WO2023247579A1 - Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus - Google Patents

Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus Download PDF

Info

Publication number
WO2023247579A1
WO2023247579A1 PCT/EP2023/066703 EP2023066703W WO2023247579A1 WO 2023247579 A1 WO2023247579 A1 WO 2023247579A1 EP 2023066703 W EP2023066703 W EP 2023066703W WO 2023247579 A1 WO2023247579 A1 WO 2023247579A1
Authority
WO
WIPO (PCT)
Prior art keywords
lactobacillus rhamnosus
bacteria
viability
lacto
regeneration
Prior art date
Application number
PCT/EP2023/066703
Other languages
English (en)
Inventor
Nicole Seifert
Wilbert SYBESMA
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2023247579A1 publication Critical patent/WO2023247579A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • A23V2250/00—Food ingredients
    • A23V2250/28—Oligosaccharides
    • A23V2250/284—Oligosaccharides, non digestible
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/113—Acidophilus
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/145—Gasseri
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/149—Jensenii
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/173—Reuteri
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/175—Rhamnosus
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/513—Adolescentes
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/517—Bifidum
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/519—Breve
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/529—Infantis
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/531—Lactis
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/533—Longum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to the use of human milk oligosaccharides (HMOs) for improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in acidic environments.
  • HMOs human milk oligosaccharides
  • HMOs were found to increase the number of viable Lactobacillus rhamnosus bacteria upon their rehydration (regeneration) in acidic liquids. This improves probiotic potential of these bacteria in food, beverages, dietary supplements, and oral pharmaceuticals, as their viability during preparation for consumption, after ingestion, and/or along the path through the gastrointestinal tract increases.
  • probiotic is a term used to describe live bacteria which, when ingested in adequate amounts, provide a benefit to the human or animal host. The viability of a probiotic is therefore of crucial importance for its efficacy.
  • Lactobacillus is a genus of gram-positive, aerotolerant anaerobes or microaerophilic, rod-shaped, non-spore- forming bacteria. Lactobacillus species constitute a significant component of the human and animal microbiota at a number of body sites. Lactobacillus rhamnosus (L. rhamnosus) is a probiotic species known for having beneficial effects in the gut and the female urogenital tract.
  • Lactobacillus rhamnosus was officially reclassified as “Lacticaseibacillus rhamnosus” in 2020, but the art still refers to it as “Lactobacillus rhamnosus”. For the purpose of this invention, both genera names are considered interchangeable.
  • Dried product forms include capsules, beadles, tablets, sachets, powders, and the like. They can be directly swallowed or dissolved in a liquid before swallowing. These products depend on their ability to regenerate (rehydrate) and deliver viable, functional bacteria in amounts which result in a health benefit. Both direct consumption of live bacteria and reconstitution (regeneration) of dehydrated probiotic preparations before application “compromise” the survival and functional characteristics of the bacteria under the stress of the upper gastro-intestinal tract, including the acidic environment of the stomach.
  • Rehydration involves an important step in the recovery of dehydrated bacteria; an inadequate rehydration/ regeneration step may lead to poor cell viability and a low final survival rate. Rehydration is therefore a highly critical step in the revitalization of a lyophilized culture.
  • Dried probiotics need to regenerate upon reconstitution/ rehydration, which is a very harsh process, dependent upon pH, temperature, osmolarity and other variables. Reconstitution is usually with excessive water, more than that removed during the dehydration process, thereby resulting in osmotic shock.
  • Many bacteria simply do not "revive”: 99% of probiotic bacteria can be killed prior to reaching their destination in the intestine when they are dissolved in an acidic liquid (such as a juice or a carbonated soft drink), or when they encounter the acidic environment of the stomach.
  • Live probiotics which are delivered e.g. in food, such as yoghurt also need to survive the stomach acid before reaching the intestine.
  • dried probiotics which are directly delivered to the female genital tract have to cope with the acid pH of the vagina.
  • probiotic bacteria which can be delivered in a safe, reliable form, can facilitate a smooth regeneration and improved viability, and is accessible to all consumers.
  • the present invention relates to the following items:
  • HMOs human milk oligosaccharides
  • the one or more HMOs is (i) selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST a, LST c, LNFP-I, and LNFP-II I ; and/or (ii) a combination of 2’-FL and DFL; and/or (iii) a combination of 2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I.
  • a method of improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in an acidic environment, wherein the Lactobacillus rhamnosus bacteria are contained in a powder composition comprising mixing the powder composition with one or more HMOs prior to or during the process of adding the powder composition to a dietary supplement, medicament, foodstuff or beverage.
  • a composition comprising Lactobacillus rhamnosus bacteria and one or more HMOs.
  • composition of item 8 wherein the Lactobacillus rhamnosus bacteria are dried, preferably wherein the Lactobacillus rhamnosus bacteria are lyophilized.
  • composition of any one of items 8-10, wherein the one or more HMOs is (i) selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST a, LST c, LNFP-I, and LNFP-III; and/or (ii) a combination of 2’-FL and DFL; and/or (iii) a combination of 2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I .
  • An acidic composition comprising the composition of any one of items 8-12.
  • beverage selected from the group consisting of: carbonated mineral water, sports drinks, carbonated soft drinks, fruit juices, fruit drinks, sodas, energy drinks, cold teas, and coffee.
  • FIGURE 1 Shows the experimental setup of the regeneration and viability assessment of lyophilized probiotics under pH 3.0 acidic conditions.
  • FIGURE 2 Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 without HMOs (control). 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Dilution steps 1 :100, 1 :1000, 1 :10’000 (E-2 - E-3 - E-4) in duplicates.
  • FIGURE 3 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 2’-FL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 2’-FL, compared to the control (L. rhamnosus only).
  • FIGURE 4 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 3'-SL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 3'-SL, compared to the control (L rhamnosus only).
  • FIGURE 5 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 6'-SL in acidic conditions. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 6'-SL in acidic conditions, compared to the control (L rhamnosus only).
  • FIGURE 6 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 2’-FL/DFL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 2 -FL/DFL, compared to the control (L. rhamnosus only).
  • FIGURE 7 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with a 5-HMO blend. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with a 5-HMO blend, compared to the control (L. rhamnosus only).
  • FIGURE 8 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LST a. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LST a, compared to the control (L. rhamnosus only).
  • FIGURE 9 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LST c. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LST c, compared to the control (L. rhamnosus only).
  • FIGURE 10 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LNT. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with LNT, compared to the control (L. rhamnosus only).
  • FIGURE 11 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus DSM 32550, incubated for 3 h at pH 3.0 in combination with 3-FL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates.
  • FIGURE 12 A) Shows the regeneration and viability of lyophilized Lactobacillus rhamnosus GG DSM 33156 (LGG®) incubated for 3 h at pH 3.0 without HMOs (control). This control was used to compare effects shown in Figures 13-18. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Dilution steps 1 :100, 1 :1000 (E-2 - E-3) in duplicates. B) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 without HMOs (control). This control was used to compare effects shown in Figures 19-23.
  • FIGURE 13 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 2’-FL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 2’-FL, compared to the control (LGG® only).
  • FIGURE 14 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNnT. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNnT, compared to the control (LGG® only).
  • FIGURE 15 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 3'-SL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 3'-SL, compared to the control (LGG® only).
  • FIGURE 16 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 6'-SL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 6'-SL, compared to the control (LGG® only).
  • FIGURE 17 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 2’-FL/DFL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 2 -FL/DFL, compared to the control (LGG® only).
  • FIGURE 18 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with a 5-HMO blend. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with a 5-HMO blend, compared to the control (LGG® only).
  • FIGURE 19 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNFP4. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNFP-I, compared to the control (LGG® only).
  • FIGURE 20 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNFP-III. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNFP-III, compared to the control (LGG® only).
  • FIGURE 21 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LST c. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LST c, compared to the control (LGG® only).
  • FIGURE 22 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNT. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-3 - E-4 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with LNT, compared to the control (LGG® only).
  • FIGURE 23 A) Shows the regeneration and viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 3-FL. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: E-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized LGG® incubated for 3 h at pH 3.0 in combination with 3-FL, compared to the control (LGG® only).
  • HMOs Human milk oligosaccharides
  • HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more p-N-acetyl-lactosaminyl and/or one or p-more lacto- N-biosyl units, and which core structure can be substituted by an a-L-fucopyranosyl (“fucosyl”) and/or an a-N- acetyl-neuraminyl (“sialyl”) moiety.
  • fucosyl a-L-fucopyranosyl
  • sialyl alyl
  • HMOs can be isolated or enriched by well-known processes from milk(s) secreted by mammals including, but not limited to human, bovine, ovine, porcine, or caprine species.
  • the HMOs can also be produced by well- known processes using microbial fermentation, enzymatic processes, chemical synthesis, or combinations of these technologies.
  • sialylated oligosaccharides can be made as described in WO 2012/113404, and mixtures of human milk oligosaccharides can be made as described in WO 2012/113405.
  • sialylated oligosaccharides can be made as described in WO 2012/007588
  • fucosylated oligosaccharides can be made as described in WO 2012/127410
  • diversified blends of human milk oligosaccharides can be made as described in WO 2012/156897 and WO 2012/156898.
  • W02001/04341 and WO 2007/101862 describe how to make core human milk oligosaccharides optionally substituted by fucose or sialic acid using genetically modified E. coli.
  • HMOs with five or more monosaccharide units produced by fermentation is described, for example, in WO2016/040531 , WO2019/008133, W02022/034067, WO2019/020707, W02020/115671 , WO2022/243312 and EP 3 848 471 .
  • EP22209675 describes the combination of fermentation and enzymatic processes to produce HMOs with five or more monosaccharide units.
  • HMOs are either neutral or acidic.
  • the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure.
  • the non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated.
  • Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II) lacto-N-tetraose (LNT), lacto- N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).
  • LNT-II lacto-N-triose II
  • LNT lacto-N-tetraose
  • LNnT lacto- N-neotetraose
  • LNnH lacto-N-neohexaose
  • pLNnH para-lacto-N-neohexaose
  • pLNH para-lacto-N-hexa
  • neutral fucosylated HMOs examples include 2'-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI), lacto-N- difucohexaose I (LNDFH-I), lacto-N-difucohexaose II (LNDFH-II), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose I (FL
  • acidic HMOs examples include 3’-sialyllactose (3’-SL), 6’-sialyllactose (6’-SL), 3-fucosyl-3’-sialyllactose (FSL), 3’-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6’-sialyllacto-N-tetraose b (LST b), fucosyl- LST b (FLST b), 6’-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3’-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), disialyl-lacto-N-tetraose (DSLNT), sialyl
  • Regeneration means the process of regaining/ restoring a dried bacteria’s viability (i.e., “reviving” the bacterial cells by rehydration, wherein “rehydration” means restoring fluid). This process is also sometimes referred to as “reconstitution”.
  • “Viability” is the ability of a bacterial cell to live and function as a living cell.
  • One way of determining the viability of bacterial cells is by spreading them on an agar plate with suitable growth medium and counting the number of colonies formed after incubation for a predefined time (plate counting). Alternatively, FACS analysis may be used.
  • “Improving the regeneration” of Lactobacillus rhamnosus bacteria means to increase the amount (number) of Lactobacillus rhamnosus bacteria successfully regenerating/ reviving compared to the respective control (i.e., the amount/ number of Lactobacillus rhamnosus bacteria without the addition of HMO).
  • “Improving the viability” of Lactobacillus rhamnosus bacteria means to increase the amount (number) of viable Lactobacillus rhamnosus bacteria compared to the respective control (i.e., the amount/ number of Lactobacillus rhamnosus bacteria without the addition of HMO).
  • Lactobacillus rhamnosus was officially reclassified as “Lacticaseibacillus rhamnosus” in 2020, but the art still refers to it as “Lactobacillus rhamnosus”. For the purpose of this invention, both genera names are considered interchangeable.
  • “Acidic” means having a pH below 7.0 (for example, having a pH ⁇ 6.0, or ⁇ 5.0, or ⁇ 4.0, or ⁇ 3.0, or in the range of 2.0-6.0, etc.).
  • the pH measured in the stomach is in the range of about 1 .5-3.5.
  • the pH measured in a healthy vagina is in the range of about 3.8-5.0.
  • the pH of fruit juices is in the range of about 2.0-4.5.
  • “Dried” means that the probiotic has been subjected to any of the following processes: lyophilization (freeze-drying), fluidized bed drying, atmospheric air drying, spray-drying, liquid-drying (L-drying), or vacuum drying. These processes are generally known in the art. A dried probiotic may be rehydrated by restoring its water content.
  • Lactobacillus rhamnosus bacteria when admixed with a human milk oligosaccharide (HMO), have a significantly increased regeneration and viability when coming into contact with a low pH (acidic) environment, such as stomach acid or an acidic beverage.
  • HMO human milk oligosaccharide
  • this combination can offer a reliable way of delivering an adequate amount of Lactobacillus rhamnosus bacteria to a host (human or animal), either in pharmaceutical-like forms, or in food-based forms.
  • the present invention relates to the use of one or more human milk oligosaccharide(s) (HMO(s)) for improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in an acidic environment.
  • HMO(s) human milk oligosaccharide(s)
  • the acidic environment is or contains an acidic liquid.
  • the acidic environment is a beverage or the stomach.
  • the acidic environment has a pH below 7.0.
  • the pH is below 6.0. More preferably, the pH is below 5.0, or even below 4.0.
  • the pH is in the range of 1 .0-6.0. In another embodiment, the pH is in the range of 1 .0-5.0. In another embodiment, the pH is in the range of 1 .0-4.0.
  • the pH range corresponds to the pH usually measured in the stomach (1 .5-3.5).
  • Another preferred pH range corresponds to the pH usually measured in the healthy vaginal tract (3.8-5.0).
  • pH ranges specifically considered are those of beverages (2.0-6.0): The pH of fruit and vegetable juices is in the range of 2.0-4.5, that of coffee in the range of 4.5-6.0. Most sodas have a pH in the range of 2.5-4.0.
  • the Lactobacillus rhamnosus bacteria are dried.
  • the dried bacteria may be the result of any known dehydration process, including freeze-drying (lyophilization), spray-drying, and liquiddrying.
  • the Lactobacillus rhamnosus bacteria are lyophilized.
  • Dried product forms include capsules, beadles, tablets, sachets, powders, and the like. They can be directly swallowed or dissolved in a liquid before swallowing.
  • the HMDs are used to improve the regeneration/ rehydration of such dried bacteria.
  • Lactobacillus rhamnosus bacteria may be live bacteria which are contained, for example, in probiotic drinks or food.
  • the Lactobacillus rhamnosus bacteria used may be any type of Lactobacillus rhamnosus bacteria.
  • the Lactobacillus rhamnosus bacteria are probiotics, more preferably probiotics known to have beneficial effects in the gut and/or vaginal tract.
  • the Lactobacillus rhamnosus bacteria of the invention may be selected from the following group of bacteria: L. rhamnosus GG, L. rhamnosus HN001, L. rhamnosus GR-1, L. rhamnosus Rosell-11, L. rhamnosus M21 , L. rhamnosus LB21, L. rhamnosus L34, L. rhamnosus 35, L.
  • L. rhamnosus strains include: L. rhamnosus GG, L. rhamnosus HN001 (available, e.g., from Howaru/IFF; Danisco/DuPont), L. rhamnosus GR-1 (available, e.g., from Chr. Hansen, Denmark), and L. rhamnosus Rosell- 11 (available, e.g., from Lallemand, Canada).
  • Lactobacillus rhamnosus GG is one of the most widely used probiotic strains.
  • L. rhamnosus GG is particularly preferred.
  • the L. rhamnosus is Lactobacillus rhamnosus DSM 32550.
  • the L. rhamnosus is Lactobacillus rhamnosus GG DSM 33156 (“LGG®”)
  • Lactobacillus rhamnosus GG DSM 33156 can be purchased, for example, from Chr. Hansen, Denmark, as LGG®. Lactobacillus rhamnosus DSM 32550 has a genomic sequence which is 99.99% identical to the genomic sequence of LGG®. It can thus be considered that L. rhamnosus DSM 32550 is equivalent to LGG®. Therefore, L. rhamnosus DSM 32550 will herein also be referred to as a Lactobacillus rhamnosus GG strain. L. rhamnosus DSM 32550 was deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, according to the Budapest Treaty on 6. July 2017, and has the accession number DSM 32550.
  • the one or more HMO used in the present invention may be any HMO.
  • HMOs which may be used in accordance with the invention are: lacto-N-triose II (LNT-II) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH), lacto- N-hexaose (LNH), 2'-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL or LDFT), lacto-N- fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N
  • the one or more HMOs is (i) one or more selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST a, LST c, LNFP-I, and LNFP-III; and/or (ii) a combination of 2’-FL and DFL; and/or (iii) a combination of 2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I . More preferably, the HMO according to the present invention is one or more selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
  • the present invention relates to a method of improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in an acidic environment, wherein the Lactobacillus rhamnosus bacteria are contained in a powder composition, said method comprising mixing the powder composition with one or more HMO(s) prior to or during the process of adding the powder composition to a dietary supplement, medicament, foodstuff or beverage.
  • the powder composition may further optionally comprise lyoprotection agents and/or processing aids.
  • the method is for improving the regeneration of probiotic blends upon reconstitution in liquid, wherein the probiotic blend comprises probiotic culture powders which are blended with carrier material and or other functional material aimed to dilute the number of probiotics and/or make the probiotic blend more functional, said method comprising adding one or more HMOs to the liquid.
  • the HMO may be added to the liquid prior to introduction of the probiotic culture powders to the liquid; substantially simultaneously to the introduction of the probiotic culture to the liquid; or after the introduction of the probiotic culture to the liquid.
  • the HMO can be added to the probiotic culture powders and the resultant mixture added to the liquid.
  • the HMO is preferably added in an effective/protective amount.
  • the effective/protective amount of the one or more HMOs may be from 0.5 g to 15 g, more preferably 1 g to 10 g.
  • the effective amount is from 2 g to 7.5 g of the one or more HMOs.
  • the probiotic comes in direct contact with the stomach acid without prior mixing with another liquid. It has been found that the HMOs will protect the Lactobacillus rhamnosus bacteria from the harsh effects of stomach acid, and allow a better regeneration and greater survival rate.
  • the present invention relates to compositions comprising Lactobacillus rhamnosus bacteria and one or more HMO(s).
  • the Lactobacillus rhamnosus bacteria of the inventive compositions may be any type of Lactobacillus rhamnosus bacteria.
  • the Lactobacillus rhamnosus bacteria are probiotics, including probiotics known to have beneficial effects in the gut and/or vaginal tract.
  • the Lactobacillus rhamnosus bacteria may be selected from the following group of bacteria: L. rhamnosus GG, L. rhamnosus HN001, L. rhamnosus GR-1, L. rhamnosus Rosell-11, L. rhamnosus M21 , L. rhamnosus LB21, L. rhamnosus L34, L. rhamnosus 35, L.
  • Preferred L. rhamnosus strains include: L. rhamnosus GG, L. rhamnosus HN001, L rhamnosus GR-1 , and L. rhamnosus Rosell-11 .
  • L. rhamnosus GG is particularly preferred.
  • the L. rhamnosus comprised in the inventive compositions is Lactobacillus rhamnosus DSM 32550.
  • the L. rhamnosus is Lactobacillus rhamnosus GG DSM 33156 (“LGG®”)
  • the HMO comprised in the inventive compositions may be any HMO.
  • HMOs which may be used in accordance with the present invention are: lacto-N-triose II (LNT-II) lacto-N-tetraose (LNT), lacto-N- neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH), lacto-N-hexaose (LNH), 2'-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-I I), lacto
  • the one or more HMOs comprised in the inventive compositions is (i) one or more selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST a, LST c, LNFP-I, and LNFP-III; and/or (ii) a combination of 2’-FL and DFL; and/or (iii) a combination of 2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I .
  • the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
  • composition comprising the probiotic and HMO may optionally contain other ingredients such as vitamins, minerals, flavorings, and further nutritional supplementation.
  • the composition of the invention may comprise a probiotic dose between 1 E+08 and 1 E+12 cfu.
  • the probiotic dose is at least 1 E+08, 2E+08, 3E+08, 4E+08, 5E+08, 6E+08, 7E+08, 8E+08, 9E+08, 1 E+09, 2E+09, 3E+09, 4E+09, 5E+09, 6E+09, 7E+09, 8E+09, 9E+09, 1 E+10, 2E+10, 3E+10, 4E+10, 5E+10, 6E+10, 7E+10, 8E+10, 9E+10, 1 E+11 , 2E+11 , 3E+11 , 4E+11 , 5E+11 , 6E+11 , 7E+11 , 8 E+11 , 9E+11 , or 1 E+12 cfu.
  • the composition of the invention preferably comprises an effective/protective amount of one or more sialylated and/or fucosylated HMO(s) with at least five monosaccharide units from 0.5 g to 15 g, more preferably 1 g to 10 g.
  • the effective amount is from 2 g to 7.5 g of the one or more human milk oligosaccharides (amount per HMO if a single HMO is used, and total HMOs if several HMOs are used, respectively).
  • the composition comprising the probiotic and the HMO(s) can be in the form of a nutritional composition.
  • the nutritional composition can be a food composition, a rehydration solution, a medical food or food for special medical purposes, a nutritional supplement, an early life nutrition product and the like.
  • the nutritional composition can contain sources of protein, lipids and/or digestible carbohydrates and can be in powdered or liquid forms.
  • the Lactobacillus rhamnosus bacteria in the inventive compositions are dried.
  • the dried bacteria may be the result of any known dehydration process, including freeze-drying (lyophilization), spray-drying, and liquid-drying.
  • the Lactobacillus rhamnosus bacteria are lyophilized.
  • the composition comprising the probiotic and the HMO(s) is in a powdery form, such as in a sachet, a dissolvable capsule or tablet, or any other convenient dry formulation.
  • the composition may also be in a liquid form, such as a liquid concentrate.
  • compositions of the invention may be used as a starter culture for fermented foods and drinks, such as spoonable dairy yoghurt, drinkable yoghurt or other fermented beverages, and spoonable non-dairy yoghurt.
  • Starter cultures obtained from probiotic providers typically contain so-called lyoprotection agents and/or processing aids added during their production. These are often proprietary to the provider and may include: disaccharides (saccharose, lactose, trehalose), polyols (mannitol, sorbitol), and polymers (maltodextrin, dextran, inulin), as well as others.
  • lyoprotection agents and/or processing aids added during their production. These are often proprietary to the provider and may include: disaccharides (saccharose, lactose, trehalose), polyols (mannitol, sorbitol), and polymers (maltodextrin, dextran, inulin), as well as others.
  • the inventive composition is consisting essentially of Lactobacillus rhamnosus bacteria and one or more HMO(s).
  • these two elements are the only bioactive ingredients; other ingredients such as binders, fillers, etc. may also be present.
  • the present invention relates to an acidic composition comprising the compositions of the present invention.
  • the acidic composition is a liquid composition.
  • acidic liquids contemplated in this invention include: carbonated mineral water, sports drinks, carbonated soft drinks (such as coca cola), fruit juices (such as orange juice or apple juice), fruit drinks, sodas, energy drinks, cold teas, and coffee.
  • one embodiment of this invention is an acidic drink comprising a reconstituted Lactobacillus rhamnosus probiotic, and a protective amount of an HMO.
  • the composition of the invention has a pH below 7.0.
  • the pH is below 6.0. More preferably, the pH is below 5.0, or even below 4.0.
  • the pH is in the range of 1 .0-6.0. In another embodiment, the pH is in the range of 1 .0-5.0. In yet another embodiment, the pH is in the range of 1 .0-4.0.
  • the preferred pH range for beverages is 2.0-6.0; for fruit and vegetable juices it is in the range of 2.0-4.5, for coffee in the range of 4.5-6.0, for sodas in the range of 2.5-4.0.
  • HMOs and Lactobacillus rhamnosus are preferred embodiments of the uses, methods, and compositions of the present invention. It is understood that any of the following combinations is suitable for the compositions, uses and methods described herein.
  • Lactobacillus rhamnosus Rosell-11 and one or more HMOs selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST a, LST c, LNFP-I, and LNFP-III; preferably, one or more HMOs selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
  • Lactobacillus rhamnosus GG DSM 33156 (LGG®) and one or more HMOs selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, LNnT, LST c, LNFP-I, and LNFP-III; and/or a combination of 2’-FL and DFL; and/or a combination of 2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I.
  • Lactobacillus rhamnosus strains were used in the Examples:
  • Example 1 Lactobacillus rhamnosus DSM 32550;
  • Example 2 Lactobacillus rhamnosus GG DSM 33156 (LGG®).
  • Lyophilized probiotics (0.4 mg/ml), alone or in combination with HMOs (5% w/v), were dissolved into sterile pH 3.0 PBS, warmed to 37°C, and vigorously mixed for about 30 seconds until no visible clumps remained. The tubes were incubated at 37°C for 3 h. The samples were further diluted, and 100 pl were spread in duplicates onto MRS agar plates which were incubated for 48 h at 37°C in anaerobic chambers. The regeneration and viability of the probiotics were determined by counting the colonies on the plates after 48h of incubation. For the experimental setup, see Figure 1 .
  • Regeneration and viability of lyophilized LGG® under pH 3.0 acidic conditions, in combination with LNnT When the lyophilized LGG® bacteria are simultaneously dissolved with 5% LNnT, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control ( Figure 14).
  • Regeneration and viability of lyophilized LGG® under pH 3.0 acidic conditions, in combination with 3’- SL When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 3’-SL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control ( Figure 15).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Polymers & Plastics (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Saccharide Compounds (AREA)

Abstract

La présente invention concerne l'utilisation d'oligosaccharides de lait humain (HMO) pour améliorer la régénération et/ou la viabilité de bactéries Lactobacillus rhamnosus dans des environnements acides. On a découvert que les HMO augmentaient le nombre de bactéries Lactobacillus rhamnosus viables lors de leur réhydratation (régénération) dans des liquides acides. Ceci améliore le potentiel probiotique de ces bactéries dans des aliments, des boissons, des compléments alimentaires et des produits pharmaceutiques oraux, comme leur viabilité pendant la préparation pour la consommation, après ingestion et/ou le long du trajet à travers le tractus gastro-intestinal augmente.
PCT/EP2023/066703 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus WO2023247579A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA202200588 2022-06-20
DKPA202200588A DK202200588A1 (en) 2022-06-20 2022-06-20 Mixture of fucosylated HMOs

Publications (1)

Publication Number Publication Date
WO2023247579A1 true WO2023247579A1 (fr) 2023-12-28

Family

ID=87059739

Family Applications (4)

Application Number Title Priority Date Filing Date
PCT/EP2023/066702 WO2023247578A1 (fr) 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de bifidobactéries
PCT/EP2023/066550 WO2023247483A1 (fr) 2022-06-20 2023-06-20 Mélange de hmo fucosylés
PCT/EP2023/066701 WO2023247577A1 (fr) 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacilles
PCT/EP2023/066703 WO2023247579A1 (fr) 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus

Family Applications Before (3)

Application Number Title Priority Date Filing Date
PCT/EP2023/066702 WO2023247578A1 (fr) 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de bifidobactéries
PCT/EP2023/066550 WO2023247483A1 (fr) 2022-06-20 2023-06-20 Mélange de hmo fucosylés
PCT/EP2023/066701 WO2023247577A1 (fr) 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de lactobacilles

Country Status (4)

Country Link
DE (1) DE202023103382U1 (fr)
DK (2) DK202200588A1 (fr)
FR (1) FR3136650A3 (fr)
WO (4) WO2023247578A1 (fr)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004341A1 (fr) 1999-07-07 2001-01-18 Centre National De La Recherche Scientifique (Cnrs) Procede de production d'oligosaccharides
WO2007101862A1 (fr) 2006-03-09 2007-09-13 Centre National De La Recherche Scientifique (Cnrs) Procédé de production d'oligosaccharides sialylés
WO2012007588A1 (fr) 2010-07-16 2012-01-19 Glycom A/S Synthèse de nouveaux dérivés sialo-oligosaccharide
WO2012113404A1 (fr) 2011-02-21 2012-08-30 Glycom A/S Nouveaux glycosyles phosphites
WO2012127410A1 (fr) 2011-03-18 2012-09-27 Glycom A/S Synthèse de nouveaux dérivés glucidiques contenant du fucose
WO2012156898A1 (fr) 2011-05-13 2012-11-22 Glycom A/S Diversification d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2012158517A1 (fr) * 2011-05-13 2012-11-22 Glycosyn LLC Utilisation de 2'-fucosyllactose, 3-fucosyllactose et lactodifucotétraose purifiés en tant que prébiotiques
WO2012156897A1 (fr) 2011-05-13 2012-11-22 Glycom A/S Procédé de préparation d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2016040531A1 (fr) 2014-09-09 2016-03-17 Glycosyn LLC Alpha (1,3) fucosyltransférases destinées à être utilisées dans la production d'oligosaccharides fucosylés
WO2019008133A1 (fr) 2017-07-07 2019-01-10 Jennewein Biotechnologie Gmbh Fucosyltransférases et leur utilisation dans la production d'oligosaccharides fucosylés
WO2019020707A1 (fr) 2017-07-26 2019-01-31 Jennewein Biotechnologie Gmbh Sialyl-transférases et leur utilisation dans la production d'oligosaccharides sialylés
WO2020115671A1 (fr) 2018-12-04 2020-06-11 Glycom A/S Synthèse de l'oligosaccharide fucosylé lnfp-v
CN112189850A (zh) * 2020-09-17 2021-01-08 合生元（广州）健康产品有限公司 益生元在促进胃肠道中益生菌存活的应用
EP3848471A1 (fr) 2020-01-10 2021-07-14 Chr. Hansen HMO GmbH Production fermentative séquentielle d'oligosaccharides
WO2022034067A1 (fr) 2020-08-10 2022-02-17 Inbiose N.V. Production d'un mélange d'oligosaccharides par une cellule
WO2022243312A1 (fr) 2021-05-17 2022-11-24 Dsm Ip Assets B.V. IDENTIFICATION D'UNE α-1,2-FUCOSYLTRANSFÉRASE POUR LA PRODUCTION IN VIVO DE LNFP-I PUR<i />

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069520A4 (fr) 2006-09-15 2010-05-05 Univ California Séquences géniques de bifidobactéries et leur utilisation
US9453230B2 (en) 2011-02-16 2016-09-27 Glycosyn LLC Biosynthesis of human milk oligosaccharides in engineered bacteria
EP2836218A4 (fr) 2012-04-13 2015-10-21 Trustees Boston College Compositions prébiotiques et leurs procédés d'utilisation
US10415021B2 (en) * 2014-10-24 2019-09-17 Glycom A/S Mutated fucosidase
EP3209673B2 (fr) 2014-10-24 2023-07-26 Glycom A/S Mélanges d'oligosaccharides de lait humain
CN116171329A (zh) * 2020-08-10 2023-05-26 因比奥斯公司 通过细胞培养或微生物发酵产生的寡糖溶液的纯化方法

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001004341A1 (fr) 1999-07-07 2001-01-18 Centre National De La Recherche Scientifique (Cnrs) Procede de production d'oligosaccharides
WO2007101862A1 (fr) 2006-03-09 2007-09-13 Centre National De La Recherche Scientifique (Cnrs) Procédé de production d'oligosaccharides sialylés
WO2012007588A1 (fr) 2010-07-16 2012-01-19 Glycom A/S Synthèse de nouveaux dérivés sialo-oligosaccharide
WO2012113404A1 (fr) 2011-02-21 2012-08-30 Glycom A/S Nouveaux glycosyles phosphites
WO2012113405A1 (fr) 2011-02-21 2012-08-30 Glycom A/S Hydrogénolyse catalytique d'une composition d'un mélange de précurseurs d'oligosaccharides et ses utilisations
WO2012127410A1 (fr) 2011-03-18 2012-09-27 Glycom A/S Synthèse de nouveaux dérivés glucidiques contenant du fucose
WO2012156897A1 (fr) 2011-05-13 2012-11-22 Glycom A/S Procédé de préparation d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2012158517A1 (fr) * 2011-05-13 2012-11-22 Glycosyn LLC Utilisation de 2'-fucosyllactose, 3-fucosyllactose et lactodifucotétraose purifiés en tant que prébiotiques
WO2012156898A1 (fr) 2011-05-13 2012-11-22 Glycom A/S Diversification d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2016040531A1 (fr) 2014-09-09 2016-03-17 Glycosyn LLC Alpha (1,3) fucosyltransférases destinées à être utilisées dans la production d'oligosaccharides fucosylés
WO2019008133A1 (fr) 2017-07-07 2019-01-10 Jennewein Biotechnologie Gmbh Fucosyltransférases et leur utilisation dans la production d'oligosaccharides fucosylés
WO2019020707A1 (fr) 2017-07-26 2019-01-31 Jennewein Biotechnologie Gmbh Sialyl-transférases et leur utilisation dans la production d'oligosaccharides sialylés
WO2020115671A1 (fr) 2018-12-04 2020-06-11 Glycom A/S Synthèse de l'oligosaccharide fucosylé lnfp-v
EP3848471A1 (fr) 2020-01-10 2021-07-14 Chr. Hansen HMO GmbH Production fermentative séquentielle d'oligosaccharides
WO2022034067A1 (fr) 2020-08-10 2022-02-17 Inbiose N.V. Production d'un mélange d'oligosaccharides par une cellule
CN112189850A (zh) * 2020-09-17 2021-01-08 合生元（广州）健康产品有限公司 益生元在促进胃肠道中益生菌存活的应用
WO2022243312A1 (fr) 2021-05-17 2022-11-24 Dsm Ip Assets B.V. IDENTIFICATION D'UNE α-1,2-FUCOSYLTRANSFÉRASE POUR LA PRODUCTION IN VIVO DE LNFP-I PUR<i />

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Milk Oligosaccharides", NOVA SCIENCE PUBLISHER, 2011
BYCH ET AL., CURRENT OPINION IN BIOTECHNOLOGY, vol. 56, 2019, pages 130 - 137
CHEN, ADV. CARBOHYDR. CHEM. BIOCHEM., vol. 72, 2015, pages 113
HANSEN CHR: "nu-trish probiotics", 12 April 2022 (2022-04-12), pages 1 - 2, XP093084149, Retrieved from the Internet <URL:https://cdn.chr-hansen.com/_/media/files/chrhansen/home/food-cultures-and-enzymes/probiotics/nu-trish_probiotic-juice_one-pager_en.pdf?rev=512df44dc88140858ff9099a0dc00edf&hash=FB7AEBCD07C4BB8D2BA4906C04A7D752> [retrieved on 20230921] *

Also Published As

Publication number Publication date
FR3136650A3 (fr) 2023-12-22
DK202300035U3 (da) 2023-09-22
DK202200588A1 (en) 2024-02-23
DE202023103382U1 (de) 2023-11-29
WO2023247578A1 (fr) 2023-12-28
WO2023247483A1 (fr) 2023-12-28
WO2023247577A1 (fr) 2023-12-28

Similar Documents

Publication Publication Date Title
Soccol et al. The potential of probiotics: a review.
Vasiljevic et al. Probiotics—from Metchnikoff to bioactives
AU2010311325B2 (en) Nutritional compositions comprising lactoferrin and probiotics and kits of parts thereof
Mckinley The nutrition and health benefits of yoghurt
EP2276357B1 (fr) Produit alimentaire probiotique de longue conservation
Xiao et al. Lactic acid bacteria in health and disease
CN102065867A (zh) 预防感染的营养物
WO2007010977A1 (fr) Nouvelle bacterie appartenant au genre bifidobacterium et utilisation de celle-ci
WO2011096808A1 (fr) Utilisation de sialyl-oligosaccharides pour gestion du poids
WO2010002241A1 (fr) Composition nutritive pour bébés nés par césarienne
WO2009151315A1 (fr) Composition nutritionnelle pour nourrissons nés par césarienne
CZ300143B6 (cs) Kmen Lactobacillus, jeho použití a výrobek jej obsahující
EP2552464A1 (fr) Utilisation de bifidobactéries pour prévenir les allergies chez les enfants nourris au sein
Misra et al. Applications of probiotics as a functional ingredient in food and gut health
Sarkar Effect of probiotics on biotechnological characteristics of yoghurt: A review
TW201105252A (en) Prevention and treatment of allergic diarrhoea
JP3784045B2 (ja) Ｌｄｌ酸化抑制飲食品及び医薬品
Tang et al. Commercial strains of lactic acid bacteria with health benefits
EP1997880A1 (fr) Bifidobactérie du lait maternel, composition correspondante, leur utilisation et milieu de culture pour les obtenir
US20230227771A1 (en) Pre-conditioning of l.reuteri
Jena et al. Bifidobacteria in Fermented Dairy Foods: A Health Beneficial Outlook
WO2023247579A1 (fr) Utilisation d&#39;oligosaccharides de lait humain pour améliorer la viabilité de lactobacillus rhamnosus
Behare et al. Prebiotic and probiotic food formulations
Hoppe et al. Commercially available human probiotic microorganisms
Nath Aparna H Nath

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23734577

Country of ref document: EP

Kind code of ref document: A1