WO2023247578A1 - Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de bifidobactéries - Google Patents

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

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WO2023247578A1
WO2023247578A1 PCT/EP2023/066702 EP2023066702W WO2023247578A1 WO 2023247578 A1 WO2023247578 A1 WO 2023247578A1 EP 2023066702 W EP2023066702 W EP 2023066702W WO 2023247578 A1 WO2023247578 A1 WO 2023247578A1
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lst
lacto
lnfp
fucopentaose
bifidobacteria
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PCT/EP2023/066702
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English (en)
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Nicole Seifert
Wilbert SYBESMA
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Dsm Ip Assets B.V.
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    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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Definitions

  • 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.
  • B. animalis ssp. animalis and B. animalis ssp. lactis were previously described as two distinct species. Presently, both are considered B. animalis, with the subspecies (abbreviated “subsp.” or “ssp.”) Bifidobacterium animals ssp. animalis and Bifidobacterium animals ssp. lactis.
  • 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.
  • 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.
  • 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.
  • Bifidobacteria are one or more selected from the following group of bacteria: Bifidobacterium (B.) bifidum, B. longum (B. longum ssp. longum), B. animalis (B. animalis ssp. animalis), B. lactis (B. animalis ssp. lactis), B. breve, B. infantis (B. longum ssp. infantis), B. adolescentis , and B. thermacidophilum. 5.
  • Bifidobacterium (B.) bifidum B. longum (B. longum ssp. longum)
  • B. animalis B. animalis ssp. animalis
  • B. lactis B. animalis ssp. lactis
  • B. breve B. infantis (B. longum ssp. infantis)
  • B. adolescentis B. thermacidophilum. 5.
  • any one of items 1-4, wherein the one or more HMO(s) is selected from the group consisting of: 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), 3’sialyllacto-N-tetraose a (LST a), 6’-sialyllacto-N-tetraose b (LST b), 6’-sialyllacto-N-neotetraose (LST c), Lacto-N- difucohexaose I (LNDFH-I), Lacto-N-difucohexaose
  • a composition comprising Bifidobacteria and one or more HMO(s), wherein the HMO is a sialylated or fucosylated HMO and has at least five monosaccharide units.
  • composition of item 8 wherein the Bifidobacteria are dried, preferably wherein the Bifidobacteria are lyophilized.
  • Bifidobacteria are one or more selected from the following group of bacteria: Bifidobacterium (B.) bifidum, B. longum (B. longum ssp. longum), B. animalis (B. animalis ssp. animalis), B. lactis (B. animalis ssp. lactis), B. breve, B. infantis (B. longum ssp. infantis), B. adolescentis , and B. thermacidophilum.
  • Bifidobacterium B.
  • B. longum B. longum ssp. longum
  • B. animalis B. animalis ssp. animalis
  • B. lactis B. animalis ssp. lactis
  • B. breve B. infantis (B. longum ssp. infantis)
  • B. adolescentis B. thermacidophilum.
  • composition of any one of items 8-10, wherein the one or more HMO(s) is selected from the group consisting of: 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), 3’sialyllacto-N-tetraose a (LST a), 6’-sialyllacto-N-tetraose b (LST b), 6’-sialyllacto-N-neotetraose (LST c), Lacto-N-difucohexaose I (LNDFH-I), Lacto-N-difucohexaose
  • composition of any one of items 8-11 wherein the one or more HMO(s) is selected from the group consisting of: LNFP-I, LNFP-III, LST a, and LST c; preferably the HMO is LST c.
  • 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, and cold teas, and coffee.
  • FIGURE 2 Shows the regeneration and viability of lyophilized Bifidobacterium breve incubated for 30 minutes at pH 3.0 without HMOs (control). The original sample was plated on agar plates and incubated for 48 h in anaerobic chamber.
  • FIGURE 3 A) Shows the regeneration and viability of lyophilized Bifidobacterium breve incubated for 30 minutes at pH 3.0 in combination with LNFP-III. The original sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates. B) Shows the viability of lyophilized Bifidobacterium breve incubated for 30 minutes at pH 3.0 in combination with LNFP-III, compared to the control (B. breve only).
  • FIGURE 5 A) Shows the regeneration and viability of lyophilized Bifidobacterium breve incubated for 30 minutes at pH 3.0 in combination with LST c. 1 :10 dilutions and the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution steps 1 :10 and 1 :100 in duplicates. B) Shows the viability of lyophilized Bifidobacterium breve incubated for 30 minutes at pH 3.0 in combination with LST c, compared to the control (B. breve only).
  • FIGURE 6 Shows the regeneration and viability of lyophilized Bifidobacterium longum incubated for 30 minutes at pH 3.0 without HMOs (control). The original sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates.
  • FIGURE 7 A) Shows the regeneration and viability of lyophilized Bifidobacterium longum incubated for 30 minutes at pH 3.0 in combination with LNFP-III. The original sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates. B) Shows the viability of lyophilized Bifidobacterium longum incubated for 30 minutes at pH 3.0 in combination with LNFP-III, compared to the control (B. longum only).
  • FIGURE 8 A) Shows the regeneration and viability of lyophilized Bifidobacterium longum incubated for 30 minutes at pH 3.0 in combination with LST c. The original sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates. B) Shows the viability of lyophilized Bifidobacterium longum incubated for 30 minutes at pH 3.0 in combination with LST c, compared to the control (B. longum only).
  • FIGURE 9 Shows the regeneration and viability of lyophilized Bifidobacterium bifidum incubated for 30 minutes at pH 3.0 without HMOs (control). 1 :10 dilutions of the original sample were plated on agar plates and incubated for 5 days in anaerobic chamber. In the picture from left to right: Dilution steps 1 :100, 1 :1000 (E-2 - E-3) in duplicates.
  • FIGURE 11 Shows the regeneration and viability of lyophilized Bifidobacterium infantis incubated for 30 minutes at pH 3.0 without HMOs (control). The sample was plated on agar plates in duplicates and incubated for 72 h in anaerobic chamber.
  • FIGURE 14 A) Shows the regeneration and viability of lyophilized Bifidobacterium infantis incubated for 30 minutes at pH 3.0 in combination with LST c. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 72 h in anaerobic chamber. In the picture from left to right: Undiluted, 1 :10 - 1 :100 - 1 :1000 in duplicates. B) Shows the viability of lyophilized Bifidobacterium infantis incubated for 30 minutes at pH 3.0 in combination with LST c, compared to the control (B. infantis only).
  • FIGURE 15 Shows the regeneration and viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 without HMOs (control). 1 :10 dilutions of the original sample were plated on agar plates and incubated for 5 days in anaerobic chamber. In the picture from left to right: Dilution steps 1 :1000, 1 :10’000 (E-3 - E-4) in duplicates.
  • FIGURE 16 A) Shows the regeneration and viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LNFP-I. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 5 days in anaerobic chamber. In the picture from left to right: E-3 - E-4 in duplicates.
  • B) Shows the viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LNFP-I, compared to the control (B. lactis only). Results are expressed as mean values (n 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from B. lactis colonies on agar plates when plated at dilution step E-4 ( Figure 16A). * indicates statistically significant difference relative to control, p 0.0439.
  • FIGURE 17 A) Shows the regeneration and viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LST a. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 5 days in anaerobic chamber. In the picture from left to right: E-3 - E-4 in duplicates.
  • B) Shows the viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LST a, compared to the control (B. lactis only). Results are expressed as mean values (n 2) with standard deviation (SD) of colonyforming units (CFU) per milliliter calculated from B. lactis colonies on agar plates when plated at dilution step E-4 ( Figure 17A). ** indicates statistically significant difference relative to control, p 0.007.
  • FIGURE 18 A) Shows the regeneration and viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LST c. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 5 days in anaerobic chamber. In the picture from left to right: E-3 - E-4 in duplicates.
  • B) Shows the viability of lyophilized Bifidobacterium lactis incubated for 1 h at pH 2.0 in combination with LST c, compared to the control (B. lactis only). Results are expressed as mean values (n 2) with standard deviation (SD) of colonyforming units (CFU) per milliliter calculated from B. lactis colonies on agar plates when plated at dilution step E-4 ( Figure 18A). ** indicates statistically significant difference relative to control, p 0.0054.
  • FIGURE 19 Shows the regeneration and viability of lyophilized Bifidobacterium animalis incubated for 2 h at pH 2.0 without HMOs (control). 1 :10 dilutions of the original sample were plated on agar plates and incubated for 4 days 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 20 A) Shows the regeneration and viability of lyophilized Bifidobacterium animalis incubated for 2 h at pH 2.0 in combination with LST a. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 4 days in anaerobic chamber. In the picture from left to right: E-2 - E-3 - E-4 in duplicates.
  • B) Shows the viability of lyophilized Bifidobacterium animalis incubated for 2 h at pH 2.0 in combination with LST a, compared to the control (B. animalis only). Results are expressed as mean values (n 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from B. animalis colonies on agar plates when plated at dilution step E-3 ( Figure 20A). * indicates statistically significant difference relative to control, p 0.0150.
  • FIGURE 22 Shows the regeneration and viability of lyophilized Bifidobacterium adolescentis incubated for 3 h at pH 3.0 without HMOs (control). The undiluted sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates.
  • FIGURE 25 A) Shows the regeneration and viability of lyophilized Bifidobacterium adolescentis incubated for 3 h at pH 3.0 in combination with LST c. The undiluted sample and a 1 :10 dilution were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture from left to right: Undiluted sample and dilution step 1 :10 in duplicates. B) Shows the viability of lyophilized Bifidobacterium adolescentis incubated for 3 h at pH 3.0 in combination with LST c, compared to the control (B. adolescentis only).
  • “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.
  • the present inventors have found that Bifidobacteria, 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 Bifidobacteria to a host (human or animal), either in pharmaceutical-like forms, or in food-based forms.
  • 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.
  • the pH is in the range of 1 .0-5.0.
  • the pH is in the range of 1 .0-4.0.
  • the Bifidobacteria may be live bacteria which are contained, for example, in probiotic drinks or food.
  • the HMDs are used to improve the viability of such live bacteria, for example by helping them survive contact with stomach acid.
  • Bifidobacteria Preferred strains of Bifidobacteria are any of the following: Bifidobacterium breve DSM 33789, Bifidobacterium longum DSM 32946, Bifidobacterium bifidum DSM 32403, Bifidobacterium infantis DSM 32687, and Bifidobacterium animalis ssp. lactis DSM 32269.
  • the HMO used may be any HMO which is sialylated and/or fucosylated and has at least five monosaccharide units.
  • fucosylated HMDs with five monosaccharide units which may be used are: lacto-N- fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N- fucopentaose V (LNFP-V), and lacto-N-fucopentaose VI (LNFP-VI).
  • sialylated HMDs with five monosaccharide units which may be used are: 3’-O-sialyllacto-N-tetraose a (LST a), 6’-O-sialyllacto-N-tetraose b (LST b), and 6’-O-sialyllacto-N-neotetraose (LST c).
  • the HMO is one or more selected from the group consisting of: LNFP-I, LNFP-III, LST a, and LST c. LST c is particularly preferred.
  • the HMO used may further be any HMO which is sialylated and/or fucosylated and has at least six monosaccharide units.
  • fucosylated HMOs with six monosaccharide units include Lacto-N- difucohexaose I (LNDFH-I), Lacto-N-difucohexaose II (LNDFH-II) and Lacto-N-difucohexaose III (LNDFH-III.
  • Examples of sialylated HMOs with six monosaccharide units include 3’,6-Disialyllacto-N-tetraose (DSLNT).
  • Examples of fucosylated and sialylated HMOs with six monosaccharide units include Sialyl-lacto-N- fucopentaose I (F-LST b), Sialyl-lacto-N-fucopentaose II (F-LST a) and Sialyl-lacto-N-fucopentaose III (F-LST c).
  • the present invention relates to a method of improving the regeneration and/or viability of Bifidobacteria in an acidic environment, wherein the Bifidobacteria 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, wherein the HMO is a sialylated and/or fucosylated HMO with at least five monosaccharide units.
  • 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 an HMO 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 HMO(s) 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 HMO(s).
  • 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 Bifidobacteria from the harsh effects of stomach acid, and allow a better regeneration and greater survival rate.
  • the present invention relates to compositions comprising Bifidobacteria and one or more HMO(s), wherein the one or more HMO(s) is a sialylated and/or fucosylated HMO with at least five monosaccharide units.
  • the Bifidobacteria of the inventive compositions may be any type of Bifidobacteria.
  • the Bifidobacteria are probiotics, including probiotics known to have beneficial effects in the gut, such as, for example, Bifidobacterium (B.) bifidum, B. longum, B. animalis, B. animalis ssp. lactis, B. breve, B. infantis, B. adolescentis, and B. thermacidophilum.
  • the Bifidobacteria are selected from the following group of bacteria: Bifidobacterium (B.) bifidum, B. longum, B. animalis, B.
  • the Bifidobacteria are selected from the group consisting of: B. bifidum, B. longum, B. animalis ssp. lactis, B. breve, and B. infantis; B. animalis ssp. lactis is particularly preferred.
  • Bifidobacteria Preferred strains of Bifidobacteria are any of the following: Bifidobacterium breve DSM 33789, Bifidobacterium longum DSM 32946, Bifidobacterium bifidum DSM 32403, Bifidobacterium infantis DSM 32687, and Bifidobacterium animalis ssp. lactis DSM 32269.
  • the HMO comprised in the inventive compositions may be any HMO which is sialylated and/or fucosylated and has at least five monosaccharide units.
  • fucosylated HMDs with five monosaccharide units include: lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-II I), lacto- N-fucopentaose V (LNFP-V), and lacto-N-fucopentaose VI (LNFP-VI).
  • sialylated HMDs with five monosaccharide units examples include: 3’-O-sialyllacto-N-tetraose a (LST a), 6’-O-sialyllacto-N-tetraose b (LST b), and 6’-O-sialyllacto-N-neotetraose (LST c).
  • 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 Bifidobacteria of 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 Bifidobacteria 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.
  • 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 Bifidobacteria probiotic, and a protective amount of an HMO.
  • the above Bifidobacterium longum is the strain Bifidobacterium longum ssp. longum DSM 32946 which 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 7 Nov 2018, and has the accession number DSM 32946.
  • the above Bifidobacterium infantis is the strain Bifidobacterium infantis DSM 32687 which 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 15 Nov 2017, and has the accession number DSM 32687.
  • Example 2 Bifidobacterium longum DSM 32946;
  • Example 4 Bifidobacterium infantis DSM 32687;
  • Example 5 Bifidobacterium animalis ssp. lactis DSM 32269;
  • Example 6 ( Figures 19-21): Bifidobacterium animalis ssp. animalis DSM 16284 (DSM Austria GmbH);
  • Example 7 ( Figures 22-25): Bifidobacterium adolescentis DSM 33750 (DSM Austria GmbH).

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Abstract

La présente invention concerne l'utilisation d'oligosaccharides de lait humain (HMO) pour améliorer la régénération et/ou la viabilité de bifidobactéries dans des environnements acides. On a découvert que les HMO augmentaient le nombre de bifidobactéries viables lors de leur réhydratation (régénération) dans des liquides acides. Ceci améliore le potentiel probiotique de ces bactéries dans des aliments, des boissons, des compléments alimentaires et des produits pharmaceutiques oraux, comme leur viabilité pendant la préparation pour la consommation, après ingestion et/ou le long du trajet à travers le tractus gastro-intestinal augmente.
PCT/EP2023/066702 2022-06-20 2023-06-20 Utilisation d'oligosaccharides de lait humain pour améliorer la viabilité de bifidobactéries WO2023247578A1 (fr)

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