FIELD OF THE INVENTION

The present invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain belonging to bifidobacteria and a prebiotic mixture of human milk oligosaccharides (HMO) consisting of 2’-fucosyllactose (2FL), difucosyllactose (DFL), 3’-sialyllactose (3SL), 6’-sialyllactose (6SL) and lacto-N- tetraose (LNT), and optionally 3-fucosyllactose (3FL) and applications of the mixtures in human health.

BACKGROUND OF THE INVENTION

HMOs (human milk oligosaccharides) have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011 , ISBN: 978- 1-61122-831-1).

Evidence is accumulating that the resident community of microbes, called the microbiota, in the human digestive tract plays a major role in health and disease. When the composition of the intestinal microbiota is thrown off balance, the human host can suffer consequences. Recent research has implicated intestinal microbiota imbalances in individual disorders as diverse as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism. Individual non-digestible fibres, including HMOs, are believed to positively modulate the microbiota, and they are of increasing interest for treating one or more of such disorders.

Infancy, especially the first weeks, 3 months, 6 months or 12 months of life is a critical period for the establishment of a balanced gut microbiota.

It is known that the modulation of the gut microbiota during infancy can prospectively have a significant influence in the future health status of the body. For example, the gut microbiome can have an influence on the development of a strong immune system later in life, as well as normal growth, and even on the development of obesity later in life.

The gut microbiome and its evolution during the development of the infant is, however, a fine balance between the presence and prevalence (amount) of many populations of gut bacteria. Some gut bacteria are classified as “generally positive” while others are “generally negative” (or pathogenic) regarding their effect on the overall health of the infant. Certain species of “generally positive” bacteria, such as bifidobacteria, may be under-represented in infants fed conventional infant formula in comparison to breastfed infants. Similarly, some bacterial populations are considered pathogenic and should remain at a low prevalence in the gut microbiota.

Bifidobacterium longum subsp. infantis has been demonstrated to predominate in the gut microbiota of breastfed infants and to benefit the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing short-chain fatty acid production. Reduced abundance of Bifidobacterium species in infants has been correlated to chronic diseases, including asthma and obesity, as well as to lower vaccine response. Researchers have postulated that loss of Bifidobacterium species in the infant gut in populations of developed countries is linked to increased incidence of allergic and autoimmune diseases.

Increasing the abundance of Bifidobacterium species or specifically Bifidobacterium longum subsp. infantis in the gut microbial ecosystem through exogenous administration may be difficult to achieve, especially in formula-fed infants.

Key metabolites produced by Bifidobacterium longum subsp. infantis are lactic acid and acetic acid. Lactic and acetic acid contribute to decreasing stool pH and providing colonization resistance against pathogens in infants (Duar RM, Kyle D and Casaburi G, Colonization Resistance in the Infant Gut: The Role of B. infantis in Reducing pH and Preventing Pathogen Growth; High-Throughput, 2020). Both acids can serve as substrates for other members of the microbiota that produce other short-chain fatty acids (SCFA). SCFA are especially produced by microbial fermentation of dietary fibers in the colon. These colonic fermentations have been known to play a role in energy supply, as trophic factors, and in immune regulation and growing evidence suggests that SCFAs also exert important physiological effects on several organs, including the brain. High abundance of bacteria producing SCFA, particularly butyrate, has been shown to be linked to milder atopic eczema in infants (Nylund L et al., Severity of Atopic disease inversely correlates with intestinal microbiota diversity and butyrate- producing bacteria, Allergy, 2015).

Due to the loss of Bifidobacterium species in the infant gut and low breast-feeding rates, there is a need to provide infants with both, HMOs and HMO-utilizing bacteria such as B. longum subsp. infantis to support a healthy microbiome for long-term health. SUMMARY OF THE INVENTION

A first aspect of this invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain being bifidobacteria and a prebiotic mixture of human milk oligosaccharides consisting of 2’- fucosyllactose (2FL), 3’-sialyllactose (3SL), difucosyllactose (DFL), 6’-sialyllactose (6SL), lacto-N-tetraose (LNT) and optionally 3-fucosyllactose (3FL).

The present inventors have surprisingly found that a synergistic effect is obtained when this HMO mix is used in combination with Bifidobacterium, in particularthe combination of the probiotic, for instance B. longum subsp. infantis, with the HMO mix synergistically promotes a healthier gut environment due to the enhancement of beneficial fermentative metabolites such as short chain fatty acids and the growth stimulation of healthy commensal gut bacteria such as bifidobacteria.

In one aspect the present invention relates to a composition wherein the bifodobacteria is Bifidobacterium animalis subsp. lactis and the prebiotic oligosaccharides mixture consists of 2'-fucosyllactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

In another aspect the present invention relates to a composition wherein the bifodobacteria is a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis and the prebiotic oligosaccharides mixture consists of 2'-fucosyllactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

In one aspect the present invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain being bifidobacteria, and a prebiotic oligosaccharides mixture consisting of 2'-fucosy I lactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL); wherein the bifidobacteria comprises Bifidobacterium longum subsp. infantis or combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis thereof; wherein at least one of the bifidobacteria is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 99.9% with this strain.

In one aspect the present invention relates to a nutritional composition selected from the list consisting of an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, growing-up-milk, a fortifier such as a human milk fortifier, or a supplement. In one aspect the present invention relates to a nutrional composition as defined above for use in: i) preventing and/or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and/or iii) preventing and/or treating allergy of an infant or a young child.

In one aspect the present invention relates to a composition as defined above for use for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and/or Bifidobacterium longum subsp. infantis being increased.

In one aspect the present invention relates to a composition as defined above for modulating the microbiota of an infant or a young child; and/or preventing and/or treating allergy of an infant or a young child by increasing intestinal short-chain fatty acids (SCFA) production in such infant or young child.

In one aspect the present invention relates to a method of modulating the microbiota of an infant or a young child to increase the abundance of Bifidobacteriacea and/or Bifidobacterium longum subsp. infantis, the method comprising:

- administering, to the infant or young child, a composition as defined.

FIGURES

Figure 1 : Total short chain fatty acid concentration after 24 h averaged over 8 faecal donors. Average concentrations increase upon supplementation of a single HMO (2FL). A larger increase occurs when supplementing an HMO mix instead of a single HMO. Further incremental increases are obtained when supplementing Bifidobacterium infantis B. Bifidobacterium infantis B is B. longum subsp. infantis LMG 11588.

Figure 2: Prevalence of the Bifidobacteriaceae operational taxonomic unit after 24 h averaged over 8 faecal donors. Average prevalence increases upon supplementation of a single HMO (2FL). A larger increase occurs when supplementing an HMO mix instead of a single HMO. Further incremental increases are obtained when supplementing Bifidobacterium infantis B. Bifidobacterium infantis B is B. longum subsp. infantis LMG 11588.

Figure 3: Total acidification after 48h (average of triplicates). Average concentrations increase upon supplementation of an HMO mix. Sequential increases are obtained when supplementing respectively Bifidobacterium infantis A and Bifidobacterium infantis C in the presence of an HMO mix. Bifidobacterium infantis A is B. longum subsp. infantis AT CO 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with B. longum subsp. infantis LMG 11588.

Figure 4: Total short chain fatty acid concentration after 48h (average of triplicates). Average concentrations increase upon supplementation of an HMO mix. Sequential increases are obtained when supplementing respectively Bifidobacterium infantis A and Bifidobacterium infantis C in the presence of an HMO mix. Bifidobacterium infantis A is B. longum subsp. infantis ATCC 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with B. longum subsp. infantis LMG 11588.

Figure 5: Prevalence of the Bifidobacterium infantis- related operational taxonomic unit after 48h (average of triplicates). Largest increase is obtained when supplementing Bifidobacterium infantis C in the presence of an HMO mix. Bifidobacterium infantis A is B. longum subsp. infantis ATCC 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with B. longum subsp. infantis LMG 11588.

Figure 6: Total short chain fatty acid concentration after 24 h averaged over 6 faecal donors at infant age (3 months old) and toddler age (12 months old). There is no notable increase in average total short chain fatty acid concentrations compared to the blank when supplementing Bifidobacterium infantis B without HMOs or Bifidobacterium lactis without HMOs. Average concentrations increase upon supplementation of an HMO mix alone. Further increases are obtained when supplementing B. infantis B with an HMO mix and with the combination of B. infantis B and B. lactis with an HMO mix. A horizontal dashed line is added to denote the blank level of total short chain fatty acid concentration for infants and toddlers. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG 11588. Bifidobacterium lactis is Bificobacterium animalis subsp. lactis CNCM I-3446.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms have the following meanings.

The term “infant” means a child under the age of 12 months.

The expression “young child” means a child aged between one and three years, also called toddler. The expressions “a composition for infants or young children” and “a composition to be administered to infants or young children” can be used interchangeably.

In some embodiments, the composition comprising the HMOs mixture according to the invention is a nutritional composition. The expression “nutritional composition” means a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously, and it usually includes a lipid or fat source and a protein source. In a particular embodiment, the nutritional composition is a synthetic nutritional composition.

The expression "infant formula" as used herein refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 91/321/EEC 2006/141/EC of 22 December 2006 on infant formulae and follow-on formulae). It also refers to a nutritional composition intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialities (incl. Food for Special Medical Purpose). The expression "infant formula" encompasses both “starter infant formula” and “follow-up formula” or “follow-on formula”.

A “follow-up formula” or “follow-on formula” is given from the 6th month onwards. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

The expression “baby food” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

The expression “infant cereal composition” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

The term “fortifier” refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula. In one embodiment, the aqueous composition according to the present invention is a milk fortifier. In such embodiment, the aqueous composition of the invention may be packed in single doses.

The term “supplement” means a foodstuff containing specific nutrients and/or probiotics intended to supplement a diet.

In one embodiment, the present invention is a supplement in form of powder or an oil. In such embodiment, the composition of the invention is administered as a standalone composition, or combined with other ingredients such as maltodextrin. The term “growing-up milk” (or GUM) refers to a milk-based drink generally with added vitamins and minerals, that is intended for young children or children.

HMO (Human Milk Oligosaccharides) are Oligosaccharide structures that naturally occur in human milk. The HMOs that are added to foodstuffs are typically obtained from cow milk, by chemical synthesis or by a biotechnological production process.

The term “SCFA” means short chain fatty acid(s).

The expression “increasing SCFA production” means that the amount of systemic and/or colonic SCFA, is higher in an individual fed with the nutritional composition according to the present invention in comparison with a standard. The SCFA production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography.

The “Average Nucleotide Identity (ANI)” is a measure of nucleotide-level genomic similarity between the coding regions of two genomes. Average Nucleotide Identity can be assessed as describe here: Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017 Oct;110(10):1281- 1286. In the present embodiment the strain Bifidobacterium longum subsp. infantis LMG 11588 (also known as ATCC 17930) represents the reference genome to which a microbial genome is compared. An example of a microorganism genome that has at least 99.9% ANI with B. longum subsp. infantis LMG 11588 can be found in PATRIC (https://www.patricbrc.org), genome ID 1678.111 (strain C). In one embodiment of the present invention, the Bifidobacterium longum subsp. infantis strain does not harbour potentially transferable antibiotic resistances.

The HMO and bifidobacteria mixture of this invention can: i) Increase the indigenous intestinal abundance of Bifidobacteriacea and ii) Increase intestinal SCFA production and prolong faecal pH decrease and iii) Support growth of B. longum subsp. infantis

The effect on bifidobacteria and SCFA of the HMO and bifidobacteria mixture is greater than expected from the effects of bifidobacteria or HMO mix alone. The HMO and bifidobacteria mixture of this invention has thus a surprising synergistic effect.

These effects can render an intestinal milieu less prone to invasion and overgrowth of harmful bacteria in the infant gut. These effects of the HMO and bifidobacteria mixture may prevent and/or treat conditions such as allergies, bacterial infections, inflammatory bowel disease, irritable bowel syndrome, obesity and other conditions associated with inflammation and impaired barrier function. In one embodiment, the bifidobacteria is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 99.9% with this strain.

In one embodiment, the bifidobacteria is Bifidobacterium animalis subsp. lactis CNCM 1-3446.

In one embodiment, the HMO mixture of this invention consists essentially of: i. 34 wt% to 85 wt% of 2FL, preferably 42wt% to 71 wt%; ii. 10 wt% to 40 wt% of LNT, preferably 14 wt% to 26 wt%; iii. 4 wt% to 14 wt% of DFL, preferably 5 wt% to 10 wt%; and iv. 9 wt% to 31 wt% of 6SL and 3SL combined, preferably 10 wt% to 28 wt%.

In another embodiment, the HMO mixture of this invention consists essentially of: i. 26 wt% to 65 wt% of 2FL, preferably 32 wt% to 54 wt%; ii. 8 wt% to 30 wt% of LNT, preferably 11 wt% to 20 wt%; iii. 3 wt% to 11 wt % of DFL, preferably 4 wt% to 8 wt%; iv. 7wt% to 23wt % of 6SL and 3SL combined, preferably 8wt % to 22wt %; and v. 12wt% to 38wt% of 3FL, preferably 17wt% to 31wt%.

In one embodiment, the HMO mixture of this invention consists essentially of: i. 40 wt% to 80 wt% of 2FL, preferably 50wt% to 70 wt%; ii. 10 wt% to 40 wt% of LNT, preferably 14 wt% to 26 wt%; iii. 4 wt% to 14 wt% of DFL, preferably 5 wt% to 10 wt%; and iv. 9 wt% to 31 wt% of 6SL and 3SL combined, preferably 10 wt% to 28 wt%.

In another embodiment, the HMO mixture of this invention consists essentially of: i. 20 wt% to 60 wt% of 2FL, preferably 25 wt% to 55 wt%; ii. 8 wt% to 30 wt% of LNT, preferably 11 wt% to 20 wt%; iii. 2 wt% to 12 wt % of DFL, preferably 4 wt% to 8 wt%; iv. 7wt% to 23wt % of 6SL and 3SL combined, preferably 8wt % to 22wt %; and v. 10wt% to 40wt% of 3FL, preferably 11wt% to 37wt%.

The HMO and bifidobacteria mixture can be administered to a human in any suitable form such as, for example, a nutritional composition in a unit dosage form (for example, a tablet, a capsule, a sachet of powder, etc.). The HMO and bifidobacteria mixture of this invention can also be added to a nutritional composition. For example, it can be added to an infant formula, a food composition, a rehydration solution, or a dietary maintenance or supplement for infants or young children. The nutritional composition can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, or a supplement. In some particular embodiments, the composition of the invention is an infant formula, a fortifier or a supplement that may be intended for the first 4 or 6 months of age. In a preferred embodiment the nutritional composition of the invention is an infant formula. In some other embodiments the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow-on/follow-up formula fortifier.

Macronutrients such as edible fats, carbohydrates and proteins can also be included to such a nutritional composition. Edible fats include, for example, coconut oil, soy oil and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolysed corn starch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e. g. calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and B complex) can also be included in such a nutritional composition.

The nutritional composition may be prepared in any suitable manner. A composition will now be described by way of example.

For example, a formula such as an infant formula may be prepared by blending together the protein source, the carbohydrate source and the fat source in appropriate proportions. If used, the emulsifiers may be included at this point. The vitamins and minerals may be added at this point but they are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The temperature of the water is conveniently in the range between about 50°C and about 80°C to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture.

The HMO mixture of the present invention may be added at this stage, especially if the final product is to have a liquid form. If the final product is to be a powder, they may likewise be added at this stage if desired.

The liquid mixture is then homogenised, for example in two stages. The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes, for example. This may be carried out by means of steam injection, an autoclave or a heat exchanger, for example a plate heat exchanger.

Then, the liquid mixture may be cooled to between about 60°C and about 85°C for example by flash cooling. The liquid mixture may then be again homogenised, for example in two stages between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenised mixture may then be further cooled to add any heat sensitive components, such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.

If the final product is to be a powder, the homogenised mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. The powder should have a moisture content of less than about 5% by weight. The HMO mixture of the present invention may also or alternatively be added at this stage by dry-mixing along with the probiotic strain(s) in powder.

Preferred features and embodiments of the invention will now be described by way of nonlimiting examples.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

Examples

Example 1 - Investigation of synbiotic effect of B. longum subsp. infantis LMG 11588 in combination with 2FL or 5 HMO mix

Material & Methods

Fecal material was collected from 10 approximately 3-month-old donors in Belgium. 4 donors were born by C-section and 6 were vaginally born. One donor was breast-fed, the other 9 donors were infant formula fed in the week before sampling. Fecal suspensions were prepared, mixed with cryoprotectant, aliquoted, flash frozen and then preserved at -80°C. Just before the experiment, fecal samples were defrosted and immediately used in the experiments.

The fecal material was used to perform short-term batch fermentation experiments that mimic the infant colon. These experiments represent a simplified simulation of the continuous Simulator of the Human Microbial Ecosystem (SHIME®). At the start of the short-term colonic incubations, test ingredients were added to a sugar-depleted nutritional medium containing basal nutrients of the colon. Because nutrients of this sugar-depleted nutritional medium will also be fermented by the colonic microbiota, a blank containing only the sugar-depleted nutritional medium (without product) was included for each donor. Finally, fecal inocula of the infant donors were added. Five treatments and one blank were tested per donor, resulting in 60 independent experiments. Treatments were as follows:

1 . Blank (control)

2. Single HMO (1.3 g/L)

3. 5 HMO mix (2.5 g/L)

4. B. longum subsp. infantis LMG 11588 (1x10 ⁷ CFU/ml)

5. Single HMO (1.3 g/L) + B. longum subsp. infantis LMG 11588 (1x10 ⁷ CFU/ml)

6. 5 HMO mix (2.5 g/L) + B. longum subsp. infantis LMG 11588 (1x10 ⁷ CFU/ml)

The single HMO was 2’-fucosyllactose (2FL). The HMO mix consisted of 5 HMOs in the following ratio in the dry mix: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL & 14% DFL. Therefore the addition rate of 2FL was the same in the single HMO and HMO mix treatment.

Reactors were incubated for 48h at 37°C, under shaking and anaerobic conditions. The incubations were performed in fully independent reactors with sufficiently high volume to not only ensure robust microbial fermentation, but also to allow the collection of multiple samples over time. Sample collection enables assessment of metabolite production and thus to understand the complex microbial interactions that are taking place.

An assessment was made amongst others on short chain fatty acids (SCFA) production at the start of the incubation and after 6h, 24h and 48h. The pattern of SCFA production is an assessment of the microbial carbohydrate metabolism.

Changes in microbial composition were analysed at the start and after 24h and 48h of incubation. Samples were analyzed with a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing to convert the proportional values obtained with Illumina into absolute cell counts.

Two donors (both vaginally born and infant formula fed) were excluded from the result evaluation as the sequencing results indicated technical or analytical problems.

Results

Figure 1 shows the short chain fatty acid (SCFA) concentration obtained in the colonic fermentations averaged over 8 faecal donors. When only B. longum subsp. infantis LMG 11588 was added, no increase in SCFA occurred compared to the control (no HMO, no B. infantis). With the single HMO 2FL, an increase versus the control could be observed; this increase was even greater with the HMO mix. However, when B. longum subsp. infantis was added on top of the single HMO or HMO mix, an additional increase in SCFA concentration was achieved compared to not adding B. longum subsp. infantis. The effect of the combinations of the single HMO or HMO mix with B. longum subsp. infantis versus the control was greater than adding up the effects of the individual ingredients versus the control, thus showing a clear synergistic (synbiotic) effect. The greatest increase in SCFA can be obtained with the combination of the HMO mix and B. longum subsp. infantis.

Figure 2 shows the cell count of the Bifidobacteriaceae family obtained in the colonic fermentations averaged over 8 faecal donors. Bifidobacterium longum subsp. infantis LMG 11588 was added at a level of 1x10 ⁷ CFU/ml in the variants where it was added. When only B. longum subsp. infantis was added, a small increase in Bifidobacteriaceae compared to the control (no HMO, no B. infantis) was observed. With the single HMO 2FL, an increase in Bifidobacteriaceae versus the control could be seen; this increase was even greater with the HMO mix. However, when B. longum subsp. infantis was added on top of the the single HMO or HMO mix, an additional increase in Bifidobacteriaceae was achieved compared to not adding B. longum subsp. infantis. For the combination of the HMO mix and B. longum subsp. infantis, the increase in Bifidobacteriaceae versus the control was greater than the individual effects of either ingredient versus the control, thus showing a clear synergistic (synbiotic) effect. Thus either B. longum subsp. infantis LMG 11588 has grown and/or its addition has stimulated growth of other members of the Bifidobacteriaceae family. The combination of the HMO mix and B. longum subsp. infantis was thus most effective in increasing the number of Bifidobacteriaceae. The overall results correlate with the SCFA data shown above.

2 - Investigation of svnbiotic effect of 2 different strains of B. infantis in combination with 5 HMO mix

Material & Methods

Fecal material was collected from a 3-month-old baby donor in Belgium. The donor was different from the 10 donors in Example 1. The experiment was similar to Example 1 , but all colonic fermentations were carried out in triplicate and the test ingredients differed. Five treatments and one blank were tested, resulting in 18 experiments in total. Treatments were as follows:

1 . Blank (Control)

2. 5 HMO mix (2.5g/L)

3. B. longum subsp. infantis A (1 E+07 cfu/ml)

4. B. longum subsp. infantis C (1 E+07 cfu/ml)

5. B. longum subsp. infantis A (1 E+07 cfu/ml) + 5 HMO mix (2.5g/L)

6. B. longum subsp. infantis C (1 E+07 cfu/ml) + 5 HMO mix (2.5g/L)

B. longum subsp. infantis A is the strain ATCC 15697, which is the type strain of the subspecies and shares less than 99.9% ANI (98.2%) with B. longum subsp. infantis LMG 11588. The strain B. longum subsp. infantis C has more than 99.9% ANI with B. longum subsp. infantis LMG 11588 and is thus closely related with the LMG 11588 strain.

The HMO mix consisted of 5 HMOs in the following ratio in the dry mix: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL & 14% DFL.

An assessment was made amongst others on pH and short chain fatty acids (SCFA) production at the start of the incubation and after 6h, 24h and 48h.

Changes in microbial composition were analysed at the start and after 24h and 48h of incubation. Samples were analyzed with a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing to convert the proportional values obtained with Illumina into absolute cell counts. Results

Figures 3 and 4 show that the addition of either B. longum subsp. infantis strain alone did not have an impact on acidification or SCFA production in this donor. The addition of the HMO mix alone resulted in a marked increase in SCFAs. Production of SCFAs could be further increased with the combination of the HMO mix with either B. longum subsp. infantis strain. The best effect was achieved with the combination of the HMO mix with B. longum subsp. infantis C - the strain that has more than 99.9% ANI with B. longum subsp. infantis LMG 11588. The effects of the HMO mix and B. longum subsp. infantis combinations versus the control (no HMO, no B. infantis) was greater than the sum of the effects of single ingredients versus the control, thus showing a clear synbiotic effect. This synbiotic effect was more pronounced with B. longum subsp. infantis C than with B. longum subsp. infantis A.

The sequencing data revealed that the infant donor had no detectable B. longum subsp. infantis- related operational taxonomic unit present in the inoculum, but it could be detected after addition of the two strains. After 48h colonic simulation (Figure 5) levels of B. longum subsp. infantis remained low for both strains when no HMO were added. However, for B. longum subsp. infantis C in combination with the HMO mix a large increase in B. longum subsp. /nfant/s-related operational taxonomic was detected, indicating a strong growth stimulation of the species. This shows again the synergistic effect that can be obtained with the combination of the HMO mix with B. longum subsp. infantis.

Example 3 - Investigation of synbiotic effect of B. longum subsp. infantis LMG 11588 in combination with 6 HMO mix with and without B. lactis in an infant and toddler colonic model

Material & Methods

Fecal material was collected from 6 donors in Belgium at about 3 months of age (infants) and from same donors at about 12 months of age (toddlers). 3 donors were born by C-section and 3 were vaginally born. One donor was breast-fed, the other 5 donors were infant formula fed in the week before the first sampling. The experiment was similar to Example 1. Five treatments and one blank were tested per donor at two ages, resulting in 72 independent experiments. Treatments were as follows:

1 . Blank (control)

2. B. longum subsp. infantis LMG 11588 (1x10 ⁸ CFU/ml)

3. B. animalis subsp. lactis CNCM I-3446 (1x10 ⁸ CFU/ml) 4. 6 HMO mix (2.5 g/L)

5. 6 HMO mix (2.5 g/L) + B. longum subsp. infantis LMG 11588 (1x10 ⁸ CFU/ml)

6. 6 HMO mix (2.5 g/L) + B. longum subsp. infantis LMG 11588 (1x10 ⁸ CFU/ml) + B. animalis subsp. lactis CNCM I-3446 (1x10 ⁸ CFU/ml)

The HMO mix consisted of 6 HMOs in the following ratio in the dry mix: for the infant study 16% LNT, 8% 6SL, 6% 3SL, 49% 2FL, 7% DFL & 14% 3FL; for the toddler study 10% LNT, 5% 6SL, 14% 3SL, 29% 2FL, 4% DFL & 37% 3FL.

An assessment was made amongst others on short chain fatty acids (SCFA) production at the start of the incubation and after 6h, 24h and 48h.

Results

Figure 6 shows the short chain fatty acid (SCFA) concentration obtained in the colonic fermentations at 24h averaged over 6 faecal donors at the two different ages. When only B. longum subsp. infantis LMG 11588 or B. animalis subsp. lactis CNCM I-3446 were added, no meaningful increase in SCFA occurred compared to the blank control for both models, infants and toddlers. With the 6 HMO mix, an increase in SCFA concentration versus the blank control could be observed; the effect was stronger in the the toddler model than in the infant model. However, when B. longum subsp. infantis was added additionally to the 6 HMO mix, an additional increase in SCFA concentration was achieved compared to not adding B. longum subsp. infantis. The additional effect of B. longum subsp. infantis addition was stronger in the infant model than in the toddler model. The effect of the combination of the HMO mix with B. longum subsp. infantis versus the control was greater than adding up the effects of the individual ingredients versus the blank control in both models, thus showing a clear synergistic (synbiotic) effect. The synbiotic effect of the HMO mix with B. longum subsp. infantis was maintained when additionally B. animalis subsp. lactis was added; addition of B. animalis subsp. lactis led to a slight further increase in SCFA concentration. The greatest increase in SCFA can be obtained with the combination of the 6 HMO mix with B. longum subsp. infantis and with B. animalis subsp. lactis in both, infant and toddler model.