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WO2024184260A2 - Composition comprenant du bifidobacterium longum subsp. infantis, du bifidobacterium breve et du 2'-fucosyllactose - Google Patents

Composition comprenant du bifidobacterium longum subsp. infantis, du bifidobacterium breve et du 2'-fucosyllactose Download PDF

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WO2024184260A2
WO2024184260A2 PCT/EP2024/055458 EP2024055458W WO2024184260A2 WO 2024184260 A2 WO2024184260 A2 WO 2024184260A2 EP 2024055458 W EP2024055458 W EP 2024055458W WO 2024184260 A2 WO2024184260 A2 WO 2024184260A2
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dsm
composition
infantis
bifidobacterium
longum subsp
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WO2024184260A3 (fr
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Anja Wellejus
Cathrine MELSAETHER
Mari Lilith LUND
Natalia JIMENEZ
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Chr Hansen AS
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Chr Hansen AS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system

Definitions

  • the present disclosure generally relates to a composition
  • a composition comprising the Bifidobacterium longum subsp. infantis strain deposited with Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ) as DSM 15953, the Bifidobacterium breve strain deposited as DSM 34541 and 2'-Fucosyllactose (2'FL).
  • DSMZ Deutsche Sammlung von Microorganismen und Zellkulturen GmbH
  • 2'FL 2'-Fucosyllactose
  • Bifidobacteria are natural inhabitants of the gastrointestinal tract possessing genetic adaptations that enable colonization of this harsh and complex habitat. Bifidobacteria are thought to interact with key elements of intestinal functioning and contribute to maintaining homeostasis. It is believed that strain-dependent interactions with the host may reduce mucosal antigen load, improve the intestinal barrier, and induce regulation of local and systemic immune responses.
  • Probiotics refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects 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).
  • Digestive health issues affect daily life for millions of people. Antibiotics, diets and travels all have the potential to disrupt the microbiome balance. In addition, increasing evidence suggest that a healthy microbiome is beneficial for more than the gastrointestinal and digestive health of an individual. The proper functioning of the immune system has been linked to a healthy microbiome and there is an interest in establishing the beneficial microbiome immediately after birth to secure healthy development and general well-being of both mother and child.
  • Probiotics may offer a solution to the challenge of digestive health issues as well as enabling establishment of an early healthy microbiome. About a dozen Bifidobacterium strains with clinically documented effects are commercially available. In particular, the use of probiotics for preterm infants and other infants born with challenges, such as very low birth weight has been evaluated.
  • probiotic compositions which could be useful in the support of gastrointestinal health including, for example, defending against intestinal tissue damage. Additionally, probiotic compositions which could have a beneficial impact on the immune system would be of interest.
  • the present disclosure relates to the Bifidobacterium longum subsp. infantis strain deposited with Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ) as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541. These strains have both been found in a MIC test to be sensitive to all relevant antibiotics according to the EFSA 2018 guideline. Consistent herewith, no antibiotic resistance genes were identified. Further, the strains have been tested for cytotoxicity by the Vero cell assay and found to be non- cytotoxic.
  • DSMZ Deutsche Sammlung von Microorganismen und Zellkulturen GmbH
  • Example 2 Based upon the in vitro results provided in Example 1 the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541 is considered safe.
  • Example 2 provides information regarding the ability of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541 to cleave human bile salts.
  • Example 3 provides information on the ability of B. longum subsp. infantis deposited as DSM 15953 to utilize HMOs as a carbon source for growth.
  • Example 4 provides information of a transepithelial electrical resistance (TEER.) assay which is indicative of the intestinal barrier integrity.
  • TEER. transepithelial electrical resistance
  • Caco-2 cells When grown on transwells, the well-established cell line, Caco-2 cells, originally derived from human colon adenocarcinoma, form a polarized monolayer which is commonly used as an intestinal permeability model.
  • Modulation of the inflammatory status of the gastrointestinal tract is possible by dosing bacteria that will accordingly activate immune cells to secrete cytokines.
  • Foligne and co-workers 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 association between bacteria-mediated in vivo protection against experimental colitis and cytokine profile induced by the bacteria in vitro, in particular the IL-10 and interleukin 12 (IL-10/IL-12) ratio.
  • IL-10/IL-12 interleukin 12
  • mice showed how in vivo protection against trinitrobenzene sulfonate (TNBS)-induced colitis in mice was mediated by bacterial strains with a capacity to induce high levels of the "anti"-inflammatory cytokine IL-10 and low levels of "pro”-inflammatory cytokine IL-12 in vitro. In contrast, strains inducing a low IL-10/IL-12 ratio were not capable of attenuating inflammatory colitis.
  • TNBS trinitrobenzene sulfonate
  • in vitro modulation of human inflammatory cells is a useful tool to predict induction of a pro- or anti-inflammatory gut environment induced by bacteria.
  • Example 5 provides information on cytokine secretion from human Peripheral blood mononuclear cells (PBMCs) stimulated with B. longum subsp. infantis (DSM 15953) and 2'- O-Fucosyllactose (2'FL).
  • PBMCs Peripheral blood mononuclear cells
  • DSM 15953 B. longum subsp. infantis
  • 2'FL 2'- O-Fucosyllactose
  • Example 6 provides information on cytokine secretion from human Peripheral blood mononuclear cells (PBMCs) stimulated with B. longum subsp. infantis (DSM 15953) or B. breve (DSM 34541).
  • PBMCs Peripheral blood mononuclear cells
  • DSM 15953 B. longum subsp. infantis
  • DSM 34541 B. breve
  • Example 7 provides the information that IL-10 secretion from both B. longum subsp. infantis (DSM 15953) and B. breve (DSM 34541) was significantly increased compared to the control, whereas IL-12 was significantly increased in DCs stimulated with B. longum subsp. infantis (DSM 15953)
  • the present probiotic compositions might be useful in support of gastrointestinal health including promoting intestinal barrier integrity and growth of beneficial microbiota in the gastrointestinal tract.
  • the present compositions might also have a beneficial impact on the immune function.
  • FIGURE 1 A first figure.
  • Figure 1 is showing bile salt hydrolase activity. Quantification of (A) glycine- and (B) taurinedeconjugation activities by Bifidobacterium longum subsp. infantis (DSM 15953) (grey, lower graphs) and Bifidobacterium breve (DSM 34541) (black, upper graphs). Standard error bars are depicted as SD ⁇ .
  • Figure 2 is showing growth measured spectrophotometrically at ODeoo of Bifidobacterium longum subsp. infantis (DSM 15953) after 96 hrs of incubation in broth containing no carbohydrate (white bar) and in broth containing 2% 2'-fucosyllactose, 2'FL (grey bar). Growth on MRS was used as positive control (black bar).
  • FIGURE 3 is showing growth measured spectrophotometrically at ODeoo of Bifidobacterium longum subsp. infantis (DSM 15953) after 96 hrs of incubation in broth containing no carbohydrate (white bar) and in broth containing 2% 2'-fucosyllactose, 2'FL (grey bar). Growth on MRS was used as positive control (black bar).
  • FIGURE 3 is showing growth measured spectrophotometrically at ODeoo of Bifidobacterium longum subsp. infantis (DSM 15953) after 96
  • Figure 3 is showing cytokine secretion from human PBMCs stimulated with 2'FL, B. longum subsp. infantis (DSM 15953) or a 2'FL+B. longum subsp. infantis (DSM 15953) combination.
  • Figure 4 is showing cytokine secretion from human PBMCs stimulated with B. breve (DSM 34541) or B. longum subsp. infantis (DSM 15953).
  • Data bars represent the mean concentrations from immune cells isolated from four healthy donors, and error bars represent Standard Error of the Mean (SEM).
  • Figure 5 is showing cytokine secretion from human dendritic cells (DCs) stimulated with B. longum subsp. infantis (DSM 15953) or B. breve (DSM 34541). Mean cytokine concentrations (pg/ml) of A. IL-10 and B. IL-12 measured in the supernatants of human DCs co-incubated with B. longum subsp. infantis (DSM 15953), B. breve (DSM 34541), or left unstimulated for 20 hours.
  • C. IL-10/IL-12 cytokine ratio Data bars represent the mean cytokine concentrations or ratio secreted from DCs isolated from six healthy donors, and error bars represent Standard Error of the Mean (SEM).
  • FIGURE 6 is showing the ability to induce a higher transepithelial electrical resistance (TEER) across a Caco-2 monolayer as studied using B. longum subsp. infantis (DSM 15953), B. breve (DSM 34541), and 2'FL in the combinations as shown.
  • A) Represents the real-time TEER measurements, whereas B) represents the area under the curve (AUC) after 8 hrs of measurements.
  • composition refers to a composition comprising a carrier and at least one bacterial strain as described herein.
  • the terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the overall health of the animal and confer benefits similar to the ones demonstrated in the examples.
  • the actual effective dosage in absolute numbers depends on factors including the state of health of the subject in question, and other ingredients present.
  • the "effective amount”, “effective concentration”, or “effective dosage” of the bacterial strains may be determined by routine assays known to those skilled in the art.
  • isolated means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.
  • a bacterial "strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied and that originates from a single isolate or pure culture. Probiotics are classified by their genus (e.g. Bifidobacterium), species (e.g. animalis subsp. lactis), and strains (e.g. DSM 15954 and/or BB-12®). FAO/WHO has stated that probiotic effects are strain specific and that most probiotic characteristics of a particular strain can therefore not be extrapolated to other strains of the same species.
  • the term "improving immune health” is intended to mean providing an increase in IL-10 secretion and/or promoting a Thl and a T regulatory response evaluated as described in Example 5-7.
  • This present disclosure provides an isolated Bifidobacterium longum subsp. infantis strain deposited with Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ) as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541. Both strains are sensitive to gentamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin when tested as described in ISO 10932:2010, has no antibiotic resistance genes when analyzed as described Example 1, and was found to be non-cytotoxic when tested for cytotoxicity by the Vero cell assay as described in Example 1.
  • the survival of the probiotic in the presence of gastric acid and bile in the upper gastrointestinal tract is critical.
  • infantis strain deposited as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541 are believed to have the ability to deconjugate human bile salts. Both glycine-, and taurine-conjugated bile salts were found to be deconjugated, leading to liberation of free glycine and taurine after incubation of the bile salts with the Bifidobacterium iongum subsp. infantis strain deposited as DSM 15953 or the Bifidobacterium breve strain deposited as DSM 34541. Thus, this example demonstrates that both the Bifidobacterium iongum subsp. infantis strain deposited as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541 have a statistically significant BSH activity compared to control, which could be beneficial for a subject ingesting the strain.
  • Epithelial and endothelial cells form barriers in the body. The strength and integrity of these barriers can be accessed via measurements of the electrical resistance across the cell layer in vitro, called TEER.
  • TEER is a well-established method of evaluating and monitoring epithelial tissue in a nondestructive assay.
  • the confluence of the monolayer is quickly determined.
  • the confluence can be tracked and monitored in real-time as the TEER measurement will rise as the gaps in the monolayer close.
  • TEER is often used with epithelial and endothelial cells in a monolayer as a strong indicator of cell barrier integrity and permeability.
  • Example 4 of the present disclosure evaluates the effect in a TEER assay of Bifidobacterium iongum subsp. infantis (DSM 15953) and Bifidobacterium breve (DSM 34541).
  • compositions comprising more than one probiotic strain together with at least one HMO may improve cell barrier integrity and permeability more than a composition comprising only one or no probiotic strain.
  • compositions of the present disclosure may comprise at least one B. iongum subsp. infantis strain, such as the B. iongum subsp. infantis strain deposited as DSM 15953.
  • the microbiome of the infant gut develops after birth to ensure a healthy functioning gastrointestinal system and an optimal maturation of the immune system.
  • Bifidobacterium are thought to be especially important for the gut health of infants. These are B. iongum subsp. infantis, B. breve, B. bifidum and B. iongum subsp. iongum. These Bifidobacterium all digest Human Milk Oligosaccharides which are present in breastmilk. By breaking down these HMOs, various metabolites which are believed to be beneficial for the maturation of the intestine and development of the immune system of the infant, such as acetate and various aromatic lactic acids, are formed.
  • B. longum subsp. infantis is efficacious in utilizing HMOs.
  • B. longum subsp. infantis such as the B. longum subsp. infantis strain deposited as DSM 15953, has the ability to utilize the HMOs as a carbon source for growth and thus B. longum subsp. infantis grows well on HMOs present in the infant colon, where other carbon sources are scarce.
  • Bifidobacterium in the infant gut microbiome it is possible to increase the amount of Bifidobacterium in the infant gut microbiome by, for example, seeding the infants itself and/or by seeding the mother with a combination of probiotic bacteria including B. longum subsp. infantis. It has been found that seeding the infant with the probiotic combination may have an effect on stool frequency, stool consistency and/or crying and fuzzing.
  • Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective aromatic lactic acids (indole-3-lactic acid, phenyllactic acid and p-hydroxyphenyllactic acid) via a previously unrecognized aromatic lactate dehydrogenase.
  • aromatic amino acids tryptophan, phenylalanine and tyrosine
  • aromatic lactic acids indole-3-lactic acid, phenyllactic acid and p-hydroxyphenyllactic acid
  • This triangular relationship between the gut microbiome, metabolites, and the developing immune system supports the impact B. longum subsp. infantis DSM 15953 may have on the immune maturation through indole-3-lactic acid production.
  • composition comprising more than one probiotic component would provide a benefit for the subject receiving such a composition, since it is believed that each probiotic bacteria added may provide different positive effects and functionalities and that these can work together and may result in synergistic effects.
  • probiotic component refers to a culture of live or freeze-dried microorganisms, dead microorganisms, fragments of microorganisms and extracts or supernatants of microorganisms which, when applied to man or animal, beneficially affects 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).
  • intestinal barrier integrity refers to the natural integrity in the intestinal wall, which functions as a barrier between the intestinal inside and the surrounding tissue and lumen.
  • One method for measuring this is the TEER. method as described in Example 4, which measured intestinal barrier tightness.
  • the present disclosure relates to a method of promoting intestinal barrier integrity, improving immune health and promoting the growth of beneficial microbiota in the gastrointestinal tract of an infant and/or child in need thereof, the method comprising administering to the infant and/or child a composition comprising Bifidobacterium longum subsp. infantis and Bifidobacterium breve and at least one Human Milk Oligosaccharide.
  • the disclosure provides a composition comprising the Bifidobacterium longum subsp. infantis deposited as DSM 15953, the Bifidobacterium breve strain deposited as DSM 34541 and at least one HMO for use as a supplement.
  • Said supplement may be used for providing beneficial effects to a subject in need thereof.
  • the present composition may promote intestinal barrier integrity in a subject in need thereof, promote the growth of beneficial microbiota in the gastrointestinal tract, or the like.
  • the supplement may also be used to regulate immune health of a subject in need thereof.
  • the present composition may increase the IL10 and/or IL12 secretion and/or promote a Thl response, or the like.
  • compositions of the present disclosure may be used to support the intestinal barrier integrity and the growth of beneficial microbiota in the gastrointestinal tract in a subject, such as a child with digestive health issues and/or an infant with an immature gastrointestinal tract, with the aim of providing a healthy gut microbiome by promoting the growth of beneficial microbiota in the gastrointestinal tract.
  • the subject may be an infant (age 0-12 month of age) or a pregnant woman, for example in the age of 18-45 years of age, with the aim of seeding the bifidogenic environment of the infant either through seeding directly by administering the composition to the infant or by seeding the expectant mother who then passes on the beneficial Bifidobacterium to her child.
  • the composition of the present disclosure may be used to help boost immunity in, for example, young children in the age of 1-6 years of age. As shown in example 5-7 the present composition may promote a Thl and a T regulatory response, which is a special benefit for infants and children since they have an immature and Th2 skewed immune system.
  • the Thl wil counteract the Th2 prenatal state and additionally it may help them fight infections faster and better.
  • the increase in IL10 may be indicative of antiinflammatory properties which may help infants and children to build tolerance towards microbiota and food antigens and thereby providing a benefit for the subject ingesting a composition comprising this strain, by regulation of immunity.
  • compositions may be used for the reduction of intestinal tissue damage, i.e. by prevention, reduction, or treatment of intestinal mucosal breaks or lesions, thereby supporting intestinal barrier function.
  • compositions comprising Bifidobacterium longum subsp. infantis deposited as DSM 15953 and Bifidobacterium breve deposited as DSM 34541 and at least one Human Milk Oligosaccharide, may be useful for administration to an infant or toddler.
  • an infant is a child from 0-12 months of age and a toddler is a child in the age of 1-6 years of age.
  • compositions may comprise additional components.
  • at least one other bacterial strain for example, at least one other bacterial strain, vitamins, minerals, prebiotics, fibers or combinations thereof.
  • the other components may be fructo-oligosaccharides (FOS), galactooligosaccharide (GOS), inulin, human milk oligosaccharides (HMO) or combinations thereof.
  • FOS fructo-oligosaccharides
  • GOS galactooligosaccharide
  • HMO human milk oligosaccharides
  • compositions may comprise bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and at least one other bacterial strain together with a suitable HMO, such as 2'-Fucosyllactose (2'FL).
  • a suitable HMO such as 2'-Fucosyllactose (2'FL).
  • compositions may comprise bacteria of the Bifidobacterium breve strain deposited as DSM 34541 and at least one other bacterial strain together with a suitable HMO, such as 2'-Fucosyllactose (2'FL).
  • a suitable HMO such as 2'-Fucosyllactose (2'FL).
  • the at least one other bacterial strain may, for example, be selected from Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, any strain belonging to the genus Lactobacillus (including but not limited to Lactobacillus acidophilus, Lactobacillus easel subsp. easel, Lactobacillus delbrueckii subsp.
  • Lactobacillus fermentum Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus plantarum
  • any strain belonging to the genus Bifidobacterium including but not limited to Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis subsp.
  • lactis Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp.
  • compositions may comprise at least one strain of a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus rhamnosus deposited as DSM 33870, Lactobacillus rhamnosus deposited as ATCC 55826, Lactobacillus reuteri deposited as ATCC 55845, Lactobacillus paracasei subsp.
  • a bacterium selected from the group comprising Bifidobacterium animalis subsp. lactis deposited as DSM 15954, Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 53103, Lactobacillus rhamnosus deposited as DSM 33870, Lactobacillus rhamnosus deposited as AT
  • the present compositions comprise bacteria of the species Bifidobacterium iongum subsp. infantis, for example bacteria of the Bifidobacterium Iongum subsp. infantis strain deposited as DSM 15953 and the bacteria of the species Bifidobacterium breve, for example bacteria of the Bifidobacterium breve strain deposited as DSM 34541.
  • the composition may comprise bacteria of these two strains as the only probiotic component.
  • the Bifidobacterium iongum subsp. infantis strain deposited as DSM 15953 is the only probiotic component in the composition.
  • the Bifidobacterium breve strain deposited as DSM 34541 is the only probiotic component in the composition.
  • composition of the present disclosure may in one aspect comprise the bacteria of the species Bifidobacterium iongum subsp. infantis, for example bacteria of the Bifidobacterium iongum subsp. infantis strain deposited as DSM 15953, and the bacteria of the species Lactobacillus rhamnosus, for example bacteria of the Lactobacillus rhamnosus deposited as DSM 33870.
  • the composition may comprise bacteria of these two strains as the only probiotic component.
  • compositions may comprise the bacteria in any suitable form for administration to the subject.
  • the compositions may comprise the bacteria in dried form, which can be obtained by freeze-drying, spray-drying, lyophilization, or the like.
  • cryoprotectant is used herein to refer to a substance that is able to improve the survival during freezing and/or drying and to improve the storage stability of bacteria.
  • the cryoprotectant used herein preferably comprises a saccharide and/or a sugar alcohol such as inositol.
  • the saccharide may be a mono-, di-, oligo- or polysaccharide, or a mixture of at least two saccharides.
  • 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 comprise one or more mono- or disaccharides, such as one, two, or three or even more different saccharides.
  • the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.
  • the cryoprotectant may further comprise a peptide, protein, protein hydrolysate or a mixture thereof.
  • peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any isolate or hydrolysate thereof.
  • Other additives e.g. antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine hydrochloride may also be present.
  • Skim milk powder and yeast extract may also be ingredients.
  • the present composition comprises bacteria of the Bifidobacterium longum subsp. infantis deposited as DSM 15953 in frozen or freeze-dried form and a cryoprotectant.
  • the cryoprotectant may comprise a saccharide.
  • the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.
  • the present composition comprises bacteria of the Bifidobacterium breve deposited as DSM 34541 in frozen or freeze-dried form and a cryoprotectant.
  • the cryoprotectant may comprise a saccharide.
  • the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.
  • the present compositions comprise bacteria of the strain Bifidobacterium longum subsp. infantis deposited as DSM 15953 and bacteria of the strain Bifidobacterium breve deposited as DSM 34541, both bacteria present in frozen or freeze-dried form, and a cryoprotectant.
  • the cryoprotectant may comprise a saccharide.
  • the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose, and a polysaccharide, such as maltodextrin.
  • the present composition may comprise at least one Human Milk Oligosaccharide (HMO).
  • HMOs Human Milk Oligosaccharide
  • the compositions may be formulated as combined or as separate compositions of the bacterial strain or strains and the HMO e.g. the HMO 2'-fucosyllactose (2'-FL).
  • HMOs fucosylated, sialylated and neutral core HMOs.
  • the composition of HMOs in breast milk is individual to each mother and varies over the period of lactation.
  • the dominant oligosaccharide in 80% of all women is 2'-fucosyllactose, which is present in human breast milk at a concentration of approximately 2.5 g/L, other abundant oligosaccharides include lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose. It has been found by numerous studies that the concentration of each individual HMO changes throughout the different periods of lactation (colostrum, transitional, mature and late milk) and depend on various factors such as the mother's genetic secretor status and length of gestation.
  • HMOs Human milk oligosaccharides
  • lactose which can be decorated by four monosaccharides (N-acetyl-D-glucosamine, D-galactose, sialic acid and/or L-fucose) to form an oligosaccharide.
  • compositions may comprise 2'-fucosyllactose.
  • the compositions may be formulated as combined or as separate compositions of the probiotic strains and the HMO.
  • the compositions may comprise other Human Milk Oligosaccharide (HMO) such as, for example, 3-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N- neotetraose, and combinations thereof.
  • HMO Human Milk Oligosaccharide
  • the HMOs may be in any suitable combination.
  • 2'-fucosyllactose may be combined with any one, two, three, four, or five of 3- fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, and lacto-N-neotetraose.
  • the HMO mix comprises 2'-fucosyllactose, 3-fucosyllactose, 3'- sialyllactose, 6'-sialyllactose, and lacto-N-tetraose.
  • HMOs are indigestible for the nursing child. However, they have a prebiotic effect and serve as food for certain intestinal bacteria, especially bifidobacteria. The abundance of these intestinal bacteria in the gut seems to reduce the colonization with pathogenic bacteria and thereby promotes a healthy intestinal microbiota and reduces the risk of dangerous intestinal infections. Studies suggest that HMOs lower the risk of viral and bacterial infections and thus diminish the chance of diarrhea and respiratory diseases.
  • HMOs This protective function of the HMOs appears to be activated when in contact with specific pathogens, such as certain bacteria or viruses which have the ability to bind to the glycan receptors (receptors for long chains of connected sugar molecules on the surface of human cells) located on the surface of the intestinal cells. This binding enables infection of the intestinal mucosa.
  • pathogens such as certain bacteria or viruses which have the ability to bind to the glycan receptors (receptors for long chains of connected sugar molecules on the surface of human cells) located on the surface of the intestinal cells.
  • glycan receptors receptors for long chains of connected sugar molecules on the surface of human cells located on the surface of the intestinal cells.
  • HMOs can mimic these glycan receptors, so the pathogens bind to the HMOs rather than the intestinal cells. This reduces the risk of an infection.
  • HMOs seem to influence the reaction of specific cells of the immune system in a way that reduces inflammatory responses.
  • HMOs such as 2'-fucosyllactose
  • pathogens such as Staphylococcus aureus.
  • Biofilm formation is one way in which pathogenic bacteria protects themselves and evade the immune system of the host infected.
  • the microorganisms in a biofilm aggregate to form a colony for metabolic cooperation.
  • This cooperative method of growth increases the pathogen's survival through improved defense, increased availability of nutrients, and better opportunities for cellular communication and transfer of genetic material.
  • Cellular defense is important to combat physical threats such as displacement by a flowing fluid or removal by the immune system.
  • Example 3 of the present disclosure shows how the Bifidobacterium longum subsp. infantis deposited as DSM 15953 can utilize the HMO, such as 2'FL, and demonstrates a higher degree of growth as measured by ODeoo when growing on HMOs in comparison to growth without a carbon source (see figure 2).
  • composition comprising at least one probiotic bacteria and at least one HMO will provide a benefit for the subject receiving such a composition, since it is believed that each component of said composition will function better when administered in combination than if administered on their own, because the components can interact and the bacterial strain may feed of the HMO.
  • the present composition may comprise HMO, such as 2'-fucosyllactose, in any suitable amount, such as, 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, at least about 0.5 g weight.
  • the compositions may, for example, comprise 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, less than about 1.5 g, of HMO, such as 2'-fucosyllactose.
  • the present composition may comprise 2 ' -fucosyllactose in any suitable amount, such as, for example, at least about 0.001 g per daily intake, at least about 0.01 g per daily intake, at least about 0.05 g per daily intake, at least about 0.1 g per daily intake, at least about 0.5 g g per daily intake.
  • the compositions may, for example, comprise less than about 20 g per daily intake, less than about 10 g per daily intake, less than about 5 g per daily intake, less than about 4 g per daily intake, less than about 3 g per daily intake, less than about 2 g per daily intake, less than about 1.5 g per daily intake, of 2 ' -fucosyllactose.
  • PSD particle size distribution
  • Particle size of an HMO may be determined using a standard method, such as using a sieve tower, which separates the powder into the different fractions after a defined time with a predefined amplitude.
  • the sieves used in such a method may be sieves which comply with DIN ISO 3310-1.
  • the 2'-fucosyllactose (or other HMO) used in the present compositions have the following particle size characteristics:
  • Percent through mesh #100 150 pm - greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.
  • Percent through mesh #45 355 pm - greater than about 95%, greater than about 92%, greater than or equal to about 90%.
  • compositions of the present disclosure are administered orally.
  • the compositions are thus typically in a form suitable for oral administration.
  • the composition may be a solid or a liquid composition.
  • the composition may be in unit dosage form.
  • the composition can be a capsule, pastille, a pill, a tablet, a soft gel, a sachet, a stick, a stick powder, or in a more general composition such as oil drops, an emulsion, or a paste, or in any other suitable carrier determined by those of skill in the art to be an effective carrier for live organisms.
  • compositions may be encapsulated for example using a suitable polymeric matrix to improve long-term stability and storage of the compositions.
  • suitable polymeric matrix to improve long-term stability and storage of the compositions.
  • composition may be included in a dietary supplement or pharmaceutical composition or may be part of a feed product or a food product such as a fermented milk product e.g. a yogurt or an infant formula.
  • a fermented milk product e.g. a yogurt or an infant formula.
  • compositions of the present disclosure may comprise bacteria of the Bifidobacterium longum subsp. infantis deposited as DSM 15953, and bacteria of the strain Bifidobacterium breve deposited as DSM 34541, in a unit dosage form or a more general composition as described above or as part of a dietary supplement or a feed or food product such as a fermented milk product e.g. a yogurt or an infant formula.
  • subject refers to any mammal, including, but not limited to, livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans.
  • livestock and other farm animals such as cattle, goats, sheep, horses, pigs and chickens
  • performance animals such as racehorses
  • companion animals such as cats and dogs
  • laboratory test animals and humans Typically, the subject is a human.
  • the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 may be administered in an amount of at least 1 x 10 5 CFU/day. Preferred amounts are at least 1 x 10 5 CFU/day, at least 1 x 10 7 CFU/day, at least 1 x 10 8 CFU/day, at least 1 x 10 9 CFU/day, at least 1 x 10 10 CFU/day, at least 1 x 10 11 CFU/day, at least 1 x 10 12 CFU/day.
  • the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the at least one other strain, such as the strain Bifidobacterium breve deposited as DSM 34541 may be administered in an amount of at least 1 x 10 5 CFU/day.
  • Preferred amounts are at least 1 x 10 5 CFU/day, at least 1 x 10 7 CFU/day, at least 1 x 10 8 CFU/day, at least 1 x 10 9 CFU/day, at least 1 x 10 10 CFU/day, at least 1 x 10 11 CFU/day, at least 1 x 10 12 CFU/day.
  • the composition of the present disclosure comprises bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 in dried, frozen or freeze-dried form and the composition is administered in an amount of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day.
  • the composition of the present disclosure comprises bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 in dried, frozen or freeze-dried form as the only probiotic product and the composition is administered in an amount of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day.
  • the composition of the present disclosure comprises bacteria of the as the only probiotic product and the composition is administered in an amount of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day.
  • the composition of the present disclosure comprises bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Lactobacillus rhamnosus deposited as DSM 33870 as the only probiotic product and the composition is administered in an amount of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day.
  • the composition comprises the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541 in a concentration of at least 10 5 CFU/g of each strain.
  • the concentration may be at least 1 x 10 5 CFU/g, at least 1 x 10 7 CFU/g, at least 1 x 10 8 CFU/g, at least 1 x 10 9 CFU/g, at least 1 x IO 10 CFU/g, at least 1 x 10 11 CFU/g or at least 1 x 10 12 CFU/g of each strain.
  • compositions disclosed herein can be carried out with dose levels and dosing regimens as required depending on the circumstances and on the condition of the subject. Suitable dosage regimes can be determined based on the teaching of the present application. Dosage regimens may be adjusted to provide the optimal support of intestinal barrier integrity of adults and/or children, and/or promoting growth of beneficial microbiota in the gastrointestinal tract of adults and/or children, and/or improving immune health of adults and/or children.
  • the composition may be administered daily for at least 1 day.
  • the composition can be administered once or more daily for at least 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.
  • the present disclosure provides a composition of the present disclosure comprising bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541 in dried, frozen or freeze-dried form wherein the composition is administered in a dosage of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day for at least 2 weeks.
  • the present disclosure provides a composition of the present disclosure comprising bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541 in dried, frozen or freeze dried from wherein the composition is orally administered in an amount of from 1 x 10 8 CFU/day to 1 x 10 11 CFU/day for at least 8 weeks.
  • a further aspect of the present disclosure provides a combination treatment comprising a composition which comprises the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541, and at least one HMO as described herein for co-administration to a subject in need thereof.
  • the compositions may be formulated as combined or as separate compositions of the bacterial strain or strains and the HMO.
  • the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541 is incorporated into a feed or food product such as an infant formula, health food, food additive, dietary supplement, pharmaceutical or over-the-counter formulation in a solid form such as a powder, a tablet, or a liquid form.
  • the strain of Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 is present in a concentration of at least 10 5 CFU/g.
  • the concentration may be at least 1 x 10 5 CFU/g, at least 1 x 10 7 CFU/g, at least 1 x 10 8 CFU/g, at least 1 x 10 9 CFU/g, at least 1 x IO 10 CFU/g, at least 1 x 10 11 CFU/g or at least 1 x 10 12 CFU/g.
  • the strain of Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the at least one other strain are present in a concentration of at least 10 5 CFU/g.
  • the concentration may be at least 1 x 10 5 CFU/g, at least 1 x 10 7 CFU/g, at least 1 x 10 8 CFU/g, at least 1 x 10 9 CFU/g, at least 1 x IO 10 CFU/g, at least 1 x 10 11 CFU/g or at least 1 x 10 12 CFU/g.
  • the present disclosure provides a method for producing a feed or food product, dietary supplement or pharmaceutical composition
  • a method for producing a feed or food product, dietary supplement or pharmaceutical composition comprising producing bacteria of the Bifidobacterium longum subsp. infantis strain deposited as DSM 15953 and the strain Bifidobacterium breve deposited as DSM 34541 and at least one HMO, and incorporating the same into a food product, supplement or pharmaceutical composition in a concentration of at least 10 6 CFU/g.
  • Table 1 The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.
  • DSM 34541 CHCC38346; DSM 34541 has been deposited at DSMZ before the priority date of the application with the identification reference CHCC38346.
  • EXAMPLE 1 Bifidobacterium longum subsp. infantis strain (DSM 15953) and Bifidobacterium breve (DSM 34541) are antibiotic susceptible
  • Antibiotic susceptibility of the Bifidobacterium longum subsp. infantis strain deposited with Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ) as DSM 15953 and the Bifidobacterium breve strain deposited as DSM 34541 was determined by measuring the minimum inhibitory concentrations (MICs) of a number of antibiotics according to the ISO 10932
  • MICs minimum inhibitory concentrations
  • the test performed was a broth microdilution method using VetMIC Lact-1 and Lact-2 panels (National Veterinary Institute of Sweden, Uppsala, Sweden) and growth in LSM medium (ISO-sensitest medium (Oxoid) supplemented with 10% MRS (de Man, Rogosa and Sharpe) broth (BD Difco 288110, UK) with 0.05% Cysteine hydrochloride (CyHCI) (Merck 102839, Germany) for 48 hours at 37°C under anaerobic conditions with three biological replicates.
  • the range of antibiotics tested complies with the European Food Safety Authority (EFSA) "Guidance on the characterisation of microorganisms used as feed
  • DSM 15953 and DSM 34541 were found to be sensitive to all antibiotics relevant for the Bifidobacterium group according to the EFSA guideline (gentamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin) with MIC values below the EFSA 2018 cut-off values.
  • DSM 15953 and DSM 34541 were analyzed for antibiotic resistance genes by screening against the curated database ResFinder which contains more than 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.
  • DSM 15953 and DSM 34541 were tested for cytotoxic activity using a Vero cell assay method based on the EFSA guidance "Guidance on the assessment of the toxigenic potential of Bacillus species used in animal nutrition" (EFSA Journal 2014, 12:3665).
  • DSM 34541 and DSM 15953 were grown at 37°C under anaerobic conditions in MRS broth (BD Difco 288110, UK) with 0.05% Cysteine hydrochloride (CyHCI) (Merck 102839, Germany). Culture supernatants were isolated after 24 and 48 hours by centrifugation and analyzed for cytotoxicity at Bioneer A/S, Hoersholm, Denmark using the Vero cell assay.
  • DSM 15953 and DSM 34541 were found to be non-cytotoxic.
  • bile salts Upon food intake bile salts are excreted from the gall bladder into the upper small intestine where they play an essential role in dietary lipid absorption. Primary bile salts are synthesized in the liver where the last step in this pathway conjugate glycine or taurine to the bile acid to increase its solubility. The pool of glycine conjugated bile salts is larger than the taurine conjugated with a ratio of glycine- to taurine-conjugated bile salts at about 3 to 1.
  • BSH bile salt hydrolase
  • BSH bile salt hydrolase
  • the Bifidobacterium longum subsp. infantis strain (DSM 15953) and Bifidobacterium breve strain (DSM 34541) were inoculated from a frozen stock from the Chr. Hansen culture collection and cultured anaerobically with AnaeroGen pads (Oxoid) at 37°C in pH 6.5 MRS (de Man, Rogosa and Sharpe) broth (Difco) overnight. A 10-fold dilution series was prepared from the overnight culture and incubated overnight, anaerobically at 37°C. Two late exponential growth phase cultures were selected based on measures of optical density at 600 nm (ODeoo) and pooled. The bacterial culture was centrifuged for 2 min at 6000x g, washed twice in Phosphate Buffered Salt Solution (PBS).
  • PBS Phosphate Buffered Salt Solution
  • the bile salts included as substrate in the bile salt hydrolyse assay were: Glycocholic acid (GCA), Glycodeoxycholic acid (GDCA), Glycochenodeoxycholic acid (GCDCA), Taurocholic acid (TCA), Taurodeoxycholic acid (TDCA), and Taurochenodeoxycholic acid (TCDCA).
  • BS total Bile Salt
  • the bacteria suspension was added together with BS mix in a 0.1 M acetate buffer and incubated at 37°C. Aliquots were sampled after 10- and 60-minutes and the reaction were immediately stopped by the addition of methanol. After rigorous mixing the samples were snap frozen in dry ice and stored in -80°C freezer until further analysis.
  • the samples Prior to analysis, the samples were diluted five times in MilliQ water and mixed with internal standard, 0.2 M borate buffer and AccQ-Tag Ultra reagent (Waters). To derivatize the samples the mix was heated for 10 min at 55°C in a thermomixer at 2000 rpm. Samples were injected into a Liquid Chromatography Triple Quadrupole Mass Spectrometer (LC-QqQ-MS) with a AccQ-Tag Ultra RP Column and amino acids, including glycine and taurine were quantified in positive mode via multiple reaction monitoring (MRM). Glycine and taurine concentrations were obtained via internal calibration with authentic standards.
  • LC-QqQ-MS Liquid Chromatography Triple Quadrupole Mass Spectrometer
  • MRM multiple reaction monitoring
  • Bifidobacterium longum subsp. infantis (DSM 15953) and Bifidobacterium breve (DSM 34541) both demonstrated BSH activity when deconjugated glycine and taurine were measured after 10- and 60-minutes of incubation (Fig. 1A and B).
  • Bifidobacterium longum subsp. infantis (DSM 15953) deconjugated glycine with a rate of 33.2 pM/h and taurine with 4.4 pM/h.
  • Bifidobacterium breve (DSM 34541) displayed a slightly higher activity with glycine deconjugation rate at 39.5 pM/h and taurine deconjugation rate at 6.6 pM/h.
  • EXAMPLE 3 Bifidobacterium longum subsp. infantis (DSM 15953) and the ability to utilize HMO for growth
  • Bifidobacterium longum subsp. infantis (DSM 15953) was inoculated from frozen stock and cultured overnight at 37°C in De Man, Rogosa and Sharpe (MRS) broth, pH 6.5 with 0.05% cysteine hydrochloride monohydrate (CyHCI), anaerobically. Ten-fold dilution series were prepared from the overnight cultures and incubated under the same conditions as described above. Late exponential/early stationary phase was selected based on measures of optical density at 600nm (ODeoo). The bacterial culture was washed twice in minimal broth containing no carbohydrate with 0.05% CyHCI added.
  • the 2'fucosyllactose, 2'FL was prepared as a 20 g/L (2% wt/vol) solution in minimal broth with 0.05% CyHCI and the solution was sterile filtered using Vacuum filtration (0.22 pm, PES filter).
  • Vacuum filtration (0.22 pm, PES filter).
  • Bacterial growth in MRS was prepared as positive control, whereas 180 pL of minimal broth w/o 2'FL and 20 pL washed bacteria was prepared as negative control.
  • the 96-well microtiter plate was incubated for 96 hrs at 37°C under anaerobic conditions. After the incubation optical density (ODeoo) was measured via a spectrophotometer. The experiment was repeated at least four times in triplicates.
  • EXAMPLE 4 B. Iongum subsp. infantis (DSM 15953), B. breve (DSM 34541) and 2'fucosyllactose (2'FL) combined introduces higher intestinal barrier tightness as evaluated by transepithelial electrical resistance (TEER) across a Caco-2 cell monolayer in vitro compared with the components tested individually or combined in pairs.
  • DSM 15953 B. breve
  • 2'fucosyllactose (2'FL) 2'fucosyllactose
  • the human intestinal epithelial Caco-2 cell line (DSMZ ACC 169, Leibniz-Institut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) was cultured in complete DMEM medium containing DMEM GlutaMax supplemented with 20% heat inactivated fetal bovine serum, 1 X MEM non-essential amino acids and 1 X Pen-Strep-Amp B at 5% CO? at 37°C. Caco-2 cell passages 7-25 were used. When the cells were approximately 50% confluent the medium was removed, and the cells were washed twice in Phosphate buffered saline (PBS).
  • PBS Phosphate buffered saline
  • the cells were trypsinized by adding 1 mL of trypsin and left for 3 min in the CO2 incubator at 37°C. Approximately 10 mL of medium was added to the trypsinized cells, they were counted and a concentration of 100.000 cells/mL in complete DMEM was prepared. A volume of 500 pL of cell suspension was used to seed each apical compartment of CelIQART 12-well Cell Culture inserts, 0.4 pm PET Clear from Greiner 12-well plates, where after 1.5 mL of supplemented DMEM was added to the basolateral compartment. Cells were cultured on the inserts for 21 days with change of medium twice a week. After 21 days the transwells were moved to the CellZscope (NanoAnalytics, Germany).
  • the bifidobacteria were cultured anaerobically overnight at 37°C. On the day of coincubation bacterial growth was evaluated by measuring optical density at 600 nm (ODeoo) and cultures representing late exponential/early stationary phase were selected. The cultures from each strain were centrifuged at 6000 x g for 5 min, to collect the bacteria pellet. The supernatants were discarded and 35 mL of 37°C warm PBS was added, and the bacteria were washed and spun down at 6000 x g for 5 min. This washing procedure was repeated twice. Bacterial cells were harvested by spinning at 6000 x g for 5 min and the supernatant was discarded. Bacterial cells were resuspended in 5 mL pre-heated complete DMEM medium without Abx and ODeoo was adjusted to a final density of 0.5.
  • the CellZscope measurements were paused, and the CellZscope was removed from the CO2 incubator and apical medium was removed from each transwell. Bacterial solution of zero, one or both bacterial strains, 2'FL solution or media control (DMEM) was added to the relevant wells (each in triplicate).
  • DMEM media control
  • DSM 15953 • Bifidobacterium longum subsp. infantis (DSM 15953) • Bifidobacterium breve (DSM 34541) + Bifidobacterium longum subsp. infantis (DSM 15953)
  • the CellZscope was transferred back to the CO2 incubator and the TEER measurements were resumed and continued overnight. Changes in TEER during bacterial stimulation were calculated relative to the latest value recorded immediately prior to the stimulation (baseline measurement, set to 100%). Area under the curve was calculated for each well after 8 hours.
  • TEER transepithelial electrical resistance
  • Table 2 results of statistical testing using the one-way ANOVA with Tukey's multiple comparisons test to compare all groups against each other (only comparisons against the group with all three components; Bifidobacterium breve (DSM 34541), B. longum subsp. infantis (DSM 15953) and 2'FL are shown).
  • EXAMPLE 5 Cytokine secretion from human Peripheral blood mononuclear cells (PBMCs) stimulated with B. longum subsp. infantis (DSM 15953) and 2'-Fucosyllactose (2'FL)
  • B. iongum subsp. infantis (DSM 15953) was inoculated from frozen stock and cultured overnight at 37°C in the De Man Rogosa and Sharpe (MRS) broth, pH 6.5 (DifcoTM) with 0.19% cysteine hydrochloride monohydrate (CyHCI) and 0.14% sodium bicarbonate under anaerobic conditions with AnaeroGen pads (Oxoid).
  • MRS De Man Rogosa and Sharpe
  • CyHCI cysteine hydrochloride monohydrate
  • AnaeroGen pads AnaeroGen pads
  • bacterial pellet was resuspended in 10 ml of modified mMRS medium supplemented with 2% (w/v) of 2'FL and incubated overnight as previously described. Subsequently, the overnight bacterial culture was used to prepare a 10-fold dilution row in mMRS supplemented with 2'FL. A culture representing late exponential growth phase was selected based on optical density ODeoo measurements (Biophotometer, Eppendorf). The culture was centrifuged, and the pellet was washed two times in HBSS and adjusted in RPMI-1640 medium with addition of 10% glycerol to ODeoo of 1.0. Finally, concentrations of the bacterial samples were determined by colony-forming units (CFU), diluted to a concentration of 5xl0 7 CFU/ml and stored at -80°C.
  • CFU colony-forming units
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs isolation was performed in SepMateTM tubes (50 ml) via Ficoll-Plague PlusTM density gradient centrifugation (1200g, 10 min, RT).
  • Four buffy coats were collected from healthy anonymous donors (Righospitalet, Blodbank, Copenhagen, Denmark) on the day of experiment.
  • the isolated PBMCs were washed twice in PBMC media consisting of RPMI medium (Sigma Aldrich), 2-mercaptoethanol (final concentration of 50 pM, Sigma Aldrich) and HEPES buffer (final concentration 10 mM, Sigma Aldrich).
  • the cells were counted using a NucleoCounter®-MC200TM and resuspended in the same medium supplemented with heat-inactivated fetal bovine serum (FBS, final 10%, Gibco).
  • FBS heat-inactivated fetal bovine serum
  • the PBMCs suspension was adjusted to 2xl0 5 cells/ml and plated at 96-well plates (lxlO 5 cells/well) for at least 1 hr (37°C, under 5% CO2) before a stimulation.
  • PBMCs from 4 healthy donors were stimulated with a) 2'FL (2%), b) B. Iongum subsp. infantis (DSM 15953) grown on 2'FL, c) a combination of B. Iongum subsp. infantis (DSM 15953) and 2'FL or d) left untreated. Additionally, the stimulated PBMCs were divided into two sets, one where no additional reagents were added (homeostatic conditions) and another were 50 pg/ml of Poly (I :C) (Sigma) was added after 3 hrs of incubation to simulate a viral challenge (referred to as challenged conditions).
  • challenged conditions referred to as challenged conditions.
  • DSM 15953 grown on 2'FL was used to stimulate PBMCs the secretion levels of IL-10 did not seem to change in comparison to B. longum subsp. infantis (DSM 15953) grown on 2'FL (53pg/ml), but the IFN-y secretion was reduced (190 pg/ml) indicating a possible anti-inflammatory role of 2'FL when combined with B. longum subsp. infantis (DSM 15953) as shown in figure 3B.
  • Stimulation of PBMCs with B. longum subsp. infantis induces the secretion of IFN-y, a key cytokine driving THI immune responses required for the clearance of different infections (viral and bacterial) and priming the immune system to act fast in case of an infection.
  • This priming is most prominent under the challenged conditions, where after the introduction of a viral-like molecule (Poly(I:C)), the PBMCs pre-stimulated with B. longum subsp. infantis (DSM 15953) increased the secretion of IFN-y by 5-folds, possibly leading to a fast anti-pathogenic response.
  • DSM 15953 also induced the secretion of IFN-y under homeostatic and challenged conditions, however at a reduced level. This cytokine attenuation might be beneficial for infants, who in general have an immature immune system, were the clearance of a pathogens through a THI response is beneficial, whereas a strong and uncontrolled inflammatory response might be harmful. Additionally, stimulation of PBMCs with B. longum subsp. infantis (DSM 15953) and the combination 2'FL and B. longum subsp. infantis (DSM 15053) under homeostatic conditions, induces the secretion of the antiinflammatory cytokine IL-10 involved in maintaining a balanced immune response. Under a viral-like challenge (Poly (I :C)) condition, the secretion of IL-10 does resemble that of homeostatic conditions, indicating that the capacity of returning to a balanced immune response is maintained.
  • a viral-like challenge Poly (I :C)
  • EXAMPLE 6 Cytokine secretion from human Peripheral blood mononuclear cells (PBMCs) stimulated with B. longum subsp. infantis (DSM 15953) or B. breve (DSM 34541)
  • PBMCs Peripheral blood mononuclear cells
  • DSM 15953 B. longum subsp. infantis
  • DSM 34541 B. breve
  • PBMCs Peripheral blood mononuclear cells
  • PBMCs isolation was performed in SepMateTM tubes (50 ml) via Ficoll-Plague PlusTM density gradient centrifugation (1200g, 10 min, RT).
  • Four buffy coats were collected from healthy anonymous donors (Righospitalet, Blodbank, Copenhagen, Denmark) on the day of experiment.
  • the isolated PBMC were washed twice in PBMC media consisting of RPMI medium (Sigma Aldrich), 2-mercaptoethanol (final concentration of 50 pM, Sigma Aldrich) and HEPES buffer (final 10 mM, Sigma Aldrich).
  • the cells were counted using a NucleoCounter®-MC200TM and resuspended in the same medium supplemented with heat- inactivated fetal bovine serum (FBS, final 10%, Gibco).
  • FBS heat- inactivated fetal bovine serum
  • the PBMCs suspension was adjusted to 2xl0 5 cells/ml and plated at 96-well plates (lxlO 5 cells/well) for at least 1 h (37°C, under 5%CO?) before a stimulation.
  • B. breve (DSM 34541) and B. longum subsp. infantis (DSM 15953) strains were inoculated from a frozen stock from the Chr. Hansen culture collection and cultured overnight anaerobically with AnaeroGen pads (Oxoid) at 37°C in pH 6.5 MRS (de Man, Rogosa and Sharpe) broth (Difco) supplemented with 0.05% cysteine hydrochloride or 0.19% cysteine hydrochloride and 0.14% sodium bicarbonate overnight respectively.
  • a 10-fold dilution series was prepared from the overnight culture and incubated overnight under the same conditions.
  • a culture representing late exponential growth phase was selected based on optical density ODeoo measurements. The bacterial culture was centrifuged for 2 min at 6000g, washed twice in Hank's Balanced Salt Solution (HBSS) and resuspended in antibiotic-free complete DC medium at a stock concentration of OD 0.05.
  • HBSS Hank's Balanced Salt
  • PBMCs from 4 healthy donors were stimulated for 20 hours with either B. breve (DSM 34541), B. longum subsp. infantis (DSM 15953), or left untreated.
  • the final concentration for the single strains was OD 0.01, corresponding to approximately 10 5 bacterial cells and an MOI of 10: 1 bacteria :PBMCs.
  • Supernatants were collected on AcroPrep filtertop plates (PallTMNew York, USA), centrifuged (1500 x g, 5 min, at Room Temperature) and stored at -80°C prior to cytokine profiling.
  • the cytokine profiles from supernatants of human PBMCs stimulated with B. breve (DSM 34541) or B. longum subsp. infantis (DSM 15953) indicate that both strains have the capacity of boosting human immune responses through the secretion of pro-inflammatory cytokines such as IFN-y, IL-10, IL-6 and TNF-a among others. These cytokines have been shown of great importance to quickly support the clearance of pathogens, therefore reducing the risk of infections.
  • the anti-inflammatory cytokine IL-10 is also induced by both strains tested, allowing the possibility of a speedy shift towards a tolerogenic immune response, and homeostasis.
  • EXAMPLE 7 Cytokine secretion from human dendritic cells stimulated with B. longum subsp. infantis (DSM 15953) and B. breve (DSM 34541).
  • PBMCs peripheral blood mononuclear cells
  • monocytes were isolated by positive selection for CD14 using magnetically activated cell sorting with CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured at a density of 2 x 10 5 cells/ml in complete DC medium (DC medium supplemented with 2mM L- glutamine [Life Technologies Ltd., Paisley, UK], 10% Heat inactivated FBS [GibcoTM, Dublin, Ireland] and 1% Penicillin-Streptomycin solution [Sigma-Aldrich, Saint Louise, MO, USA]).
  • DC medium DC medium supplemented with 2mM L- glutamine [Life Technologies Ltd., Paisley, UK], 10% Heat inactivated FBS [GibcoTM, Dublin, Ireland] and 1% Penicillin-Streptomycin solution [Sigma-Aldrich, Saint Louise, MO, 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, MO, USA). Cells were kept at 37°C with 5% CO2. Fresh complete DC medium containing IL-4 and GM-CSF was added after 3 days of culture. At day 6, differentiation to immature DCs was verified by surface marker expression analysis (CD14, ⁇ 12%; CDllc, >97% expression; CDla, >85%). Immature DCs were resuspended in fresh complete DC medium containing no antibiotics, least 1 hour before bacterial stimulation.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • DSM 34541 and B. longum subsp. infantis (DSM 15953) strains were inoculated from a frozen stock from the Chr. Hansen culture collection and cultured anaerobically with AnaeroGen pads (Oxoid) at 37°C in pH 6.5 MRS (de Man, Rogosa and Sharpe) broth (Difco) supplemented with 0.05% cysteine hydrochloride overnight or with 0.19% cysteine hydrochloride monohydrate (CyHCI) and 0.14% sodium bicarbonate, respectively. 10-fold dilution series were prepared from the overnight cultures and incubated overnight under the same conditions.
  • Cultures representing late exponential growth phase were selected based on optical density ODeoo measurements (Biophotometer, Eppendorf). The bacterial cultures were centrifuged for 2 min at 6000g, washed twice in Hank's Balanced Salt Solution (HBSS) and resuspended in antibiotic-free complete DC medium at a stock concentration of ODeoo 0.05.
  • HBSS Hank's Balanced Salt Solution
  • DCs were stimulated for 20 hours with either B. breve (DSM 34541), B. longum subsp. infantis (DSM 15953) or left untreated.
  • the final concentration for the single strains was ODeoo 0.01, corresponding to approximately 10 5 bacterial cells and an MOI of 10: 1 bacteria :DCs.
  • Cells were incubated at 37°C in an atmosphere of 5% CO2. After stimulation, DC supernatants were sterile-filtered through a 0.2-pm Acro-Prep Advance 96-well filter plate (Pall Corporation, Ann Arbor, MI, USA) and stored at -80°C until cytokine quantification.
  • IL-12p70 and IL-10 secreted from DCs were quantified using U-plex Proinflammatory Combo 1 Human from Meso Scale Discovery (MSD) (catalog number K15049K) (MSD, Rockville, MD, USA) according to the manufacturer's instructions. Data are expressed in picograms per milliliter (pg/ml), or as the IL-10/IL-12 ratio.
  • DCs Dendritic cells
  • T helper 1 T helper 1
  • IL-12 The main THI cytokine secreted by DCs, IL-12, was significantly increased after stimulation with B. longum subsp. infantis (DSM 15953). However, B. longum subsp. infantis (DSM 15953) also significantly induced increased secretion of the anti-inflammatory cytokine IL-10.
  • Dendritic cell cytokine profiles induced by B. breve indicate a more antiinflammatory role, evidenced by a higher IL-10/IL-12 ratio and the reduced secretion of IL- 12. This milieu might be highly beneficial under homeostatic conditions, to maintain a balanced immune system and to avoid unnecessary inflammation.

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Abstract

La présente divulgation concerne de manière générale une composition comprenant du Bifidobacterium longum subsp. infantis déposé sous DSM 15953, le Bifidobacterium breve déposé sous DSM 34541 et le 2'-Fucosyllactose (2'FL). L'utilisation des compositions dans le support de la santé gastro-intestinale, comprenant la promotion de l'intégrité de la barrière intestinale et la croissance du microbiote bénéfique dans le tractus gastro-intestinal, tout en présentant également un impact bénéfique sur la fonction immunitaire, fait également partie de la présente divulgation.
PCT/EP2024/055458 2023-03-03 2024-03-01 Composition comprenant du bifidobacterium longum subsp. infantis, du bifidobacterium breve et du 2'-fucosyllactose Pending WO2024184260A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025233455A1 (fr) * 2024-05-08 2025-11-13 N.V. Nutricia Composition nutritionnelle pour renforcer la barrière intestinale
CN120000695A (zh) * 2025-02-24 2025-05-16 合生元（广州）健康产品有限公司 2’-岩藻糖基乳糖和益生菌的组合物及其应用和产品
CN120699849A (zh) * 2025-08-14 2025-09-26 内蒙古蒙牛乳业（集团）股份有限公司 一株短双歧杆菌Imu-02及其应用

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