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 bacteria in acidic environments. HMOs were found to increase the number of viable Lactobacillus 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, including the digestive system and the female genital tract. There are probiotic species within the Lactobacillus genus which are well-known for exerting their beneficial effects in the gut, such as Lactobacillus (L.) rhamnosus, L. plantarum, L. casei, L. paracasei, L. reuteri, and L. acidophilus. Other Lactobacillus species are known to predominate the healthy vaginal microbiota, e.g., L. crispatus, L. jensenii, L. gasseri, and L. iners.

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 Lactobacilli 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 oligosaccharide(s) (HMO(s)) for improving the regeneration and/or viability of Lactobacillus bacteria in an acidic environment, wherein the HMO is a sialylated and/or fucosylated HMO with at least five monosaccharide units.

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 bacteria are dried, preferably wherein the Lactobacillus bacteria are lyophilized.

4. The use of any one of items 1 -3, wherein the Lactobacillus bacteria are one or more selected from the following group of bacteria: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. 5. The use of 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 II (LNDFH-II), Lacto-N-difucohexaose III (LNDFH- III), 3’,6-Disialyllacto-N-tetraose (DSLNT), 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).

6. The use of any one of items 1-5, wherein the one or more HMO(s) is selected from the group consisting of: LNFP-I, LNFP-II I , LST a, and LST c; preferably, the HMO is LST a and/or LST c.

7. A method of improving the regeneration and/or viability of Lactobacillus bacteria in an acidic environment, wherein the Lactobacillus 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, wherein the one or more HMO(s) is a sialylated and/or fucosylated HMO with at least five monosaccharide units.

8. A composition comprising Lactobacillus bacteria and one or more HMO(s), wherein the HMO is a sialylated and/or fucosylated HMO with at least five monosaccharide units.

9. The composition of item 8, wherein the Lactobacillus bacteria are dried, preferably wherein the Lactobacillus bacteria are lyophilized.

10. The composition of item 8 or 9, wherein the Lactobacillus bacteria are one or more selected from the following group of bacteria: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners.

11 . The 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 II (LNDFH-II), Lacto-N- difucohexaose III (LNDFH-III), 3’,6-Disialyllacto-N-tetraose (DSLNT), 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).

12. The 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 a and/or LST c.

13. An acidic composition comprising the composition of any one of items 8-12.

14. The acidic composition of item 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, and 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 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, 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 3A). *** indicates statistically significant difference relative to control, p 0.0004.

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 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, 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 4A). ** indicates statistically significant difference relative to control, p 0.0021 .

FIGURE 5: Shows the regeneration and viability of lyophilized Lactobacillus plantarum TIFN101 (DSM 34532) incubated for 1 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 :10, 1 :100 (E-1 - E-2) in duplicates.

FIGURE 6: A) Shows the regeneration and viability of lyophilized Lactobacillus plantarum TIFN101 (DSM 34532), incubated for 1 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-1 - E-2 - E-3 in duplicates. B) Shows the viability of lyophilized Lactobacillus plantarum TIFN101 , incubated for 1 h at pH 3.0 in combination with LST a, compared to the control (L. plantarum only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. plantarum colonies on agar plates when plated at dilution step E-1 (Figure 6A). indicates statistically significant difference relative to control, p 0.0002.

FIGURE 7: A) Shows the regeneration and viability of lyophilized Lactobacillus plantarum TIFN101 (DSM 34532), incubated for 1 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-1 - E-2 - E-3 - E-4 in duplicates. B) Shows the viability of lyophilized Lactobacillus plantarum TIFN101 , incubated for 1 h at pH 3.0 in combination with LST c, compared to the control (L. plantarum only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. plantarum colonies on agar plates when plated at dilution step E-4 (Figure 7A). *** indicates statistically significant difference relative to control, p 0.0009.

FIGURE 8: Shows the regeneration and viability of lyophilized Lactobacillus casei (DSM 32382), incubated for 3 h at pH 3.0 without HMDs (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 :1000, 1 :10’000 (E-3 - E-4) in duplicates.

FIGURE 9: A) Shows the regeneration and viability of lyophilized Lactobacillus casei (DSM 32382), incubated for 3 h at pH 3.0 in combination with LNFP-L A 1 :1000 dilution of the original sample was plated on agar plates in duplicates and incubated for 48 h in anaerobic chamber. B) Shows the viability of lyophilized Lactobacillus casei, incubated for 3 h at pH 3.0 in combination with LNFP-I, compared to the control (L. casei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. casei colonies on agar plates when plated at dilution step E-3 (Figure 9A). * indicates statistically significant difference relative to control, p 0.0188.

FIGURE 10: A) Shows the regeneration and viability of lyophilized Lactobacillus casei (DSM 32382), incubated for 3 h at pH 3.0 in combination with LNFP-III. A 1 :1000 dilution of the original sample was plated on agar plates in duplicates and incubated for 48 h in anaerobic chamber. B) Shows the viability of lyophilized Lactobacillus casei incubated for 3 h at pH 3.0 in combination with LNFP-III, compared to the control (L. casei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. casei colonies on agar plates when plated at dilution step E-3 (Figure 10A). * indicates statistically significant difference relative to control, p 0.0217.

FIGURE 11 : A) Shows the regeneration and viability of lyophilized Lactobacillus casei (DSM 32382), 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-3 - E-4 - E-5 in duplicates. B) Shows the viability of lyophilized Lactobacillus casei incubated for 3 h at pH 3.0 in combination with LST a, compared to the control (L. casei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. casei colonies on agar plates when plated at dilution step E-4 (Figure 11 A). *** indicates statistically significant difference relative to control, p 0.0008.

FIGURE 12: A) Shows the regeneration and viability of lyophilized Lactobacillus casei (DSM 32382), 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 Lactobacillus casei incubated for 3 h at pH 3.0 in combination with LST c, compared to the control (L casei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. casei colonies on agar plates when plated at dilution step E-5 (Figure 12A). ** indicates statistically significant difference relative to control, p 0.0027.

FIGURE 13: A) Shows the regeneration and viability of lyophilized Lactobacillus paracasei incubated for 3 h at pH 3.0 without HMOs (control). This control was used to compare effects shown in Figures 14 and 16. 1 :10 dilutions of the original sample were plated on agar plates and incubated for 48 h in anaerobic chamber. In the picture: Dilution step 1 :100 (E-2) in duplicates. B) Shows the regeneration and viability of lyophilized Lactobacillus paracasei incubated for 3 h at pH 3.0 without HMOs (control). This control was used to compare effects shown in Figure 15. 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 :1 ’000, 1 :10’00 (E-2 - E-3 - E-4) in duplicates.

FIGURE 14: A) Shows the regeneration and viability of lyophilized Lactobacillus paracasei incubated for 3 h at pH 3.0 in combination with LNFP-III. A 1 :100 dilution of the original sample was plated on agar plates in duplicates and incubated for 48 h in anaerobic chamber. B) Shows the viability of lyophilized Lactobacillus paracase/ incubated for 3 h at pH 3.0 in combination with LNFP-III, compared to the control (L. paracasei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. paracasei colonies on agar plates when plated at dilution step E-2 (Figure 14A). * indicates statistically significant difference relative to control, p 0.0160.

FIGURE 15: A) Shows the regeneration and viability of lyophilized Lactobacillus paracasei 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 paracasei incubated for 3 h at pH 3.0 in combination with LST a, compared to the control (L. paracasei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. paracasei colonies on agar plates when plated at dilution step E-2 (Figure 15A). ** indicates statistically significant difference relative to control, p 0.0020.

FIGURE 16: A) Shows the regeneration and viability of lyophilized Lactobacillus paracasei 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 - E-5 in duplicates. B) Shows the viability of lyophilized Lactobacillus paracasei incubated for 3 h at pH 3.0 in combination with LST c, compared to the control (L. paracasei only). Results are expressed as mean values (n = 2) with standard deviation (SD) of colony-forming units (CFU) per milliliter calculated from L. paracasei colonies on agar plates when plated at dilution step E-5 (Figure 16A). * indicates statistically significant difference relative to control, p 0.0144.

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.

Examples of fucosylated HMOs with five monosaccharide units include lacto-N-fucopentaose (a1-2)-Gal-(b1-3)-GlcNAc-(b1-3)-Gal-(b1-4)-Glc), lacto-N-fucopentaose (b1- GlcNAc-(b1-3)-Gal-(b1-4)-Glc), lacto-N-fucopentaose III (LNFP-III, Gal-(b GlcN (b1-4)-Glc), lacto-N-fucopentaose V (LNFP-V, Gal-(b1-3)-GlcNAc-(b1- c-(a lacto-N-fucopentaose VI (LNFP-VI, Gal-(b1-4)-GlcNAc-(b1-3)-Gal-(b1 lc). Examples of sialylated HMOs with five monosaccharide units include 3’-sialyllacto-N-tetraose a (LST a, Neu5Ac-(a2-3)-Gal- (b1-3)-GlcNAc-(b1-3)-Gal-(b1-4)-Glc), 6’-sialyllacto-N-tetraose b (LST b, Gal-(b1-3)-[Neu5Ac-(a2-6)]-GlcNAc- (b1-3)-Gal-(b1-4)-Glc), and 6’-sialyllacto-N-neotetraose( LST c, Neu5Ac-(a2-6)-Gal-(b1-4)-GlcNAc-(b1-3)-Gal- (b1-4)-Glc).

Examples offucosylated HMOs with six monosaccharide units include Lacto-N-difucohexaose I (LNDFH-I, Fuc- (a1-2)-Gal-(b1-3)-[Fuc-(a1-4)]-GlcNAc-(b1-3)-Gal-(b1-4)-Glc) , Lacto-N-difucohexaose II (LNDFH-II, Gal-(b1-

3)-[Fuc-(a1-4)]-GlcNAc-(b1-3)-Gal-(b1-4)-[Fuc-(a1-3)]-Glc ) and Lacto-N-difucohexaose III (LNDFH-III, Gal-(b1-

4)-[Fuc-(a1-3)]-GlcNAc-(b1-3)-Gal-(b1-4)-[Fuc-(a1-3)]-Glc ). Examples of sialylated HMOs with six monosaccharide units include 3’,6-Disialyllacto-N-tetraose (DSLNT, Neu5Ac-(a2-3)-Gal-(b1-3)-[Neu5Ac-(a2- 6)]-GlcNAc-(b1-3)-Gal-(b1-4)-Glc). Examples of fucosylated and sialylated HMOs with six monosaccharide units include Sialyl-lacto-N-fucopentaose I (F-LST b, Fuc-(a1-2)-Gal-(b1-3)-[Neu5Ac-(a2-6)]-GlcNAc-(b1-3)- Gal-(b1-4)-Glc), Sialyl-lacto-N-fucopentaose II (F-LST a, Neu5Ac-(a2-3)-Gal-(b1-3)-[Fuc-(a1-4)]-GlcNAc-(b1-

3)-Gal-(b1-4)-Glc), Sialyl-lacto-N-fucopentaose III (F-LST c, Neu5Ac-(a2-6)-Gal-(b1-4)-GlcNAc-(b1-3)-Gal-(b1-

4)-[Fuc-a1-4)]-Glc).

“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 bacteria means to increase the amount (number) of Lactobacillus bacteria successfully regenerating/ reviving compared to the respective control (i.e., the amount/ number of Lactobacillus bacteria without the addition of HMO).

“Improving the viability” of Lactobacillus bacteria means to increase the amount (number) of viable Lactobacillus bacteria compared to the respective control (i.e., the amount/ number of Lactobacillus bacteria without the addition of HMO).

“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 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 bacteria to a host (human or animal), either in pharmaceuticallike 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 bacteria in an acidic environment. The HMOs of the invention are sialylated and/or fucosylated HMOs with at least five monosaccharide units.

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 bacteria 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 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 HMOs are used to improve the regeneration/ rehydration of such dried bacteria.

Alternatively, the Lactobacillus bacteria may be live bacteria which are contained, for example, in probiotic drinks or food. The HMOs are used to improve the viability of such live bacteria, for example by helping them survive contact with stomach acid.

The Lactobacillus bacteria used may be any type of Lactobacillus bacteria. In a preferred embodiment, the Lactobacillus bacteria are probiotics, including probiotics known to have beneficial effects in the gut and/or vaginal tract, such as, for example, Lactobacillus rhamnosus (L rhamnosus), L. plantarum, L. casei, L. paracasei, L. reuteri, L. acidophilus, L. crispatus, L. jensenii, L. gasseri, and L. iners. Preferably, the Lactobacillus bacteria are selected from the following group of bacteria: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus paracasei. Preferred strains of Lactobacillus bacteria are: Lactobacillus rhamnosus DSM 32550; Lactobacillus rhamnosus GG DSM 33156 (“LGG®”); Lactobacillus plantarum TIFN101 CBS 138100 or DSM 34532; and Lactobacillus casei DSM 32382.

The HMO used may be any HMO which is sialylated and/or fucosylated and has at least five monosaccharide units. Examples of fucosylated HMOs 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). Examples of sialylated HMOs 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). Preferably, the HMO is one or more selected from the group consisting of: LNFP-I, LNFP-III, LST a, and LST c. LST a and LST c are particularly preferred.

The HMO used may further be any HMO which is sialylated and/or fucosylated and has at least six monosaccharide units. Examples of 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).

In another aspect, the present invention relates to a method of improving the regeneration and/or viability of Lactobacillus bacteria in an acidic environment, wherein the Lactobacillus 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, 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.

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 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. 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 HMO(s) 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 HMO(s). 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 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 bacteria 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 Lactobacillus bacteria of the inventive compositions may be any type of Lactobacillus bacteria. In a preferred embodiment, the Lactobacillus bacteria are probiotics, including probiotics known to have beneficial effects in the gut and/or vaginal tract, such as, for example Lactobacillus rhamnosus (L. rhamnosus), L. plantarum, L. casei, L. paracasei, L. reuteri, L. acidophilus, L. crispatus, L. jensenii, L. gasseri, and L. iners. Preferably, the Lactobacillus bacteria are selected from the following group of bacteria: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus paracasei. Preferred strains of Lactobacillus bacteria are any of the following: Lactobacillus rhamnosus DSM 32550; Lactobacillus rhamnosus GG DSM 33156 (“LGG®”); Lactobacillus plantarum TIFN101 CBS 138100 or DSM 34532; and Lactobacillus casei DSM 32382.

The HMO comprised in the inventive compositions may be any HMO which is sialylated and/or fucosylated and has at least five monosaccharide units. Examples of fucosylated HMOs 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). Examples of sialylated HMOs with five monosaccharide units 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 HMO may further be any HMO which is sialylated and/or fucosylated and has at least six monosaccharide units. Examples of 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).

Preferably, the HMO is one or more selected from the group consisting of: LNFP-I, LNFP-I 11 , LST a, and LST c. LST a and LST c are particularly preferred.

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).

It is understood that any of the above-described compositions are suitable for the uses and methods described herein.

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 bacteria of the inventive compositions are dried. The dried bacteria may be the result of any known dehydration process, including freeze-drying (lyophilization), spraydrying, and liquid-drying. Preferably, the Lactobacillus 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 bacteria and one or more HMO(s), wherein the HMO is a sialylated and/or fucosylated HMO with at least five monosaccharide units. 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 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 Lactobacilli are preferred embodiments of the uses, methods, and compositions of the present invention:

• Lactobacillus rhamnosus and at least one HMO selected from LST a and LST c.

• Lactobacillus plantarum and at least one HMO selected from LST a and LST c.

• Lactobacillus casei and at least one HMO selected from the group consisting of: LST a, LST c, LNFP-I, and LNFP-III.

• Lactobacillus paracasei and at least one HMO selected from the group consisting of: LST a, LST c, and LNFP-III.

Preferably, the above Lactobacillus rhamnosus is the strain Lactobacillus rhamnosus DSM 32550 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 6 July 2017, and has the accession number DSM 32550.

Preferably, the above Lactobacillus plantarum is the strain Lactobacillus plantarum TIFN101 (deposited under deposit number CBS 138100), or the Lactobacillus plantarum strain DSM 34532.

Preferably, the above Lactobacillus casei is the strain Lactobacillus casei DSM 32382 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 18 Oct 2016, and has the accession number DSM 32382.

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 probiotic Lactobacillus strains.

The following Lactobacillus strains were used in the Examples:

Example 1 (Figures 2-4): Lactobacillus rhamnosus DSM 32550;

Example 2 (Figures 5-7): Lactobacillus plantarum TIFN101 CBS 138100;

Example 3 (Figures 8-12): Lactobacillus easel DSM 32382); and

Example 4 (Figures 13-16): Lactobacillus paracasei (Biocare Copenhagen, strain IN 0106).

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 notable growth of bacteria (no colonies visible; see Figure 2).

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 (Figures 3 A and B).

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 (Figures 4 A and B). Example 2

Regeneration and viability of lyophilized Lactobacillus plantarum alone under pH 3.0 acidic conditions: When the lyophilized Lactobacillus plantarum TIFN101 bacteria are dissolved without HMOs, only very few bacterial colonies resulting from viable cells are observed (Figure 5).

Regeneration and viability of lyophilized Lactobacillus plantarum under pH 3.0 acidic conditions, in combination with LST a: When the lyophilized Lactobacillus plantarum TIFN101 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 (Figures 6 A and B).

Regeneration and viability of lyophilized Lactobacillus plantarum under pH 3.0 acidic conditions, in combination with LST c: When the lyophilized Lactobacillus plantarum TIFN101 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 (Figures 7 A and B).

Example 3

Regeneration and viability of lyophilized Lactobacillus easel alone under pH 3.0 acidic conditions:

When the lyophilized Lactobacillus casei bacteria are dissolved without HMOs, only very few bacterial colonies resulting from viable cells are observed (Figure 8).

Regeneration and viability of lyophilized Lactobacillus casei under pH 3.0 acidic conditions, in combination with LNFP-I: When the lyophilized Lactobacillus casei 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 (Figures 9 A and B).

Regeneration and viability of lyophilized Lactobacillus casei under pH 3.0 acidic conditions, in combination with LNFP- III: When the lyophilized Lactobacillus casei 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 (Figures 10 A and B).

Regeneration and viability of lyophilized Lactobacillus casei under pH 3.0 acidic conditions, in combination with LST a: When the lyophilized Lactobacillus casei 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 (Figures 11 A and B).

Regeneration and viability of lyophilized Lactobacillus casei under pH 3.0 acidic conditions, in combination with LST c: When the lyophilized Lactobacillus casei 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 (Figures 12 A and B). Example 4

Regeneration and viability of lyophilized Lactobacillus paracasei alone under pH 3.0 acidic conditions: When the lyophilized Lactobacillus paracasei bacteria are dissolved without HMOs, only very few bacterial colonies resulting from viable cells are observed (Figures 13 A and B).

Regeneration and viability of lyophilized Lactobacillus paracasei under pH 3.0 acidic conditions, in combination with LNFP- III: When the lyophilized Lactobacillus paracasei 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 (Figures 14 A and B).

Regeneration and viability of lyophilized Lactobacillus paracasei under pH 3.0 acidic conditions, in combination with LST a: When the lyophilized Lactobacillus paracasei 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 (Figures 15 A and B).

Regeneration and viability of lyophilized Lactobacillus paracasei under pH 3.0 acidic conditions, in combination with LST c: When the lyophilized Lactobacillus paracasei 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 (Figures 16 A and B).