071] This composition, comprising Bifidobacterium animalis subsp. lactis as deposited in DSM 15954, at least one human milk oligosaccharide, and optionally at least one Bifidobacterium longum subsp. infantis, can be useful for administration to infants or young children. In this embodiment, infants are children aged 0-12 months, and young children are children aged 1-6 years.

[0072] This composition may contain other components. For example, at least one other bacterial strain, vitamins, minerals, prebiotics, fiber, or combinations thereof. For example, other components may be fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, human milk oligosaccharides (HMO), or combinations thereof.

[0073] This composition may contain a strain of Bifidobacterium animalis subsp. lactis preserved as DSM 15954 and at least one other bacterial strain, as well as a suitable HMO, such as 2'-fucosylated lactose (2'FL).

[0074] At least one other bacterial strain may be selected, for example, from *Lactococcus lactis* subsp. *diacetyl*. Lactococcus lactis subsp. lactis biovar. diacetylactis Lactococcus lactis subsp. milk fat ( Lactococcus lactis subsp. cream Lactococcus lactis subsp. lactis Lactococcus milk subsp. milk Any strain belonging to the genus Lactobacillus (including (but not limited to) Lactobacillus acidophilus) Lactobacillus acidophilus Lactobacillus casei subsp. casei ( Lactobacillus cheese subsp. cheese Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. Bulgarian ), Lactobacillus fermentum ( Lactobacillus fermentum Lactobacillus gasseri ( Lactobacillus gasseri ), Swiss thermophilic bacteria ( Lactobacillus helveticus Lactobacillus acidophilus ( Lactobacillus lactis Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus salivarius ( Lactobacillus salivarius Lactobacillus plantarum ( Lactobacillus plantarum Any strain belonging to the genus Bifidobacterium (including (but not limited to) Bifidobacterium adolescentis) Bifidobacterium adolescentis ), Bifidobacterium angularis ( Bifidobacterium angulatum Bifidobacterium animalis subsp. lactis ( Bifidobacterium animal subsp. milk Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium breve Bifidobacterium breve ), Bifidobacterium chain ( Bifidobacterium catenulatum Bifidobacterium densiflorum ( Bifidobacterium dentium Bifidobacterium longum infantis subspecies ( Bifidobacterium long subsp. child Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. long Bifidobacterium longum swine subspecies ( Bifidobacterium longum subsp. their own Bifidobacterium macrocarpa ( Bifidobacterium great Bifidobacterium pseudosporidis ( ), Bifidobacterium pseudosporidis ( ) Bifidobacterium pseudocatenulatum (), or any strain from Akkermania, Corynebacterium, Butyricococcus, Christensenella, Clostridium, Cocci, Dorea, Eubacterium, Faecalibacterium, or Rosette or Rhodotorulaceae, and appropriate combinations thereof.

[0075] This composition may contain at least one bacterial strain selected from the following: *Bifidobacterium animalis* subsp. *lactamase* preserved as DSM 15954, *Lactobacillus acidophilus* preserved as DSM 13241, *Lactobacillus rhamnosus* preserved as ATCC 53103, *Lactobacillus rhamnosus* preserved as DSM 33870, *Lactobacillus rhamnosus* preserved as ATCC 55826, and *Lactobacillus reuteri* preserved as ATCC 55845. Lactobacillus reuteri Lactobacillus casei subsp. casei, preserved as ATCC 55544 Lactobacillus paracasei subsp. cheese Lactobacillus casei preserved as LMG-17806 Lactobacillus paracasei Thermophilic Streptococcus preserved as DSM 15957 Streptococcus thermophilic Lactobacillus fermentum preserved as NM02 / 31074, Lactobacillus casei subsp. casei preserved as CCTCC M204012, and appropriate combinations thereof.

[0076] In some respects, the compositions according to this disclosure contain only one, two, three, four or five different strains.

[0077] In one aspect, the composition comprises bacteria of *Bifidobacterium animalis* subsp. *lactamase*, for example, as a strain of *Bifidobacterium animalis* deposited under DSM 15954, and bacteria of *Bifidobacterium longum* subsp. *infant*, for example, as a strain of *Bifidobacterium longum* deposited under DSM 15953. The composition may comprise bacteria of both strains as the sole probiotic component.

[0078] In one respect, the Bifidobacterium animalis subsp. lactis strain preserved in DSM 15954 is the only probiotic component in the composition.

[0079] This composition may contain any suitable form of bacteria for administration to a subject. In a preferred embodiment, the composition may contain bacteria in a dried form, which may be obtained by freeze-drying, spray drying, lyophilization, etc.

[0080] If bacteria are freeze-dried, they are typically mixed with a cryoprotectant before freeze-drying. The term "cryoprotectant" is used herein to refer to a substance that can improve survival rates and enhance the storage stability of bacteria during freezing and / or drying. Cryoprotectants as used herein preferably comprise sugars and / or sugar alcohols, such as inositol.

[0081] The sugar may be a monosaccharide, disaccharide, oligosaccharide, or polysaccharide, or a mixture of at least two sugars. Useful monosaccharides include, for example, glucose (also known as dextrose), fructose, ribose, and galactose, and useful disaccharides include, for example, sucrose, trehalose, maltose, and lactose. The composition may contain one or more monosaccharides or disaccharides, such as one, two, three, or even more different sugars.

[0082] Cryoprotectants can contain a mixture of disaccharides (such as sucrose) and polysaccharides (such as maltodextrin).

[0083] Cryoprotectants may also contain peptides, proteins, protein hydrolysates, or mixtures thereof. Examples of peptides and proteins to be used are casein, pea protein, whey, albumin, glutamic acid, or gelatin, and any isolates or hydrolysates thereof. Other additives may also be present, such as antioxidants like sodium ascorbate, sodium citrate, trisodium citrate dihydrate, and cysteine ​​hydrochloride. Skim milk powder and yeast extract may also be ingredients.

[0084] In one embodiment, the composition comprises a freeze-dried or lyophilized form of *Bifidobacterium animalis* subsp. *lactamase* preserved under DSM 15954 and a cryoprotectant. The cryoprotectant may contain sugars. In a preferred aspect of this embodiment, the cryoprotectant may contain a mixture of disaccharides (such as sucrose) and polysaccharides (such as maltodextrin).

[0085] In one embodiment, the composition comprises bacteria as *Bifidobacterium animalis* subsp. *lactamase* preserved under DSM 15954 and bacteria as *Bifidobacterium longum* subsp. *infant* preserved under DSM 15953, and a cryoprotectant, both bacteria being present in frozen or lyophilized form. The cryoprotectant may contain sugars. In a particularly preferred aspect of this embodiment, the cryoprotectant may contain a mixture of disaccharides (such as sucrose) and polysaccharides (such as maltodextrin).

[0086] This composition may contain at least one human milk oligosaccharide (HMO). According to this disclosure, examples of HMOs that can be combined with bacteria such as *Bifidobacterium animalis* subsp. *lactamase* deposited under DSM 15954 and optionally *Bifidobacterium longum* subsp. *infant* deposited under DSM 15953 may be one or more of the following HMOs: 2′-fucosylvose, 3-fucosylvose, 3′-sialyl lactose, 6′-sialyl lactose, lact-N-neotetrasaccharide, lact-N-tetrasaccharide (type I and / or type II), and combinations thereof. The composition may be formulated as a combination or a single composition of one or more bacterial strains and an HMO (e.g., HMO 2′-fucosylvose (2′-FL)).

[0087] Currently, there are approximately 200 known structurally diverse HMOs. They can be categorized into fucosylated, sialylated, and neutral-core HMOs. The composition of HMOs in breast milk varies from mother to mother and changes during lactation. In 80% of women, the dominant oligosaccharide is 2'-fucosylated lactose, which is present in breast milk at a concentration of approximately 2.5 g / L. Other abundant oligosaccharides include lact-N-tetrasaccharide, lact-N-neotetrasaccharide, and lact-N-fucopentose. Numerous studies have found that the concentration of each HMO varies at different stages of lactation (colostrum, transitional milk, mature milk, and late milk) and depends on various factors, such as the mother's genetic secretory status and the length of gestation.

[0088] Typically, human milk oligosaccharides (HMOs) are derived from lactose, which can be modified by four monosaccharides (N-acetyl-D-glucosamine, D-galactose, sialic acid, and / or L-fucose) to form oligosaccharides.

[0089] This composition may contain 2'-fucosyllactose. The composition may be formulated as a combination or a single composition of probiotic strains and HMOs. The composition may contain other human lactose oligosaccharides (HMOs), such as, for example, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lact-N-tetrasaccharide, lact-N-neotetrasaccharide, and combinations thereof. HMOs can be any suitable combination. For example, 2'-fucosyllactose may be combined with any one, two, three, four, or five of 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lact-N-tetrasaccharide, and lact-N-neotetrasaccharide. In one embodiment, the HMO mixture contains 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, and lact-N-tetrasaccharide.

[0090] Unlike other components of breast milk that are absorbed by infants through breastfeeding, human malnourishing organisms (HMOs) are difficult for breastfed infants to digest. However, they have prebiotic effects and serve as food for certain gut bacteria, particularly Bifidobacteria. The abundance of these gut bacteria in the gut appears to reduce the colonization of pathogens, thereby promoting a healthy gut microbiota and reducing the risk of dangerous intestinal infections. Studies have shown that HMOs reduce the risk of viral and bacterial infections, and thus reduce the chances of diarrhea and respiratory illnesses. This protective function of HMOs appears to be activated upon contact with specific pathogens, such as certain bacteria or viruses, which are able to bind to glycan receptors (receptors to long chains of sugar molecules attached to the surface of human cells) located on the surface of intestinal cells. This binding can infect the intestinal mucosa. Researchers have found that HMOs can mimic these glycan receptors, causing pathogens to bind to HMOs rather than to intestinal cells. This reduces the risk of infection. In addition, HMOs appear to affect the response of specific cells in the immune system in a way that reduces the inflammatory response. Furthermore, HMOs, such as 2'-fucosylated lactose, may have inhibitory effects on pathogens (such as Staphylococcus aureus). Staphylococcus aureus The ability to form biofilms. Biofilm formation is a way for pathogens to protect themselves and evade the immune system of the infected host. Microorganisms aggregate in biofilms to form colonies for coordinated metabolism. This synergistic growth method improves pathogen survival by enhancing defense, increasing nutrient availability, and providing better opportunities for cell communication and transfer of genetic material. Cellular defense is important for resisting physical threats, such as displacement by flowing fluids or clearance by the immune system.

[0091] Some symbiotic bacteria may possess the ability to utilize HMOs as carbon sources. Example 4 of this disclosure demonstrates how *Bifidobacterium longum* subsp. infantis* preserved as DSM 15953 can utilize HMOs, such as 2'FL, and, when grown on HMOs, exhibits similar growth characteristics as when grown without a carbon source, as via OD. 600 The measured higher degree of growth (see Figure 5 ).

[0092] While not wishing to be bound by theory, it is believed that providing a composition containing at least one probiotic and at least one HMO would provide benefit to subjects receiving such a composition, because each component of the composition is believed to work better when administered in combination than when administered alone, since the components can interact and the bacterial strains can be fed with HMOs.

[0093] This composition may contain any suitable amount of HMO, such as 2'-fucosyllactose, for example, at least about 0.001 g, at least about 0.01 g, at least about 0.05 g, at least about 0.1 g, or at least about 0.5 g by weight. The composition may, for example, contain less than about 20 g, less than about 10 g, less than about 5 g, less than about 4 g, less than about 3 g, less than about 2 g, or less than about 1.5 g of HMO, such as 2'-fucosyllactose.

[0094] Formulating compositions using HMOs can present challenges. It has been found that more reproducible and consistent compositions can be achieved by controlling the particle size distribution (PSD) of the HMOs. While not wishing to be bound by theory, it is believed that a slightly narrower PSD improves the flowability of the HMOs, allowing for more efficient mixing with other components. Furthermore, a PSD within a specific range is believed to provide a better solubility profile. The particle size of the HMOs can be determined using standard methods, such as using a sieve tower, which separates the powder into different fractions at a predetermined amplitude after a defined time. The sieves used in this method can conform to DIN ISO 3310-1.

[0095] Preferably, the 2'-fucosylated lactose (or other HMO) used in this composition has the following particle size characteristics: Percentage passing through #230 mesh (63μm) — less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

[0096] Percentage passing through #100 mesh (150μm) — greater than approximately 75%, greater than approximately 70%, greater than approximately 65%, greater than or equal to approximately 60%.

[0097] Percentage passing through #45 mesh (355μm) — greater than approximately 95%, greater than approximately 92%, greater than or equal to approximately 90%.

[0098] Percentage passing through #20 mesh (850μm) — 100%.

[0099] Preferably, the compositions of this disclosure are administered orally. Therefore, the compositions are generally in a form suitable for oral administration. The compositions may be solid or liquid compositions. The compositions may be in unit dosage form. For example, the compositions may be capsules, lozenges, pills, tablets, soft gels, powder packets, sticks, stick powders, or in more general compositions such as oil drops, emulsions, or ointments, or in any other suitable carrier that is determined by those skilled in the art to be an effective carrier for living organisms.

[0100] The composition can be encapsulated, for example, using a suitable polymer matrix to improve its long-term stability and storage. Those skilled in the art will understand that any suitable encapsulation material or matrix, as well as encapsulation methods and techniques known to them, can be used.

[0101] The composition may be included in a dietary supplement or pharmaceutical composition, or may be part of a feeding product or food, such as fermented dairy products, like yogurt or infant formula.

[0102] The compositions disclosed herein may be in unit dose form or in a more general composition as described above or as part of a dietary supplement or feeding product or food, such as fermented dairy products, such as yogurt or infant formula, comprising bacteria as Bifidobacterium animalis subsp. lactis deposited under DSM 15954 and optionally as Bifidobacterium longum subsp. infantis deposited under DSM 15953.

[0103] As used herein, the term "subject" refers to any mammal, including (but not limited to) livestock and other agricultural animals (such as cattle, goats, sheep, horses, pigs, and chickens), performing animals (such as racehorses), companion animals (such as cats and dogs), laboratory animals, and humans. Typically, the subject is a human.

[0104] In one implementation, it can be at least 1×10 6 The dosage is CFU / day for *Bifidobacterium animalis* subsp. *lactamase* preserved in DSM 15954. The preferred dosage is at least 1 × 10⁻⁶ CFU / day. 6 CFU / day, at least 1×10 7 CFU / day, at least 1×10 8 CFU / day, at least 1×10 9 CFU / day, at least 1×10 10 CFU / day, at least 1×10 11 CFU / day, at least 1×10 12 CFU / day.

[0105] In one implementation, it can be at least 1×10 6 The dosage is CFU / day as Bifidobacterium animalis subsp. lactis, preserved in DSM 15954, and at least one other strain, such as Bifidobacterium longum subsp. infantis, preserved in DSM 15953. The preferred dosage is at least 1 × 10⁻⁶ CFU / day. 6 CFU / day, at least 1×10 7 CFU / day, at least 1×10 8 CFU / day, at least 1×10 9 CFU / day, at least 1×10 10 CFU / day, at least 1×10 11 CFU / day, at least 1×10 12 CFU / day.

[0106] In one embodiment, the composition of this disclosure comprises *Bifidobacterium animalis* subsp. *lactamase* in dried, frozen, or lyophilized form as preserved under DSM 15954, and at a concentration of 1 × 10⁻⁶. 8 CFU / day up to 1×10 11 The composition is administered at a dose of CFU / day.

[0107] In another embodiment, the composition of this disclosure comprises, in dried, frozen, or lyophilized form, *Bifidobacterium animalis* subsp. *lactamase* preserved under DSM15954 as the sole probiotic product, and is packaged at 1×10⁻⁶. 8 CFU / day up to 1×10 11 The composition is administered at a dose of CFU / day.

[0108] In one embodiment, the composition of this disclosure comprises *Bifidobacterium animalis* subsp. *lactamase*, deposited under DSM 15954, and *Bifidobacterium longum* subsp. *infanthi*, deposited under DSM 15953, as the sole probiotic product, and is packaged at 1×10⁻⁶. 8 CFU / day up to 1×10 11 The composition is administered at a dose of CFU / day.

[0109] Those skilled in the art will understand that the administration of the compositions disclosed herein can be performed at the required dose levels and dosage regimens, depending on the environment and the condition of the subject. Suitable dosage regimens can be determined based on the teachings of this application. Dosage regimens can be adjusted to provide optimal support for intestinal barrier integrity in adults and / or children, and / or promote the growth of beneficial microbiota in the gastrointestinal tract of adults and / or children, and / or improve immune health in adults and / or children.

[0110] Those skilled in the art will understand that the precise amount and rate of administration of *Bifidobacterium animalis* subsp. *lactamase* (DSM 15954) and optionally *Bifidobacterium longum* subsp. *infant* (DSM 15953) will depend on factors such as the subject's age, weight, general health condition, sex, and dietary requirements. Based on the teachings herein, those skilled in the art can determine the appropriate dosage regimen for each specific situation through routine trials and experiments.

[0111] In one exemplary embodiment, the composition may be applied daily for at least one day. Alternatively, the composition may be applied once or more daily for at least one day, two days, four days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, or longer.

[0112] Therefore, this disclosure provides a composition comprising, in dried, frozen, or lyophilized form, *Bifidobacterium animalis* subsp. *lactamase* strain preserved under DSM 15954 and at least one HMO, and optionally, *Bifidobacterium longum* subsp. *infant* strain preserved under DSM 15953, wherein the composition comprises 1 × 10⁻⁶ bacteria. 8 CFU / day up to 1×10 11 The composition was administered at a dose of CFU / day for at least 2 weeks.

[0113] In one embodiment, this disclosure provides a composition of this disclosure in dried, freeze-dried, or lyophilized form comprising bacteria as a DSM 15954-preserved *Bifidobacterium animalis* subsp. *lactamase* and at least one HMO, and optionally a DSM 15953-preserved *Bifidobacterium longum* subsp. *infant*, wherein the composition comprises 1 × 10⁻⁶ bacteria. 8 CFU / day up to 1×10 11 The composition is administered orally at a dose of CFU / day for at least 8 weeks.

[0114] Other aspects of this disclosure provide combination therapies comprising a composition comprising, as described herein, a strain of *Bifidobacterium animalis* subsp. *lactamase* deposited as DSM 15954 and optionally a strain of *Bifidobacterium longum* subsp. *infant* deposited as DSM 15953, and at least one HMO for co-administration to a subject in need thereof. The composition may be formulated as a combination or a single composition of one or more bacterial strains and HMOs.

[0115] In one embodiment, a strain of Bifidobacterium animalis subsp. lactis preserved as DSM 15954 and at least one HMO and optionally a strain of Bifidobacterium longum subsp. infantis preserved as DSM 15953 are introduced into a feeding product or food, such as an infant formula, health food, food additive, dietary supplement, drug or over-the-counter preparation, in solid form such as powder, tablet or liquid.

[0116] In a preferred embodiment, the strain of *Bifidobacterium animalis* subsp. *lactamase* preserved in DSM 15954 is used at least 10 6 A concentration of CFU / g is present. Specifically, the concentration may be at least 1 × 10⁻⁶. 6 CFU / g, at least 1×10 7 CFU / g, at least 1×10 8 CFU / g, at least 1×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g or at least 1×10⁻⁶ 12 CFU / g.

[0117] In one embodiment, the strain of *Bifidobacterium animalis* subsp. *lactamase* preserved in DSM 15954 and at least one other strain are used in a ratio of at least 10 6 A concentration of CFU / g is present. Specifically, the concentration may be at least 1 × 10⁻⁶. 6 CFU / g, at least 1×10 7 CFU / g, at least 1×10 8 CFU / g, at least 1×10 9 CFU / g, at least 1×10 10 CFU / g, at least 1×10 11 CFU / g or at least 1×10⁻⁶ 12 CFU / g.

[0118] In another aspect, this disclosure provides a method for producing a feeding product or food, dietary supplement, or pharmaceutical composition, the method comprising producing bacteria as a strain of Bifidobacterium animalis subsp. lactis preserved in DSM 15954 and at least one HMO and optionally a strain of Bifidobacterium longum subsp. infantis preserved in DSM 15953, and incorporating them into the food, supplement, or pharmaceutical composition at a concentration of at least 10⁶ CFU / g.

[0119] Preservation and expert solutions The applicant requests that, in accordance with the existing regulations of the Industrial Property Office of the Contracting State to the Budapest Treaty, the following deposited microbial samples may only be provided to experts prior to the patent grant date.

[0120] Table 1: For patent procedural purposes, the applicant has made deposits with the following institutions that have obtained international depositary status under the Budapest Treaty, in connection with the international recognition of microbial deposits: Leibniz Institute DSMZ-German Center for the Preservation of Microbial Cultures ( Leibniz Institute DSMZ-German Collection of Microorganisms and Cell CulturesInhoffenstr. 7B, 38124 Brunswick, Germany.

[0121]

[0122] Example Example 1—Antibiotic sensitivity of Bifidobacterium animalis subsp. lactis (DSM 15954) and Bifidobacterium longum subsp. infantis (DSM15953) strains. Antibiotic susceptibility and cytotoxic activity analysis According to the international standard ISO 10932 | IDF 223, the antibiotic susceptibility of *Bifidobacterium animalis* subsp. lactis strain deposited by the German Microbial Culture Collection (DSMZ) as DSM 15954 and *Bifidobacterium longum* subsp. infantis strain deposited as DSM 15953 was determined by measuring the minimum inhibitory concentration (MIC) of some antibiotics.

[0123] The tests performed were conducted using the microbroth dilution method. This method used VetMIC Lact-1 and Lact-2 groups (National Veterinary Institute of Sweden, Uppsala, Sweden) and LSM medium (ISO-sensitest medium (Oxoid)) supplemented with 10% MRS (de Man, Rogosa and Sharpe) broth (BD Difco 288110, UK) and 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany), grown anaerobically at 37°C for 48 hours. The tests were performed in triplicate. The range of antibiotics tested complied with the European Food Safety Authority (EFSA) guidelines for Bifidobacterium (Bifidobacterium). Bifidobacterium The group’s “Guidance on the characterisation of microorganisms used as feed additives or as production organisms” (EFSAJournal 2018, 16:5206).

[0124] According to the EFSA guidelines (gentamicin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin), DSM 15954 and DSM 15953 were found to be sensitive to all antibiotics associated with the Bifidobacterium group, with MIC values ​​below the EFSA 2018 cutoff values.

[0125] Antibiotic resistance genes in the genomes of DSM15954 and DSM 15953 were analyzed by screening the ResFinder database, a curated database containing over 2,200 resistance genes (Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O. et al. (2012) Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 67: 2640–2644). The database was downloaded and imported into CLC Main Workbench, version 8.0.1, on February 8, 2019. Genomic resistance genes were screened against the ResFinder database using megaBLAST settings (expected threshold 10, word length 28). Consistent with the sensitivity of DSM 15954 and DSM 15953 to all tested antibiotic phenotypes, no antibiotic resistance genes were identified in the genomes of DSM 15954 and DSM 15953.

[0126] Following the EFSA guideline "Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition" (EFSA Journal 2014, 12:3665), the cytotoxic activity of DSM 15954 and DSM 15953 was tested using the Vero cell assay. DSM 15954 and DSM 15953 were grown at 37°C under anaerobic conditions in MRS broth (BD Difco 288110, UK) containing 0.05% cysteine ​​hydrochloride (CyHCl) (Merck 102839, Germany). After 24 and 48 hours, the culture supernatant was separated by centrifugation, and cytotoxicity was analyzed using the Vero cell assay at Bioneer A / S in Helsholm, Denmark. DSM 15954 and DSM 15953 were found to be non-cytotoxic.

[0127] Example 2—Bifidobacterium animalis subsp. lactis (DSM 15954) (BB-12) ® Studies on bile salt debinding of Bifidobacterium longum subsp. infantis (DSM 15953) Bile salts are secreted from the gallbladder into the upper small intestine after dietary intake, where they play an important role in the absorption of dietary lipids. Original bile salts are synthesized in the liver, where the final step in this pathway involves binding glycine or taurine to bile acids to increase their solubility. The total amount of glycine-bound bile salts is greater than that of taurine-bound bile salts, with a ratio of approximately 3:1.

[0128] Specific mammalian gut bacteria and probiotic strains express bile salt hydrolase (BSH), which debindes secreted bile salts with variable efficiency and selectivity.

[0129] Bile salt hydrolytic enzyme assay Bile salt hydrolase (BSH) activity assays were used to investigate whether specific bacterial strains possess the ability to debind bile salts. In this study, live bacteria were co-incubated with bile acids bound to taurine and glycine under physiological conditions to explore possible amino acid debinding. Subsequently, the assay was stopped, and downstream analysis was performed to quantify the debinded “free” taurine and glycine amino acids.

[0130] Preparation of bacteria Inoculate with Bifidobacterium animalis subsp. lactis (BB-12) from the cryopreservation of Chr. Hansen culture as DSM 15954. ® Bifidobacterium longum subsp. infantis (DSM 15953) and another related strain included in the assay were used and anaerobically cultured overnight in MRS (Derman-Rogosa-Sharp) broth (Difco) at 37°C using an AnaeroGen pad (Oxoid). Ten-fold dilution series were prepared from the overnight culture and anaerobically incubated overnight at 37°C. Results were based on optical density (OD) at 600 nm. 600 Measure the values, select two late exponential growth phase cultures and merge them. Centrifuge the bacterial cultures at 6000×g for 2 min and wash twice in phosphate-buffered saline (PBS).

[0131] The bile salts included as substrates in the bile salt hydrolysis assay are: Glycinecholic acid (GCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA). All were purchased from Sigma Aldrich and dissolved in MilliQ water. Mixtures of the above bile salts were designed in proportions relevant to human small intestine physiology and tested at a total concentration of 5.2 mM to minimize toxicity.

[0132] The 5.2 mM total bile salt (BS) mixture contains 1.5 mM GCA; 1 mM GDCA; 1.5 mM GCDCA; 0.5 mM TC; 0.2 mM TDCA; and 0.5 mM TCDCA.

[0133] As control samples, the following samples were included: total BS mixture, with / without bacteria; culture medium containing only bacteria; positive analysis control (0.1 mM glycine + 0.01 mM taurine); and BSH-negative bacteria control.

[0134] At time 0, the bacterial suspension and BS mixture were added to 0.1 M acetate buffer and incubated at 37°C. After 10 and 60 minutes, aliquots were taken, and the reaction was immediately stopped by adding methanol. After vigorous mixing, the samples were rapidly frozen in dry ice and stored at -80°C until further analysis.

[0135] Amino acid quantification Prior to analysis, the sample was diluted 5-fold in MilliQ water and mixed with an internal standard, 0.2 M borate buffer, and AccQ-Tag Ultra reagent (Waters). To derivatize the sample, the mixture was heated in a hot mixer at 2000 rpm at 55 °C for 10 min. The sample was injected into a liquid chromatography-triple quadrupole mass spectrometer (LC-QqQ-MS) equipped with an AccQ-Tag Ultra RP column, and amino acids, including glycine and taurine, were quantified in positive ion mode via multiple reaction monitoring (MRM). The concentrations of glycine and taurine were obtained through internal calibration using real standards.

[0136] Data Analysis Subtract the levels of free glycine and taurine from the bacterial-free control from the sample data to remove background. Plot replicates on an XY plot to compare the BSH activity of the strains under discussion. The deviation of the replicate data (standard error bars SD±) is sometimes too small to be displayed on the plot.

[0137] result The debinding of glycine and taurine was quantified after 10 minutes and 60 minutes of incubation. Figure 1 During incubation, no spontaneous debinding of taurine or glycine from bile salts was observed in the controls with / without bacteria. Furthermore, free glycine or taurine was not detected in the controls with / without a BS mixture.

[0138] In the determination of a mixture containing 5.2 mM total BS, Bifidobacterium animalis subsp. lactis (BB-12), preserved as DSM 15954, was...® It exhibits robust glycine debinding ability, with an activity of 782 μM / h. Figure 1 A). It is known that taurine debinding is less effective than glycine debinding; however, this is the case for Bifidobacterium animalis subsp. lactis (BB-12), preserved in DSM 15954. ® Significant activity was observed in the debinding of a 5.2 mM MBS mixture at a rate of 34 μM / h. Figure 1 B). When the BS mixture was incubated with a known BSH-negative strain, no significant increase in deconjugated glycine or taurine could be measured after 60 minutes. Figure 1 A and B). Furthermore, when deconjugated glycine (33.2 μM / h) and taurine (4.4 μM / h) were measured after 10 and 60 minutes of incubation, *Bifidobacterium longum* subsp. *infantica* (DSM 15953) exhibited BSH activity (…). Figure 1 (C and D).

[0139] Compared to *Lactobacillus rhamnosus*, which was included in the same BSH activity assay, *Bifidobacterium animalis* subsp. *lactamase* (BB-12), preserved as DSM 15954, showed significantly higher activity levels. ® This demonstrates excellent BSH activity in the debinding of glycine and taurine. Figure 1 E and F).

[0140] Example 3—Study on the inoculation capacity of Bifidobacterium longum subsp. infantis (DSM 15953).

[0141] Two proof-of-principle studies investigated the effects of two different dosing regimens on the recovery rate of the administered strains, the total abundance of Bifidobacteria in infant stool samples, and gastrointestinal tolerance symptoms such as crying and irritability, stool frequency, and consistency. These studies were conducted in accordance with current versions of Good Clinical Practice and the Declaration of Helsinki. Infant-mother duos were recruited from midwifery consultations between July and September 2021. Prior to participant recruitment, these studies were registered on ClinicalTrials.gov (identified by NCT04987593 and NCT04994834).

[0142] For both studies, the mother-infant duo was included based on the following criteria: eligible infants were healthy female and male infants, born at full term (gestational age ≥37 weeks and ≤42 weeks), singleton birth, with a birth weight of 2500–4500g, and an APGAR score of at least 7 within 10 minutes of birth. Infants with any congenital conditions that could affect study results or participant safety, or those transferred to the neonatal intensive care unit within 5 hours of birth, were excluded from the study. Eligible pregnant women were those with simple pregnancies who intended vaginal delivery and breastfeeding, and who could avoid consuming other probiotics.

[0143] Research Design Two open-label proof-of-concept studies were conducted at the same time and location, with generally similar designs and evaluations, except for the participants (mothers or infants) receiving the intervention. The studies were conducted in three phases: a prenatal phase, in which pregnant women recruited for the maternal vaccination study took the study product for at least 4 weeks before 37 weeks of gestation; a 4-week postnatal intervention phase, in which infants were given the study product for 28 days (approximately 4 weeks), and the mothers receiving the maternal vaccination continued the intervention; and a final phase, a 2-week follow-up period, during which no study product was taken.

[0144] In the maternal vaccination study, mothers began taking the study product from week 33 of gestation (GA) to ensure at least 4 weeks of intervention before week 37 of GA, and continued taking it for 28 days postpartum. These mothers received a 1g daily dose containing at least 0.1 × 10⁻⁶ ppm of the product. 9 CFU Bifidobacterium infantis DSM 15953 (ISTILOS™) sticks and containing at least 2×10 9 CFU Bifidobacterium animalis subsp. lactis DSM 15954 (BB-12) ® ), 2×10 9 CFU Lactobacillus rhamnosus LGG DSM 33156 (LGG) ® ) and 2×10 9 CFU (Lactobacillus acidophilus LA-5) ® (ISTILOS™, BB-12) ® LGG ® and LA-5 ® Capsules (a registered trademark of Chr. Hansen A / S).

[0145] All statistical analyses were performed using SAS v9.4 Windows (SAS Institute Inc., Carey, NATO, USA). The Safety dataset (SS) consisted of all participants. The Intention-to-Treat dataset (ITT) included all mother-infant pairs who completed the second visit and met the final eligibility criteria. The Compliance-to-Protocol dataset (PPS) included all mother-infant pairs from the ITT without any significant bias. Statistical significance was considered to be p < 0.05. Due to the proof-of-principle and open-label design of both studies, no formal efficacy considerations or sample size calculations were performed. To use one of the studies as a control, we applied the stepped rules of thumb for the required pilot study sample size, explaining why a sample size of 25 mother-infant pairs per study was considered appropriate (Whitehead et al., 2016).

[0146] result Probiotic strains for mothers and infants: Bifidobacterium infantis DSM 15953, DSM 15954 and LGG ® This led to the recovery of these strains in infant stool samples. Figure 2 (and Figure 3), although the degree and amount of recovery differed. When supplemented directly to infants, Bifidobacterium infantis DSM 15953 was recovered in 100% of infants 14 days after ingestion and throughout the study period, compared to 20.8%, 22.7%, and 25.0% on days 14, 28, and 42 for infants administered via maternal dose, respectively. For LGG ® The same pattern was observed; in the infant vaccination group, 100% recovery was shown, compared to 20.8%, 30.4%, and 20.8% in the maternal vaccination group, respectively. In the infant vaccination group, DSM15954 was recovered in all infants on days 14 and 28, then decreased to 35% of infants on day 42. In the maternal vaccination group, DSM 15954 was detected in only 8.3%, 17.3%, and 8.3% of infants on days 14, 28, and 42, respectively. Three subjects in the infant vaccination group received the first dose of the study product before stool samples could be collected, which is reflected in the presence of all three doses of the strain in the first stool sample.

[0147] Regarding the quantity recovered from infant fecal samples, different patterns were observed within groups and between the two inoculation regimens, and among the three doses of the strain. For *Bifidobacterium infantis* DSM 15953, a steady increase in quantity was observed in infants receiving direct doses throughout the study period, even up to 14 days after intervention termination, indicating colonization of *Bifidobacterium infantis* DSM 15953. A similar trend was observed in infants receiving maternal inoculation, although the amounts reached were much lower compared to infants receiving direct doses. LGG ® Both LA-5 and DSM 15954 transiently colonized the infant intestine, and the numbers of these strains were significantly lower than those observed in infants who received direct inoculation. ® The dose was administered only in the maternal vaccination group and recovered in one infant on day 28 and day 42 (data not shown).

[0148] Strain-specific qPCR analyses were also performed in placental, umbilical cord, cord blood, maternal fecal, and breast milk samples. The administered strains were not detected in placental, umbilical cord, or cord blood samples by strain-specific qPCR. At all three time points, the administered strains were recovered from maternal fecal samples from the maternal vaccination group, but at varying frequencies. In a small number of subjects, the administered strains were detected in breast milk samples from the maternal vaccination group, while significant levels of recovery were detected in breast milk samples from the infant vaccination group. This may be due to cross-contamination of the administered strains from the infant's mouth to the nipple and areola during breastfeeding.

[0149] Direct supplementation to infants resulted in a significant increase in Bifidobacteria in the infant gut microbiome after 14 days of supplementation. Figure 2 (and 3). A similar trend was observed in infants who were maternally vaccinated; however, the increase in Bifidobacteria was gradual and only became significant at the end of the study (5th visit, day 42).

[0150] Alpha diversity analysis of samples from the infant and maternal vaccination groups showed that the postnatal microbiome was characterized by higher abundance and diversity compared to day 42 (p<0.05). In the infant vaccination group, the relative abundance of Bifidobacteria was higher at all time points than at birth. The difference in Bifidobacteria abundance between the two groups decreased over time. Compared to the infant vaccination group, the maternal vaccination group showed a higher abundance of Bifidobacteria brevis (infants) and a lower abundance of Bifidobacteria longum (infants). B. breve ) and Bifidobacterium adolescentis ( B. adolescentisOTU (Operational Taxonomic Unit) compensation. In summary, the increase in Bifidobacteria in the maternal vaccination group was slower compared to the infant vaccination group, and did not become significant until day 28 and beyond (days 28 and 42, respectively). Although the increase in Bifidobacteria in the infant vaccination group from birth to day 14 was greater than that in the maternal vaccination group, there was no significant difference in total Bifidobacteria abundance between the two interventions at day 14 due to large inter-individual variability and corrections for multiple tests. In the first fecal sample collected shortly after birth, the microbiota contained bacteria typically associated with the birth canal, vagina, and skin, including Lactobacillus ( Lactobacillaceae ),staphylococcus( Staphylococcaceae ) and Enterobacteriaceae ( Enterobacteriaceae These are all aerobic and aerobic / microaerobic species, and the compositions were similar across studies. In the infant vaccination group, the compositional changes were primarily due to a significant increase in Bifidobacteria, Lactobacillus, and Streptococcus, along with a decrease in typical skin bacteria such as Corynebacterium, Epidermobacterium, and Staphylococcus, as well as several Proteobacteria. Although the most significant effects occurred from birth to day 14, these dynamics continued to develop even after dose discontinuation. In contrast, in the maternal vaccination group, these dynamics were delayed by 14 days or longer, mainly because some infants still had Bifidobacteria below the detection threshold (while most infants in the infant vaccination group had levels above 40%). In the maternal vaccination group, the significant increase in the aforementioned Bifidobacteria, Lactobacillus, and Streptococcus, along with a decrease in typical skin bacteria such as Corynebacterium, Epidermobacterium, and Staphylococcus, as well as several Proteobacteria, was not observed until day 28.

[0151] Table 2

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Table 2: Comparison of microbial composition in fecal samples from infants at birth, day 14, day 28, and day 42 in studies of infant vaccination versus maternal vaccination (see Table 2). Figure 3B (for visual display) Infant gastrointestinal signs and symptoms During the first week after birth, the frequency of bowel movements (number of bowel movements per day) was the same in both the infant vaccination and maternal vaccination groups, averaging 3.9 ± 1.4 times and 3.4 ± 1.6 times per day, respectively. From the second week after birth, the average bowel movement frequency was significantly lower in the infant vaccination group compared to the maternal vaccination group (p = 0.0058). In the first 28 days of the intervention, infants receiving the direct dose had fewer bowel movements per day (p = 0.0027 on day 14, p = 0.0231 on day 28, AUC (area under the curve) p = 0.0052). Figure 4 a)).

[0158] Regarding other gastrointestinal signs and symptoms, no differences were observed between the two groups compared to infants who exhibited struggling, gurgling or crying before defecation, straining or gas during defecation.

[0159] Significant differences in stool consistency were also observed between the two dosage regimens, with infants receiving maternal vaccination passing softer stools more frequently than those in the infant vaccination group (p=0.0058). More frequent occurrences of hard stools were observed in the maternal vaccination group (p=0.0163). Throughout the intervention, the mean number of watery and formed stools was similar in both groups. Figure 4 b).

[0160] Compared with infants who received direct vaccination, infants who received maternal vaccination showed a significantly steeper developmental slope for crying and irritability during the intervention period (p<0.0001). Figure 4 c).

[0161] Production of microbial characteristic metabolites—conversion of aromatic amino acids to aromatic lactic acid No difference was observed in the levels of three aromatic amino acids (precursors to aromatic lactic acid) in fecal samples from both groups of infants. Therefore, an upregulation of the conversion of aromatic amino acids to aromatic lactic acid amino acids was observed in infants receiving probiotics, as measured by the presence of indole-3-lactic acid, 3-phenyllactic acid, and p-hydroxyphenyllactic acid. Figure 4 d).

[0162] Upregulation of several amino acid metabolites was also observed in the infant vaccination group, including elevated levels of tryptophan metabolites and purine nucleosides adenosine, N-acetyl-alanine, N-acetyl-serine, N-acetyl-lysine, N-acetyl-cysteine, N-acetyl-leucine, N-acetyl-isoleucine, and N-acetyl-tyrosine. This suggests that the administered strain promotes intestinal immune tolerance and the proliferation of beneficial gut microbiota (for N-acetyl-lysine, see [link to relevant documentation]). Figure 4 e).

[0163] Upregulation of methionine was observed in infants receiving direct doses, particularly on day 14 (N-acetyl-methionine, N-formyl-methionine, and S-methyl-methionine). Methionine belongs to the class of organic compounds known as methionines and their derivatives. Methionines and their derivatives are compounds containing methionine or its derivatives, produced by the reaction of methionine at the amino or carboxyl group. It is a precursor to the important antioxidant glutathione, and methionine plays a crucial role in metabolism and health in many species. Figure 4 f).

[0164] in conclusion These studies indicate that increasing the abundance of Bifidobacteria, either directly or via maternal inoculation, has a beneficial effect on the infant microbiome. Oral administration of Bifidobacterium infantis DSM 15953 and Bifidobacterium animalis subsp. lactis DSM 15954 (BB-12)... ® ) and Lactobacillus rhamnosus LGG DSM 33156 (LGG ® It causes beneficial changes in bowel movement frequency, crying, and irritability.

[0165] By administering Bifidobacterium directly to infants, we have shown that Bifidobacterium is a powerful regulator of the immune system, a “shaper” of the gut microbiota, and a modulator of the fecal metabolomics profile, each of which may help improve intestinal inflammation in early life and health later in life.

[0166] Example 4—Bifidobacterium longum subsp. infantis (DSM 15953) and its ability to grow using HMOs Materials and methods Bifidobacterium longum subsp. infantis (DSM 15953) was inoculated from the frozen primary culture and anaerobic cultured overnight at 37°C in Derman-Rogosa-Sharp (MRS) broth with 0.05% cysteine ​​hydrochloride (CyHCl), pH 6.5. Ten-fold dilutions were prepared from the overnight culture and incubated under the same conditions as described above. Based on the optical density (OD) at 600 nm... 600 The late exponential phase / early stationary phase was selected by measuring OD450. Bacterial cultures were washed twice in a carbohydrate-free broth supplemented with 0.05% CyHCl. 2'-fucosylated lactose (2'FL) was prepared as a 20 g / L (2% wt / vol) solution in a broth containing 0.05% CyHCl and sterilely filtered using a vacuum filter (0.22 μm, PES filter). In a 96-well microtiter plate, 180 μL of the 2'FL solution and 20 μL of the washed OD450 solution were added. 600 A solution of regulated bacteria was added to each well, repeated three times (final OD).600 =0.1). Bacterial growth preparations in the MRS were used as positive controls, while 180 μL of basal broth with / without 2'FL and 20 μL of washed bacteria were used as negative controls. 96-well microtiter plates were incubated at 37°C under anaerobic conditions for 96 hours. After incubation, the optical density (OD) was measured using a spectrophotometer. 600 The experiment should be repeated at least four times, with each repetition consisting of three repetitions.

[0167] Intergroup comparisons were performed using one-way ANOVA, followed by Dunnett's multiple comparisons test for broths without carbohydrates.

[0168] result The growth of *Bifidobacterium longum* subsp. *infantii* (DSM 15953) on 2'-fucosylated lactose (2'FL) was studied in vitro. Figure 5 a). Compared with growth on carbohydrate-free broth (white column), the growth of *Bifidobacterium longum* subsp. infantis (DSM15953) on 2% 2'FL (grey column) was significantly enhanced. The results suggest that HMOs and probiotics may benefit from combined administration.

[0169] Example 5—Compared to components tested individually or in pairs, Bifidobacterium animalis subsp. lactis BB-12 ® The combination of (DSM 15954), *Bifidobacterium longum* subsp. *infantitidis* (DSM 15953), and 2'-fucosylated lactose (2'FL) introduced a higher intestinal barrier tightness, as evaluated by transepithelial electrical resistance (TEER) across the Caco-2 cell monolayer in vitro. Epithelial and endothelial cells form barriers in the body. The strength and integrity of these barriers can be obtained by measuring transcellular resistance (TEER) in vitro.

[0170] TEER is a recognized method for evaluating and monitoring epithelial tissue in a non-destructive assay. Specifically, it rapidly identifies the confluence of monolayers. Confluence can be tracked and monitored in real time because TEER measurements rise as gaps in the monolayer close. TEER is commonly used as a strong indicator of cellular barrier integrity and permeability for both epithelial and endothelial cells within the monolayer.

[0171] Caco-2 cell culture Human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz Institute DSMZ-German Culture Collection, Braunschweig, Germany) was cultured in complete DMEM medium containing DMEM GlutaMax (Gibco 21885) supplemented with 20% heat-inactivated fetal bovine serum (Gibco 10500), 1×MEM non-essential amino acids (Biowest X0557-100), and 1×Pen-Strep-Amp B (Biological Industries 03-033-1B) at 5% CO2 and 37°C. Caco-2 cells were used from passages 7 to 25. When the cells reached approximately 50% confluence, the medium was removed, and the cells were washed twice in phosphate-buffered saline (PBS). Cells were trypsinized by adding 1 mL of trypsin (TrypLE, Gibco 12604) and incubated at 37°C in a CO2 incubator for 3 min. Approximately 10 mL of culture medium was added to trypsinized cells, and they were counted to prepare a concentration of 100,000 cells / mL in complete DMEM. Each top compartment of a CellQART 12-well cell culture insert (0.4 μM PET Clear (9310412)) from a Greiner 12-well plate (M40192) was seeded with 500 μL of cell suspension, followed by adding 1.5 mL of supplemental DMEM to the basal outer compartment. Cells were cultured on the insert for 21 days, with the medium changed twice weekly. After 21 days, the transfer plate was transferred to a CellZscope (NanoAnalytics, Germany). 1.65 mL and 0.8 mL of antibiotic-free (Abx) complete culture medium were added to the basal outer compartment and top compartment, respectively. The CellZscope was incubated overnight in a 37°C CO2 incubator (5%), and TEER was measured hourly using automated data collection. Before the experiment began, this overnight measurement of TEER enabled the determination of the baseline TEER in each hole and served as a quality control for the stabilizing resistance.

[0172] Preparation of 2'-fucosylated lactose (2'FL) in DMEM medium Prepare an 8% 2'-fucosylated lactose (2'FL) solution in a complete DMEM medium without Abx.

[0173] Bifidobacterium animalis subspecies BB-12 ® (DSM 15954) and Bifidobacterium longum subsp. infantis (DSM 15953) preparation One day before co-incubation with Caco-2 cells, Bifidobacterium animalis subsp. BB-12 was introduced. ®Frozen primary cultures of *Bifidobacterium animalis* (DSM 15954) were inoculated into MRS containing 0.05% CyHCl, while frozen primary cultures of *Bifidobacterium longum* subsp. *infanthi* (DSM 15953) were inoculated into MRS containing 0.19% CyHCl and 0.14% sodium carbonate. The inoculation was completed by transferring 1 mL of the mixed inoculated culture and 9 mL of MRS containing 0.05% CyHCl (for *Bifidobacterium animalis* subsp. *infanthi* BB-12). ® (DSM 15954) or 0.19% CyHCl and 0.14% sodium carbonate (for Bifidobacterium longum subsp. infantis (DSM 15953)) MRS, to produce dilution rows for each strain. Repeat this 5 times. Incubate Bifidobacterium anaerobically at 37°C overnight. On the day of co-incubation, measure the optical density (OD) at 600 nm. 600 To evaluate bacterial growth, cultures representing the late exponential phase / early stationary phase were selected. Cultures from each strain were centrifuged at 6000×g for 5 min to collect bacterial pellet particles. The supernatant was discarded, and 35 mL of warm (37°C) PBS was added. The bacteria were washed and centrifuged at 6000×g for 5 min. This washing procedure was repeated twice. Bacterial cells were harvested by centrifuging at 6000×g for 5 min, and the supernatant was discarded. The bacterial cells were resuspended in 5 mL of pre-warmed, Abx-free complete DMEM medium, and OD200 was increased. 600 Adjust to 4.

[0174] Bifidobacterium lactis subspecies BB-12 ® (DSM 15954), Bifidobacterium longum subsp. infantis (DSM 15953) and 2'FL alone or in combination stimulates Caco-2 cells To stimulate Caco-2 cells, pause CellZscope measurements, remove the CellZscope from the CO2 incubator, and remove 300 μL of top culture medium from each transfer plate. Add 100 μL of sterile bacterial solution containing one or two bacteria (final OD). 600 Add 0.5% FL, 2% FL solution or culture medium control (DMEM) to the relevant wells as described below (each repeated three times).

[0175] The following conditions were tested three times: • Culture medium control • Bifidobacterium animalis subsp. lactis BB-12 ® (DSM 15954) • Bifidobacterium longum subsp. infantis (DSM 15953) • 2% 2'FL+ Bifidobacterium longum infantis subspecies (DSM 15953) • Bifidobacterium animalis subsp. lactis DSM 15954 (BB-12) ®Bifidobacterium longum infantis subspecies (DSM 15953) • 2% 2'FL+ Bifidobacterium animalis subsp. lactis BB-12 ® (DSM 15954) + Bifidobacterium longum infantis subspecies (DSM15953) Transfer the CellZscope back to the CO2 incubator, resume TEER measurements, and incubate overnight. Calculate the change in TEER during bacterial stimulation relative to the latest value recorded immediately before stimulation (baseline measurement, set to 100%). Calculate the area under the curve for each well and compare different bacterial / 2'FL stimulations using one-way ANOVA, including Tukey's multiple comparison test.

[0176] result Bifidobacterium animalis subsp. lactis BB-12 was added to the top of the Caco-2 monolayer. ® Transepithelial electrical resistance (TEER) was measured after testing for (DSM 15954), *Bifidobacterium longum* subsp. infantis (DSM 15953), and 2'FL. Two bacteria, alone or in combination, caused moderate TEER readings; however, if all three components were present: *Bifidobacterium animalis* subsp. lactis BB-12... ® When (DSM 15954), *Bifidobacterium longum* subsp. infantis (DSM 15953), and 2'FL were added together, as shown by the dashed line, it resulted in a significantly higher TEER reading compared to all other groups. Figure 6 B). These results indicate that the combination can improve cell barrier integrity and permeability. See Table 3 for statistical tests.

[0177]

[0178] **** indicates that the adjusted p-value is <0.0001. *** indicates that the adjusted p-value is <0.001. Table 3: Statistical test results for comparisons between all groups using one-way ANOVA and Tukey's multiple comparison test (only comparisons of groups with all three components are shown: Bifidobacterium animalis subsp. lactis BB-12). ® (DSM15954), Bifidobacterium longum subsp. infantis (DSM 15953) and 2'FL).

[0179] Example 6—Cytokine secretion from human peripheral blood mononuclear cells (PBMCs) stimulated with Bifidobacterium longum subsp. infantis (DSM 15953) and 2'-fucosylated lactose (2'FL) To investigate the immunomodulatory capabilities of Bifidobacterium longum subsp. infantis (DSM 15953) and 2'-fucosylated lactose (2'FL), alone or in combination, during homeostasis and challenge conditions, the secretion of cytokines from human PBMCs was identified.

[0180] method Preparation of Bifidobacterium longum subsp. infantis (DSM 15953) Bifidobacterium longum subsp. infantis (DSM 15953) was inoculated from the frozen primary culture and cultured overnight at 37°C in Difco™ (MRS) pH 6.5 broth containing 0.19% cysteine ​​hydrochloride (CyHCl) and 0.14% sodium bicarbonate using AnaeroGen pads (Oxoid). The overnight bacterial culture was centrifuged at 3500×g for 10 min at room temperature, and the precipitate was washed twice with preheated Hanks Balanced Salt Solution (HBSS; Gibco™) and once with modified MRS (mMRS, carbohydrate-free) pH 6.9±0.2. Finally, the bacterial precipitate was resuspended in 10 ml of modified mMRS medium supplemented with 2% (w / v) 2'FL and incubated overnight as previously described. Subsequently, the overnight bacterial culture was used to prepare 10-fold dilutions in mMRS supplemented with 2'FL. Based on optical density OD... 600 Measurements were taken using a biophotometer (Epdorf), selecting cultures representing the late exponential growth phase. The cultures were centrifuged, and the precipitate particles were washed twice in HBSS and adjusted to OD in RPMI-1640 medium by adding 10% glycerol. 600 The concentration was 1.0. Finally, the bacterial sample concentration was determined by colony forming units (CFU) and diluted to 5 × 10⁻⁶. 7 The concentration is set at CFU / ml and stored at -80°C.

[0181] Isolation of peripheral blood mononuclear cells (PBMCs) PBMCs were isolated by density gradient centrifugation (1200 g, 10 min, RT) in SepMate™ tubes (50 ml). On the day of the experiment, four erythrocyte sedimentation rate (ESR) amber layers were collected from healthy, anonymous donors (Righospitalet, Blodbank, Copenhagen, Denmark). The isolated PBMCs were washed twice in PBMC medium consisting of RPMI (Sigma Aldrich), 2-mercaptoethanol (final concentration 50 μM, Sigma Aldrich), and HEPES buffer (final concentration 10 mM, Sigma Aldrich). Subsequently, they were analyzed using NucleoCounter... ®-MC200 TM Cells were counted and resuspended in the same medium supplemented with heat-inactivated fetal bovine serum (FBS, final 10%, Gibco). The PBMC suspension was adjusted to 2 × 10⁶ cells / mL. 6 Cells / ml, and pre-stimulation assays were performed in 96-well plates (1×10⁻⁶ cells / ml). 5 (1 cell / well) plated on a plate for at least 1 hour (37°C, at 5% CO2).

[0182] Stimulating PBMC PBMCs from four healthy donors were stimulated with a) 2'FL (2%), b) *Bifidobacterium longum* subsp. infantis (DSM 15953) grown on 2'FL, c) a combination of *Bifidobacterium longum* subsp. infantis (DSM 15953) and 2'FL, or d) left untreated. The stimulated PBMCs were divided into two groups: one group received no additional reagents (steady-state conditions), while the other group received 50 μg / ml poly(I:C) (Sigma) after 3 hours of incubation to simulate viral challenge (referred to as challenge conditions). Cells were incubated for 17 hours after either adding poly(I:C) to the challenge conditions or adding RPMI medium to the steady-state conditions, resulting in a total incubation time of 20 hours. The supernatants were collected and centrifuged (1500 × g, 5 min, RT) using an AcroPrep filtertop plate (Pall™ New York, USA) and then stored at -80°C until cytokine profiling analysis.

[0183] Cytokine quantification.

[0184] According to the manufacturer's instructions, the levels of IFN-γ and IL-10 secreted from stimulated PBMCs were quantified using U-plex Proinflammatory Combo 1 Human (catalog number K15049K) from Meso Scale Discovery (MSD) (MSD, Rockwell, MD, USA). Data are as follows: Figure 7 As shown, and expressed in picograms per milliliter (pg / ml).

[0185] result Under steady-state (untreated) conditions, stimulation of human PBMCs with 2'FL does not induce IFN-γ (a type of T... H 1. The secretion of response characteristic cytokines) or IL-10 (a tolerance-inducing / anti-inflammatory cytokine) Figure 7A). On the other hand, stimulation of PBMCs with *Bifidobacterium longum* subsp. infantis grown on 2'FL (DSM 15953) induced the secretion of IFN-γ (567 pg / ml) and IL-10 (68 pg / ml). When the combination of 2'FL and *Bifidobacterium longum* subsp. infantis grown on 2'FL (DSM 15953) was used to stimulate PBMCs, the IL-10 secretion level did not appear to change compared to *Bifidobacterium longum* subsp. infantis grown on 2'FL (DSM 15953) alone (53 pg / ml), but when combined with *Bifidobacterium longum* subsp. infantis (DSM 15953), IFN-γ secretion decreased (190 pg / mL), suggesting a possible anti-inflammatory effect of 2'FL, such as... Figure 7 As shown in Figure A.

[0186] In addition, to simulate viral challenge, synthetic virus-like particle aggregates (I:C) were used to challenge PBMCs 3 hours after stimulation with 2'FL, Bifidobacterium longum subsp. infantis (DSM 15953), or a combination of both. Figure 7 B). There was no significant difference in IL-10 secretion between the different groups compared to challenged PBMCs. However, IFN-γ secretion was enhanced (3165 pg / ml) in challenged PBMCs pre-stimulated with *Bifidobacterium longum* subsp. infantis grown on 2'FL. Although still enhanced, this secretion was decreased (1105 pg / ml) in challenged PBMCs pre-stimulated with a combination of 2'FL and *Bifidobacterium longum* subsp. infantis grown on 2'FL, suggesting a possible anti-inflammatory effect of 2'FL. Figure 7 As shown in B, there was no significant difference in PBMCs challenged with 2'FL pre-stimulation alone compared to the challenged control (65 pg / ml vs. 45 pg / ml).

[0187] in conclusion Stimulation of PBMCs with *Bifidobacterium longum* subsp. infantis (DSM 15953) induced IFN-γ secretion, a driver of T cells required to clear various infections (viruses and bacteria). H 1. A key cytokine in the immune response, and it initiates a rapid action of the immune system in the event of infection. This initiation is most pronounced under challenge conditions, where PBMCs pre-stimulated with *Bifidobacterium longum* subsp. infantis* (DSM 15953) increased IFN-γ secretion 5-fold after the introduction of virus-like molecules (poly(I:C)), potentially leading to a rapid antipathogen response. 2. The combination of IFN-γ and *Bifidobacterium longum* subsp. infantis* (DSM 15953) also induced IFN-γ secretion under both homeostatic and challenge conditions, although at reduced levels. If via T... HWhile a strong and uncontrolled inflammatory response is beneficial in clearing pathogens, it can be detrimental. Therefore, the reduction of this cytokine may be beneficial for infants, who typically have immature immune systems. Furthermore, under homeostatic conditions, stimulation of PBMCs with a combination of *Bifidobacterium longum* subsp. infantis (DSM 15953) and 2'FL and *Bifidobacterium longum* subsp. infantis (DSM 15053) induced the secretion of the anti-inflammatory cytokine IL-10, which is involved in maintaining immune response homeostasis. Under virus-like challenge (poly(I:C)) conditions, IL-10 secretion indeed resembled that under homeostatic conditions, indicating a maintenance of the ability to restore a balanced immune response.

[0188] Example 7—Cytokines derived from Bifidobacterium longum subsp. infantis (DSM 15953) or Bifidobacterium animalis subsp. lactis BB-12 ® (DSM 15954) Stimulation of secretion in human peripheral blood mononuclear cells (PBMCs) To identify the animal bifidobacterium lactis subspecies BB-12 ® The effects of DSM 15954 and Bifidobacterium longum infantis subsp. infantis (DSM 15953) on general immune regulatory responses induced by human PBMCs and on cytokine secretion were identified.

[0189] method As described in Example 5, peripheral blood mononuclear cells (PBMCs) were isolated.

[0190] Preparation of bacteria.

[0191] Cryogenic primary strains from Chr. Hansen culture preservation were inoculated with Bifidobacterium lactis subsp. BB-12. ® Strains of *Bifidobacterium longum* (DSM 15954) and *Bifidobacterium infantis* (DSM 15953) were cultured overnight anaerobically in AnaeroGen pads (Oxoid) at 37°C in pH 6.5 MRS (Derman-Rogosa-Sharp) broth (Difco) supplemented with 0.05% cysteine ​​hydrochloride or 0.19% cysteine ​​hydrochloride and 0.14% sodium bicarbonate, respectively. Ten-fold dilutions were prepared from the overnight cultures and incubated overnight under the same conditions. Results were based on optical density (OD). 600 Measure and select cultures representing the late exponential growth phase. Centrifuge bacterial cultures at 6000g for 2 min, wash twice in Hanks balanced salt solution (HBSS), and measure by OD. 600 The stock solution was resuspended in antibiotic-free complete DC medium at a concentration of 0.05.

[0192] Stimulation of PBMC Using a) Bifidobacterium animalis subsp. lactis BB-12 ® (a) Stimulation of PBMCs from 4 healthy donors with *Bifidobacterium longum* subsp. *infantitidis* (DSM15953) for 20 hours or (b) left untreated. The final concentration of a single strain was OD0.01, corresponding to approximately 10⁻⁶. 6 10 bacterial cells and 10:1 bacteria: PBMC MOI. Supernatant was collected on AcroPrep filtertop plates (Pall™ New York, USA), centrifuged (1500×G, 5 min, room temperature), and stored at -80°C prior to cytokine profiling analysis.

[0193] Cytokine quantification.

[0194] According to the manufacturer's instructions, the levels of IFN-γ, IL-12p70, IL-1β, IL-23, IL-6, IL-8, TNF-α, and IL-10 secreted from dendritic cells (DCs) were quantified using U-plexProinflammatory Combo 1 Human (catalog number K15049K) (MSD, Rockwell, MD, USA) from Meso Scale Discovery (MSD). Data are expressed in picograms per milliliter (pg / ml).

[0195] result Human PBMCs and Bifidobacterium lactis subsp. BB-12 ® Co-incubation with either *Bifidobacterium longum* subsp. *infanthi* (DSM 15954) or *Bifidobacterium longum* subsp. *infanthi* (DSM 15953) stimulated the secretion of common pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α. Compared to *Bifidobacterium longum* subsp. *infanthi* (DSM 15953) (400, 1329, and 1785 pg / ml, respectively), the levels of *Bifidobacterium animalis* subsp. *lactobacterium* BB-12 were significantly higher. ® PBMCs stimulated with (DSM 15954) showed an enhanced response (1940, 2040, and 6512 pg / ml, respectively). Furthermore, IFN-γ (a characteristic cytokine of the Th1 response essential for the clearance of viral and bacterial infections) was also induced by both strains, although this was less pronounced compared to stimulation with *Bifidobacterium infantis* (200 pg / ml) with *Bifidobacterium animalis* subsp. *lactam* BB-12. ® Following stimulation with (DSM15954), IFN-γ secretion was significantly increased (1077 pg / ml). Finally, the secretion of the anti-inflammatory cytokine IL-10 was also detected in PBMCs stimulated with both strains, and it is associated with Bifidobacterium animalis subsp. lactis BB-12. ®(DSM 15954) was 97 pg / ml, and for Bifidobacterium longum subsp. infantis (DSM 15953) it was 50 pg / ml, such as Figure 8 As shown.

[0196] in conclusion Overall, using Bifidobacterium animalis subsp. lactis BB-12 ® Cytokine profiles in the supernatant of human PBMCs stimulated with either *Bifidobacterium longum* subsp. *infantii* (DSM 15954) or *Bifidobacterium longum* subsp. *infantii* (DSM 15953) suggest that both strains appear to enhance immune responses that promote Th1 responses. Th1 cells play a crucial role in enhancing cell-mediated immune responses, particularly against intracellular viruses, bacteria, and protozoa; therefore, enhanced Th1 responses improve readiness for rapid infection challenge. Notably, however, compared to those stimulated with *Bifidobacterium animalis* subsp. *lactoum* BB-12... ® The cytokine response induced by *Bifidobacterium longum* subsp. *infantitidis* (DSM 15953) was not significantly greater than that induced by the response induced by DSM 15954, thus leaving room for a possible shift towards a more tolerogenic immune response driven by cytokines such as IL-10. This suggests that providing a composition containing both strains may be beneficial to those receiving the composition.

[0197] Example 8—Cytokines derived from Bifidobacterium longum infantis subsp. (DSM 15953) and Bifidobacterium animalis lactis subsp. BB-12 ® (DSM 15954) and their combinations stimulate the secretion of human dendritic cells.

[0198] To further investigate whether it is possible to provide Bifidobacterium animalis subspecies BB-12. ® The benefits associated with the combination of *Bifidobacterium longum* subsp. *infantii* (DSM 15954) and *Bifidobacterium animalis* subsp. *lactobacterium* (DSM 15953) were investigated by analyzing the cytokine profiles of dendritic cells after stimulation with the strains alone or in combination. ® (DSM 15954) and their combinations have the potential to drive specific adaptive T helper immune responses.

[0199] method Production of DCs derived from human monocytes For the in vitro production of human dendritic cells (DCs), Ficoll-Paque Plus (GE Healthcare, Freiburg, Germany) and SepMate were used. TMHuman peripheral blood mononuclear cells (PBMCs) were isolated from the erythrocyte sedimentation rate (ESR) amber layer of seven healthy donors using tubes (Stemcell Technologies, Cambridge, UK) via density gradient separation. PBMCs were then isolated by positive selection of CD14-positive cells using magnetically activated cell sorting with CD14 microbeads (Miltenyi Biotec, Bergschgladbach, Germany), and were separated at 2 × 10⁻⁶ tubes. 6 Cells / ml density in complete DC medium (DC medium supplemented with 2 mM L-glutamine [Life Technologies Ltd., Paisley, UK], 10% heat-inactivated FBS [Gibco]) TM Cells were cultured in 1% penicillin-streptomycin solution [Sigma-Aldrich, St. Louis, Missouri, USA]. The DC medium also contained 30 ng / ml human recombinant IL-4 and 20 ng / ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from Sigma-Aldrich, St. Louis, Missouri, USA). Cells were maintained at 37°C and 5% CO2. After 3 days of culture, fresh complete DC medium containing IL-4 and GM-CSF was added. On day 6, differentiation into immature DCs was confirmed by surface marker expression analysis (CD14, <12%; CD11c, >97% expression; CD1a, >85%). Immature DCs were resuspended in fresh, antibiotic-free complete DC medium at least 1 hour before bacterial stimulation.

[0200] Preparation of bacteria.

[0201] Cryogenic primary strains from Chr. Hansen culture preservation were inoculated with Bifidobacterium lactis subsp. BB-12. ® (DSM15954) and *Bifidobacterium longum* subsp. *infantitidis* (DSM 15953) strains were cultured anaerobically overnight in pH 6.5 MRS (Derman-Rogosa-Sharp) broth (Difco) supplemented with 0.05% cysteine ​​hydrochloride using an AnaeroGen pad (Oxoid). Ten-fold dilutions were prepared from the overnight cultures and incubated overnight under the same conditions. Optical density (OD) at 600 nm was used as a basis for analysis. 600 ) Measure and select cultures representing the late exponential growth phase. Centrifuge the bacterial cultures at 6000g for 2 min, wash twice in Hanks balanced salt solution (HBSS), and measure the OD value. 600 The stock solution was resuspended in antibiotic-free complete DC medium at a concentration of 0.05.

[0202] Bacterial irritation from DCs.

[0203] Using a) Bifidobacterium animalis subsp. lactis BB-12 ® (a) *Bifidobacterium longum* subsp. *infantii* (DSM15953), (b) a combination of two strains (1:1) stimulated DCs for 20 hours, or (d) left untreated. The final concentration of a single strain was OD 0.01, corresponding to approximately 10⁶ bacterial cells and a 10:1 bacteria:DC MOI. For combinations, the final MOI was 20:1. Cells were incubated at 37°C in a 5% CO₂ atmosphere. After stimulation, the DC supernatant was aseptically filtered through a 0.2 μm Acro-Prep Advance 96-well plate (Pall Corporation, Ann Arbor, Michigan, USA) and stored at -80°C until cytokine quantification.

[0204] Cytokine quantification.

[0205] According to the manufacturer's instructions, the levels of IL-12p70 and IL-10 secreted from DCs were quantified using U-plexProinflammatory Combo 1 Human (catalog number K15049K) from Meso Scale Discovery (MSD) (MSD, Rockwell, MD, USA). Data are expressed in picograms per milliliter (pg / ml) or as an IL-10 / IL-12 ratio.

[0206] result Human DC and Bifidobacterium lactis subsp. BB-12 ® (DSM 15954), Bifidobacterium longum subsp. infantis (DSM15953), or Bifidobacterium animalis subsp. lactis BB-12 ® Co-incubation with a combination of *Bifidobacterium longum* subsp. *infantii* (DSM 15954) and *Bifidobacterium animalis* subsp. *infantii* (DSM 15953) did not induce the secretion of IL-12, a key cytokine inducing the TH1 response. Conversely, co-incubation with a combination of *Bifidobacterium longum* subsp. *infantii* (DSM 15953) and *Bifidobacterium animalis* subsp. *mammary* BB-12 did not induce the secretion of IL-12. ® (DSM 15954) Both stimulated dendritic cells (DCs) to secrete the anti-inflammatory cytokine IL-10 (318 pg / ml and 120 pg / ml, respectively), and this was mediated by Bifidobacterium animalis subsp. lactis BB-12. ® The combination of DSM 15954 and Bifidobacterium longum subsp. infantis (DSM 15953) significantly increased the anti-inflammatory cytokine IL-10 (498 pg / ml). The combination of DSM 15954 and Bifidobacterium animalis subsp. lactis (BB-12) also significantly increased the levels of the anti-inflammatory cytokine IL-10. ®In DCs stimulated with a combination of DSM 15954 and Bifidobacterium longum subsp. infantis (DSM 15953), the IL-10 / IL-12 ratio was significantly increased. This effect appears to be driven by the Bifidobacterium longum subsp. infantis (DSM 15953) strain, thus indicating anti-inflammatory properties (see [link to article]). Figure 9 ).

[0207] in conclusion Dendritic cells (DCs) are central to coordinating various forms of immunity and tolerance. DCs are distributed throughout the body and have been shown to be involved in both innate (rapid and general) and adaptive (more specialized) immune responses. Through antigen presentation and the secretion of diverse cytokine profiles, DCs initiate antigen-specific immune responses and promote the development of different T cell responses, such as T helper cell 1 (T2). H 1) Or regulatory T cell response. The main T cells secreted by dendritic cells (DCs) H One cytokine is IL-12, which is used in the treatment of Bifidobacterium longum subsp. infantis (DSM 15953) and Bifidobacterium animalis subsp. lactis BB-12. ® (DSM 15954) or their combination did not improve the results. However, stimulation with Bifidobacterium longum subsp. infantis (DSM 15953) and Bifidobacterium animalis subsp. lactis BB-12 did not improve the results. ® The increased IL-10 secretion by DCs stimulated by the combination of DSM 15954 and DSM 15954 indicates a move toward a more tolerogenic response driven by Bifidobacterium longum infantis (DSM 15953). Tolerogenic responses are crucial for maintaining a balanced immune system and thus preventing excessive or persistent inflammation.

Claims

1. A composition comprising at least one subspecies of Bifidobacterium animalis (Bifidobacterium lactis) Bifidobacterium animal issubsp. lactis ) and at least one human milk oligosaccharide and optionally at least one subspecies of Bifidobacterium longum (infant) Bifidobacterium longum subsp. infantis The composition is intended for use as a supplement to promote intestinal barrier integrity in adults and / or children.

2. The composition for use according to the preceding claims, comprising 1×10 8 CFU / day up to 1×10 11 At least one subsp. Bifidobacterium animalis and at least 0.01 g of at least one human milk oligosaccharide per CFU / day, and optionally 1 × 10⁻⁶ CFU / day. 8 CFU / day up to 1×10 11 At least one subspecies of Bifidobacterium longum infantis CFU / day, the composition is intended for use as a supplement for adults and / or children.

3. The composition for use according to any one of the preceding claims, comprising 1×10 8 CFU / day up to 1×10 11 At least one subsp. *Bifidobacterium animalis* and at least 0.01 g of at least one human lactose oligosaccharide per day (CFU / day) and 1 × 10⁻⁶ CFU / day. 8 CFU / day up to 1×10 11 At least one subspecies of Bifidobacterium infantis per CFU / day.

4. The composition for use according to any one of the preceding claims, wherein the human milk oligosaccharides have the following particle size distribution as measured by DIN ISO 3310-1: less than about 20% passing through #230 mesh (63 μm), more than about 65% passing through #100 mesh (150 μm), more than about 92% passing through #45 mesh (355 μm), and 100% passing through #20 mesh (850 μm).

5. The composition for use according to any one of the preceding claims, wherein the composition is used as a supplement to promote the colonization of beneficial microbiota in the gastrointestinal tract of adults and / or children.

6. The composition for use according to any one of the preceding claims, wherein the composition is used as a supplement to improve the immune health of adults and / or children.

7. The composition for use according to any one of the preceding claims, wherein, The adults who participated were subjects aged 18 to 45.

8. The composition for use according to claims 1-6, wherein, The children eligible for acceptance are infants aged 0-12 months or toddlers aged 1-6 years.

9. The composition for use according to any one of the preceding claims, wherein, The human milk oligosaccharide is selected from 2'-fucosylated lactose, 3-fucosylated lactose, 3'-sialylated lactose, 6'-sialylated lactose, lact-N-neotetrasaccharide, lact-N-tetrasaccharide, and combinations thereof.

10. The composition for use according to any one of the preceding claims, wherein, The human milk oligosaccharide is 2'-fucosylated lactose.

11. The composition for use according to any one of the preceding claims, wherein, The human milk oligosaccharide has an average particle size of about 150 μm to about 850 μm.

12. The composition for use according to any one of the preceding claims, wherein, The aforementioned *Bifidobacterium animalis* subsp. *lactamase* is the strain *Bifidobacterium animalis* subsp. *lactamase* BB-12, preserved under accession number DSM 15954. ® .

13. The composition for use according to any one of the preceding claims, wherein, The *Bifidobacterium longum* subsp. *infantitidis* is a strain deposited under accession number DSM 15953.

14. The composition for use according to any one of the preceding claims, wherein, The composition comprises the strain *Bifidobacterium animalis* subsp. *lactamase*, BB-12, deposited under accession number DSM 15954. ® Bifidobacterium longum subsp. infantis and 2'-fucosylated lactose, deposited under accession number DSM 15953.

15. The composition for use according to any one of the preceding claims, wherein, The composition is in oral dosage form.

16. A method for promoting intestinal barrier integrity, improving immune health, and / or promoting the growth of beneficial microbiota in the gastrointestinal tract in adults and / or children in need thereof, the method comprising administering to the adult or child a composition comprising Bifidobacterium animalis subsp. lactis and at least one human milk oligosaccharide and optionally at least one Bifidobacterium longum subsp. infantis.

17. The method according to claim 16, wherein, The adult is a mother who is about to give birth and / or is breastfeeding.

18. The method according to claim 16, wherein, The child in question is an infant or toddler.

19. A method for producing feeding products or foods, dietary supplements, or pharmaceutical compositions, the method comprising producing the strain of *Bifidobacterium animalis* subsp. *lactamase* preserved under DSM 15954 according to claim 1, and optionally at least one strain of *Bifidobacterium longum* subsp. *infantile* preserved under DSM 15953, and feeding the bacteria with at least 10... 6 The concentration of CFU / g is incorporated into feeding products or foods, dietary supplements or pharmaceutical compositions together with at least one human milk oligosaccharide.

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