EP4539680A1 - Use of human milk oligosaccharides for improving the viability of lactobacillus rhamnosus - Google Patents

Use of human milk oligosaccharides for improving the viability of lactobacillus rhamnosus

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Publication number
EP4539680A1
EP4539680A1 EP23734577.2A EP23734577A EP4539680A1 EP 4539680 A1 EP4539680 A1 EP 4539680A1 EP 23734577 A EP23734577 A EP 23734577A EP 4539680 A1 EP4539680 A1 EP 4539680A1
Authority
EP
European Patent Office
Prior art keywords
lactobacillus rhamnosus
bacteria
viability
lacto
regeneration
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP23734577.2A
Other languages
German (de)
French (fr)
Inventor
Nicole Seifert
Wilbert SYBESMA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
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 BV filed Critical DSM IP Assets BV
Publication of EP4539680A1 publication Critical patent/EP4539680A1/en
Pending legal-status Critical Current

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    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • 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
    • 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 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
    • 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; 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; 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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.
  • 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.
  • 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®).
  • 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).

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Abstract

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 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.

Description

USE OF HUMAN MILK OLIGOSACCHARIDES
FOR IMPROVING THE VIABILITY OF LACTOBACILLUS RHAMNOSUS
FIELD OF THE INVENTION
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 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.
BACKGROUND OF THE INVENTION
There is a consensus that certain live microorganisms have beneficial effects on human health. "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.
There are two main forms of delivering probiotics to the host:
1) Delivery of live bacteria. This includes the use of foods to deliver a live probiotic. The most common carriers are fresh cheeses, yogurts, fermented milks, fermented cereals, fermented vegetables and fermented meats. While some of these products deliver the probiotic to the human intestine, and are therefore effective, many do not. Further, not all consumers are able to enjoy dairy based foods: some people do not like the taste of these foods; some people have problems with lactose intolerance; and some people hold religious or political beliefs which do not permit such foods to be consumed.
2) Delivery of a dried product. This includes using dried (e.g. lyophilized) bacteria. 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.
Independent of the drying method, 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. Similarly, dried probiotics which are directly delivered to the female genital tract have to cope with the acid pH of the vagina.
It would be desirable to have a formulation of 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. In particular, it would be desirable to be able to improve the regeneration and/or viability of Lactobacillus rhamnosus bacteria in low pH (acidic) environments.
SUMMARY OF THE INVENTION
The present invention relates to the following items:
1 . Use of one or more human milk oligosaccharides (HMOs) for improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in an acidic environment.
2. The use of item 1 , wherein the acidic environment is or contains an acidic liquid, preferably wherein the acidic environment is a beverage or the stomach.
3. The use of item 1 or 2, wherein the Lactobacillus rhamnosus bacteria are dried, preferably wherein the Lactobacillus rhamnosus bacteria are lyophilized.
4. The use of any one of items 1-3, wherein the Lactobacillus rhamnosus is a Lactobacillus rhamnosus GG strain. 5. The use of any one of items 1-4, 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-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.
6. The use of any one of items 1 -5, wherein the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
7. 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 HMOs prior to or during the process of adding the powder composition to a dietary supplement, medicament, foodstuff or beverage.
8. A composition comprising Lactobacillus rhamnosus bacteria and one or more HMOs.
9. The composition of item 8, wherein the Lactobacillus rhamnosus bacteria are dried, preferably wherein the Lactobacillus rhamnosus bacteria are lyophilized.
10. The composition of item 8 or 9, wherein the Lactobacillus rhamnosus is a Lactobacillus rhamnosus GG strain.
11 . The 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 .
12. The composition of any one of items 8-11 , wherein the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
13. An acidic composition comprising the composition of any one of items 8-12.
14. The acidic composition of claim 13, wherein the acidic composition is a liquid composition, preferably a beverage.
15. The acidic composition of item 14, wherein the beverage is 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.
BRIEF DESCRIPTION OF THE DRAWINGS
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-2 (Figure 3A). *** indicates statistically significant difference relative to control, p 0.0007.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-3 (Figure 4A). ** indicates statistically significant difference relative to control, p 0.0031 .
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-4 (Figure 5A). *** indicates statistically significant difference relative to control, p 0.0010.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-4 (Figure 6A). * indicates statistically significant difference relative to control, p 0.0192.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-4 (Figure 7A). ** indicates statistically significant difference relative to control, p 0.0030.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-2 (Figure 8A). *** indicates statistically significant difference relative to control, p 0.0004.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-3 (Figure 9A). ** indicates statistically significant difference relative to control, p 0.0021 .
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-2 (Figure 10A). ** indicates statistically significant difference relative to control, p 0.0020. 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. B) Shows the viability of lyophilized Lactobacillus rhamnosus incubated for 3 h at pH 3.0 in combination with 3-FL, compared to the control (L. rhamnosus only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. rhamnosus colonies on agar plates when plated at dilution step E-2 (Figure 11 A). * indicates statistically significant difference relative to control, p 0.0249.
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. 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 :1000, 1 :10’000 (E-3 - E-4) in duplicates.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-4 (Figure 13A). ** indicates statistically significant difference relative to control, p 0.0034.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-4 (Figure 14A). **** indicates statistically significant difference relative to control, p <0.0001 .
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-5 (Figure 15A). ** indicates statistically significant difference relative to control, p 0.0020.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-5 (Figure 16A). **** indicates statistically significant difference relative to control, p <0.0001 .
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-5 (Figure 17A). * indicates statistically significant difference relative to control, p 0.0134.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colonyforming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E- 5 (Figure 18A). * indicates statistically significant difference relative to control, p 0.0199.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-5 (Figure 19A). ** indicates statistically significant difference relative to control, p 0.0052.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-4 (Figure 20A). *** indicates statistically significant difference relative to control, p 0.0004.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-4 (Figure 21 A). * indicates statistically significant difference relative to control, p 0.0208.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-4 (Figure 22A). *** indicates statistically significant difference relative to control, p 0.0002.
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). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from LGG® colonies on agar plates when plated at dilution step E-5 (Figure 23A). **** indicates statistically significant difference relative to control, p <0.0001 .
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used throughout the specification and claims, the following definitions apply:
“Human milk oligosaccharides” (“HMOs”, also known as human milk glycans) are complex carbohydrates that can be found, for example, in human bovine breast milk. 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. See, e.g., Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); and Chen, Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). 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. For example, 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. As examples of enzymatic production, sialylated oligosaccharides can be made as described in WO 2012/007588, fucosylated oligosaccharides can be made as described in WO 2012/127410, and diversified blends of human milk oligosaccharides can be made as described in WO 2012/156897 and WO 2012/156898. Further, 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. Production of 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. In this regard, 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).
Examples of neutral fucosylated HMOs 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 (FLNH-I), fucosyl-lacto-N-hexaose II (FLNH-II), fucosyl-lacto-N-hexaose III (FLNH- III), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (FpLNnH II), Difucosyl- Lacto-N-hexaose I (DF-LNF-I or DF-LNHa), Difucosyl-Lacto-N-hexaose II (DF-LNF-II or DF-LNHb), Difucosyl- Lacto-N-hexaose III (DF-LNF-III or DF-LNHc), Difucosyl-para-lacto-N-hexaose (DF-para-LNH), Difucosyl-para- lacto-N-neohexaose (DF-para LNnH), fucosyl-lacto-N-neohexaose a (FLNnHa), fucosyl-lacto-N-neohexaose b (FLNnHb) difucosyl-lacto-N-neohexaose (DFLNnH) and trifucosyl-lacto-N-hexaose (TF-LNH).
Examples of acidic HMOs 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-lacto-N-hexaose (SLNH), fucosyl-sialyl- lacto-N-hexaose (FSLNH), sialyl-lacto-N-neohexaose I (SLNnH-l), fucosyl-sialyl-lacto-N-neohexaose I (FSLNnH-l), sialyl-lacto-N-neohexaose II (SLNnH-ll) and Disialyl-fucosyl-lacto-N-hexaose II (DS-FLNH-II). See for example Bych et al 2019 Current Opinion in Biotechnology 56:130-137 for a review on HMO production.
“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” (or “dehydrated”) 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.
Description of the invention and preferred embodiments
The present inventors have found that 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. Thus, 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.
Hence, in a first aspect, 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.
In a preferred embodiment, the acidic environment is or contains an acidic liquid. Preferably, the acidic environment is a beverage or the stomach.
The acidic environment has a pH below 7.0. Preferably, the pH is below 6.0. More preferably, the pH is below 5.0, or even below 4.0. In one embodiment, 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.
Preferably, 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).
Other 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.
In a preferred embodiment, 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. Preferably, 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.
Alternatively, the 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. In a preferred embodiment, 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. rhamnosus CNCM i-3690, L. rhamnosus CTC1679, and L. rhamnosus CGMCC 1.3724. Preferred 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. Various health effects for Lactobacillus rhamnosus GG are well documented, including the prevention and treatment of gastro-intestinal infections and diarrhea. Therefore, L. rhamnosus GG is particularly preferred. In a preferred embodiment, the L. rhamnosus is Lactobacillus rhamnosus DSM 32550. In another preferred embodiment, 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. Examples of 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-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 (FLNH-I), fucosyl-lacto-N-hexaose II (FLNH-II), fucosyl-lacto-N-hexaose III (FLNH-III), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (FpLNnH II), Difucosyl-Lacto-N-hexaose I (DF-LNF-I or DF- LNHa), Difucosyl-Lacto-N-hexaose II (DF-LNF-II or DF-LNHb), Difucosyl-Lacto-N-hexaose III (DF-LNF-III or DF-LNHc), Difucosyl-para-lacto-N-hexaose (DF-para-LNH), Difucosyl-para-lacto-N-neohexaose (DF-para LNnH), fucosyl-lacto-N-neohexaose a (FLNnHa), fucosyl-lacto-N-neohexaose b (FLNnHb) difucosyl-lacto-N- neohexaose (DFLNnH), trifucosyl-lacto-N-hexaose (TF-LNH), 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-lacto-N- hexaose (SLNH), fucosyl-sialyl-lacto-N-hexaose (FSLNH), sialyl-lacto-N-neohexaose I (SLNnH-l), fucosyl- sialyl-lacto-N-neohexaose I (FSLNnH-l), sialyl-lacto-N-neohexaose II (SLNnH-ll) and Disialyl-fucosyl-lacto-N- hexaose II (DS-FLNH-II).
Preferably, 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.
In another aspect, 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.
In one embodiment, 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. Alternatively, 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. For example, the effective amount is from 2 g to 7.5 g of the one or more HMOs.
In another embodiment, 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.
In yet another aspect, 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. In a preferred embodiment, 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. rhamnosus CNCM i-3690, L. rhamnosus CTC1679, and L. rhamnosus CGMCC 1.3724. 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. In a preferred embodiment, the L. rhamnosus comprised in the inventive compositions is Lactobacillus rhamnosus DSM 32550. In another preferred embodiment, the L. rhamnosus is Lactobacillus rhamnosus GG DSM 33156 (“LGG®”)
The HMO comprised in the inventive compositions may be any HMO. Examples of 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-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-II I), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-lacto-N-hexaose II (FLNH-II), fucosyl-lacto-N-hexaose III (FLNH-III), fucosyl-para-lacto-N- hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (FpLNnH II), Difucosyl-Lacto-N-hexaose I (DF-LNF- I or DF-LNHa), Difucosyl-Lacto-N-hexaose II (DF-LNF-II or DF-LNHb), Difucosyl-Lacto-N-hexaose III (DF-LNF- III or DF-LNHc), Difucosyl-para-lacto-N-hexaose (DF-para-LNH), Difucosyl-para-lacto-N-neohexaose (DF-para LNnH), fucosyl-lacto-N-neohexaose a (FLNnHa), fucosyl-lacto-N-neohexaose b (FLNnHb) difucosyl-lacto-N- neohexaose (DFLNnH), trifucosyl-lacto-N-hexaose (TF-LNH), 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-lacto-N- hexaose (SLNH), fucosyl-sialyl-lacto-N-hexaose (FSLNH), sialyl-lacto-N-neohexaose I (SLNnH-l), fucosyl- sialyl-lacto-N-neohexaose I (FSLNnH-l), sialyl-lacto-N-neohexaose II (SLNnH-ll) and Disialyl-fucosyl-lacto-N- hexaose II (DS-FLNH-II).
Preferably, 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 .
More preferably, the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
The 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. Preferably, 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. For example, 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. For example, 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.
In a preferred embodiment, 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. Preferably, the Lactobacillus rhamnosus bacteria are lyophilized. Preferably, 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.
The 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. We have found however, that the addition of an HMO, according to this invention, increases the reconstitution of these probiotic cultures which contain the producers' lyoprotection and/or processing aids.
In another embodiment, the inventive composition is consisting essentially of Lactobacillus rhamnosus bacteria and one or more HMO(s). In this embodiment, these two elements (bacteria and HMOs) are the only bioactive ingredients; other ingredients such as binders, fillers, etc. may also be present.
In a further aspect, the present invention relates to an acidic composition comprising the compositions of the present invention. Preferably, the acidic composition is a liquid composition. Examples of 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. Thus, 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. Preferably, the pH is below 6.0. More preferably, the pH is below 5.0, or even below 4.0. In one embodiment, 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.
The following combinations of 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 GR-1 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 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 HN001 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 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; 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 GG and one or more HMOs selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
• Lactobacillus rhamnosus DSM 32550 and one or more HMOs selected from the group consisting of: 2’- FL, 3-FL, 3’-SL, 6’-SL, LNT, LST a, and LST c; 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 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.
The following non-limiting examples are presented to better illustrate the invention.
EXAMPLES
In acidic environments, such as the stomach or an acidic beverage, the regeneration and viability of probiotics decrease significantly after exposure to the low pH conditions. In the following experiments, the effect of HMOs on the regeneration and viability under acidic conditions was studied for different Lactobacillus rhamnosus strains. Y1
The following Lactobacillus rhamnosus strains were used in the Examples:
Example 1 (Figures 2-11): Lactobacillus rhamnosus DSM 32550;
Example 2 (Figures 12-23): Lactobacillus rhamnosus GG DSM 33156 (LGG®).
Experimental Setup
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 .
Results
Example 1
Regeneration and viability of lyophilized Lactobacillus rhamnosus alone under pH 3.0 acidic conditions: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are dissolved without HMOs, there is no visible growth of bacteria (Figure 2).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with 2’-FL: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% 2’-FL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 3).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with 3’-SL: When the lyophilized Lactobacillus rhamnosus DSM 32550 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 4).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with 6’-SL: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% 6’-SL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 5).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with 2’-FL/DFL: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% 2 -FL/DFL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 6). Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with a 5-HMO blend: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% 5-HMO blend (2’FL, 3-FL, DFL, LNDFH-I, and LNFP-I), the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 7).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with LST a: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% LST a, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 8).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with LST c: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% LST c, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 9).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with LNT: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% LNT a, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 10).
Regeneration and viability of lyophilized Lactobacillus rhamnosus under pH 3.0 acidic conditions, in combination with 3-FL: When the lyophilized Lactobacillus rhamnosus DSM 32550 bacteria are simultaneously dissolved with 5% 3-FL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 11).
Example 2
Regeneration and viability of lyophilized LGG9 alone under pH 3.0 acidic condition: When the lyophilized Lactobacillus rhamnosus DSM 33156 (LGG®) bacteria are dissolved without HMOs, the regeneration and viability of the bacteria in acidic conditions is significantly decreased compared to the HMO conditions (Figure 12 A and B).
Regeneration and viability of lyophilized LGG® under pH 3.0 acidic conditions, in combination with 2’- FL: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 2’-FL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 13).
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).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with 6’- SL: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 6’-SL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 16).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with 2’- FUDFL: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 2 -FL/DFL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 17).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with a 5- HMO blend: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 5-HMO blend (2’-FL, 3-FL, DFL, LNDFH-I, and LNFP-I), the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 18).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with LNFP-I: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% LNFP-I, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 19).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with LNFP-lll: Wnen the lyophilized LGG® bacteria are simultaneously dissolved with 5% LNFP-III, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 20).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with LST c: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% LST c, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 21).
Regeneration and viability of lyophilized LGG9 under pH 3.0 acidic conditions, in combination with LNT: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% LNT a, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 22).
Regeneration and viability of lyophilized LGG® under pH 3.0 acidic conditions, in combination with 3- FL: When the lyophilized LGG® bacteria are simultaneously dissolved with 5% 3-FL, the regeneration and viability of the bacteria in acidic conditions is significantly increased compared to the control (Figure 23).

Claims

Claims
1 . Use of one or more human milk oligosaccharides (HMOs) for improving the regeneration and/or viability of Lactobacillus rhamnosus bacteria in an acidic environment.
2. The use of claim 1 , wherein the acidic environment is or contains an acidic liquid, preferably wherein the acidic environment is a beverage or the stomach.
3. The use of claim 1 or 2, wherein the Lactobacillus rhamnosus bacteria are dried, preferably wherein the Lactobacillus rhamnosus bacteria are lyophilized.
4. The use of any one of claims 1-3, wherein the Lactobacillus rhamnosus is a Lactobacillus rhamnosus GG strain.
5. The use of any one of claims 1-4, wherein the 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-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 .
6. The use of any one of claims 1-5, wherein the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c.
7. 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 HMOs prior to or during the process of adding the powder composition to a dietary supplement, medicament, foodstuff or beverage.
8. A composition comprising Lactobacillus rhamnosus bacteria and one or more HMOs.
9. The composition of claim 8, wherein the Lactobacillus rhamnosus bacteria are dried, preferably wherein the Lactobacillus rhamnosus bacteria are lyophilized.
10. The composition of claim 8 or 9, wherein the Lactobacillus rhamnosus is a Lactobacillus rhamnosus GG strain. The composition of any one of claims 8-10, wherein 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-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 . The composition of any one of claims 8-11 , wherein the one or more HMOs is selected from the group consisting of: 2’-FL, 3-FL, 3’-SL, 6’-SL, LNT, and LST c. An acidic composition comprising the composition of any one of claims 8-12. The acidic composition of claim 13, wherein the acidic composition is a liquid composition, preferably a beverage. The acidic composition of claim 14, wherein the beverage is 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.
EP23734577.2A 2022-06-20 2023-06-20 Use of human milk oligosaccharides for improving the viability of lactobacillus rhamnosus Pending EP4539680A1 (en)

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EP23734575.6A Pending EP4539678A1 (en) 2022-06-20 2023-06-20 Use of human milk oligosaccharides for improving the viability of lactobacilli
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