Mixture of HMO and Bifidobacterium

Abstract

The present invention relates to a composition for infants or toddlers, the composition comprising at least one probiotic strain and a prebiotic mixture of human milk oligosaccharides, the probiotic strain being Bifidobacterium, the prebiotic mixture of human milk oligosaccharides consisting of 2'-fucosyllactose (2FL), lacto-difucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL), the composition being useful for (i) preventing and / or treating bacterial infections in infants or toddlers; (ii) modulating the microbiota of infants or toddlers; and / or (iii) preventing and / or treating allergies in infants or toddlers.

Description

Technical Field

[0001] The present invention relates to a composition for infants or young children, which comprises at least one probiotic strain and a prebiotic mixture of human milk oligosaccharides (HMOs), wherein the probiotic strain belongs to Bifidobacterium, and the prebiotic mixture of HMOs consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL) and lacto-N-tetraose (LNT) and optionally 3-fucosyllactose (3FL), and also relates to the use of these mixtures in human health. Background Art

[0002] In recent years, HMOs (human milk oligosaccharides) have attracted much attention due to their roles in various biological processes occurring in the human body. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashim et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1 (Urashima et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1)).

[0003] It has been shown that the symbiotic microbial community (referred to as the microbiota) in the human digestive tract plays an important role in health and disease. When the composition of the gut microbiota is out of balance, the human host may bear the consequences. Recent studies have shown that gut microbiota imbalance is the cause of various individual diseases such as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism. It is believed that unique non-digestible fibers (including HMOs) positively regulate the microbiota, and they are attracting increasing attention due to the treatment of one or more of such diseases.

[0004] The infancy of life, especially the first few weeks, first 3 months, first 6 months or first 12 months, is a crucial period for establishing a balanced gut microbiota.

[0005] It is known that the regulation of the gut microbiota in infancy can be expected to have a significant impact on the future health of the body. For example, the gut microbiome can affect the development of a robust immune system in the future, as well as normal growth, and even the development of obesity in the future.

[0006] However, during the development of an infant, the gut microbiome and its evolution are a delicate balance between the presence and proliferation (numbers) of many gut bacterial populations. Regarding the effects of gut bacteria on the overall health of an infant, some gut bacteria are classified as "generally positive", while other gut bacteria are "generally negative" (or pathogenic). Certain species of "overall positive" bacteria, such as Bifidobacterium, may be less abundant in infants fed conventional infant formula compared to breastfed infants. Similarly, some bacterial populations are considered pathogenic and should be kept at low levels of proliferation in the gut microbiota.

[0007] Bifidobacterium longum subsp. infantis has been shown to be dominant in the gut microbiota of breastfed infants and benefits the host by accelerating the maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing short-chain fatty acid production. A decrease in the abundance of Bifidobacterium species in infants is associated with chronic diseases, including asthma and obesity, as well as reduced vaccine responses. Researchers have hypothesized that the loss of Bifidobacterium species in the guts of infants in populations in developed countries is associated with an increased incidence of allergic and autoimmune diseases.

[0008] It may be difficult to achieve an increase in the abundance of Bifidobacterium species or particularly Bifidobacterium longum subsp. infantis in the gut microbial ecosystem through exogenous administration, especially in infants fed infant formula.

[0009] The key metabolites produced by Bifidobacterium longum subsp. infantis are lactic acid and acetic acid. Lactic acid and acetic acid contribute to reducing fecal 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 (SCFAs). The microbial fermentation of dietary fiber in the colon produces SCFAs in particular. These colonic fermentations are known to play roles in energy supply, as nutritional factors, and immunomodulation, and increasing evidence suggests that SCFAs also exert important physiological effects on several organs, including the brain. It has been shown that a high abundance of SCFA-producing (especially butyrate-producing) bacteria is associated with 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).

[0010] Due to the loss of bifidobacterial species in the infant gut and the low breastfeeding rate, it is necessary to provide infants with both human milk oligosaccharides (HMOs) and HMO-utilizing bacteria, such as Bifidobacterium longum subsp. infantis, to support a healthy microbiome for long-term health. Summary of the Invention

[0011] A first aspect of the present invention relates to a composition for an infant or toddler, the composition comprising at least one probiotic strain and a prebiotic mixture of human milk oligosaccharides, the probiotic strain being a bifidobacterium, the 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).

[0012] The present inventors have surprisingly found that a synergistic effect is obtained when the HMO mixture is used in combination with the following: Bifidobacterium, especially in combination with probiotics, for example, Bifidobacterium longum subsp. infantis synergistically promotes a healthier gut environment in combination with the HMO mixture due to the enhancement of beneficial fermentation metabolites such as short-chain fatty acids and the growth stimulation of healthy commensal gut bacteria such as Bifidobacterium.

[0013] In one aspect, the present invention relates to a composition, wherein the Bifidobacterium is Bifidobacterium animalis subsp. lactis and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

[0014] In another aspect, the present invention relates to a composition, wherein the Bifidobacterium is a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

[0015] In one aspect, the present invention relates to a composition for infants or toddlers, the composition comprising at least one probiotic strain and a prebiotic oligosaccharide mixture, the probiotic strain being Bifidobacterium, the prebiotic oligosaccharide mixture consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and optionally 3-fucosyllactose (3FL);

[0016] wherein the Bifidobacterium comprises Bifidobacterium longum subsp. infantis or a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis;

[0017] wherein at least one of the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having at least 99.9% average nucleotide identity (ANI) with this strain.

[0018] In one aspect, the present invention relates to a nutritional composition selected from the list consisting of: infant formula, stage 1 infant formula, follow-on formula or stage 2 infant formula, baby food, infant cereal composition, growing-up milk, fortifiers such as human milk fortifier, or supplements.

[0019] In one aspect, the present invention relates to a nutritional composition as defined above, which is used for: i) preventing and / or treating bacterial infections in infants or young children; ii) modulating the microbiota of infants or young children; and / or iii) preventing and / or treating allergic reactions in infants or young children.

[0020] In one aspect, the present invention relates to a composition as defined above, which is used for modulating the microbiota of infants or young children, and administering the composition results in an increase in the abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis.

[0021] In one aspect, the present invention relates to a composition as defined above, which is used for modulating the microbiota of infants or young children; and / or preventing and / or treating allergic reactions in such infants or young children by increasing the production of gut short-chain fatty acids (SCFAs) in infants or young children.

[0022] In one aspect, the present invention relates to a method for modulating the microbiota of infants or young children to increase the abundance of Bifidobacteriacea and / or Bifidobacterium longum subsp. infantis, the method comprising:

[0023] - administering the defined composition to an infant or young child. Description of the Drawings

[0024] Figure 1 : Average total short-chain fatty acid concentration of 8 fecal donors after 24 hours. The average concentration increased after supplementation with a single HMO (2FL). A greater increase occurred when supplementing with an HMO mixture rather than a single HMO. A further incremental increase was obtained when supplementing with Bifidobacterium infantis B. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG 11588.

[0025] Figure 2 : Average proliferation rate of Bifidobacteriaceae operational taxonomic units of 8 fecal donors after 24 hours. The average proliferation rate increased after supplementation with a single HMO (2FL). A greater increase occurred when supplementing with an HMO mixture rather than a single HMO. A further incremental increase was obtained when supplementing with Bifidobacterium infantis B. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG11588.

[0026] Figure 3 : Complete acidification after 48 hours (average of three replicates). The average concentration increased after supplementation with an HMO mixture. Successive increases were obtained when supplementing with Bifidobacterium infantis A and Bifidobacterium infantis C respectively in the presence of the HMO mixture. Bifidobacterium infantis A is Bifidobacterium longum subsp. infantis ATCC 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with Bifidobacterium longum subsp. infantis LMG 11588.

[0027] Figure 4 : Total short-chain fatty acid concentration after 48 hours (average of three replicates). The average concentration increased after supplementation with the HMO mixture. Successive increases were obtained when Bifidobacterium infantis A and Bifidobacterium infantis C were supplemented separately in the presence of the HMO mixture. Bifidobacterium infantis A is Bifidobacterium longum subsp. infantis ATCC 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with Bifidobacterium longum subsp. infantis LMG 11588.

[0028] Figure 5 : Proliferation rate of Bifidobacterium infantis - related operational taxonomic units after 48 hours (average of three replicates). The greatest increase was obtained when Bifidobacterium infantis C was supplemented in the presence of the HMO mixture. Bifidobacterium infantis A is Bifidobacterium longum subsp. infantis ATCC 15697. Bifidobacterium infantis C is a strain with more than 99.9% ANI with Bifidobacterium longum subsp. infantis LMG 11588.

[0029] Figure 6 : Average total short-chain fatty acid concentration of 6 fecal donors at infant age (3 months old) and toddler age (12 months old) after 24 hours. When Bifidobacterium infantis B without HMO or Bifidobacterium lactis without HMO was supplemented, there was no significant increase in the average total short-chain fatty acid concentration compared to the blank. The average concentration increased when the HMO mixture was supplemented alone. Further increases were obtained when Bifidobacterium infantis B was supplemented with the HMO mixture, and when combinations of Bifidobacterium infantis B and Bifidobacterium lactis with the HMO mixture were supplemented. Horizontal dotted lines were added to indicate the blank content of total short-chain fatty acid concentration in infants and toddlers. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG 11588. Bifidobacterium lactis is Bifidobacterium animalis subsp. lactis CNCM I - 3446. Detailed Description

[0030] As used herein, the following terms have the following meanings.

[0031] The term "infant" refers to a child under 12 months of age.

[0032] The expression "toddler" refers to a child between one and three years of age, also known as a young child learning to walk.

[0033] The expressions "composition for an infant or toddler" and "composition to be administered to an infant or toddler" are used interchangeably.

[0034] In some embodiments, a composition comprising an HMO mixture according to the invention is a nutritional composition. The expression "nutritional composition" refers to a composition that supplies nutrients to an individual. Such nutritional compositions are typically administered orally or intravenously and generally include a lipid or fat source and a protein source. In a specific embodiment, the nutritional composition is a synthetic nutritional composition.

[0035] As used herein, the expression "infant formula" refers to a foodstuff (meeting the requirements of Article 2(c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formulae and follow-on formulae) intended specifically for the nutrition of infants during the first months of life and which by itself satisfies the various nutritional requirements of such persons. It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and in infant specialties (including foods for special medical purposes). The expression "infant formula" covers both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0036] "Follow-up formula" or "follow-on formula" is given from the 6th month. Infant formula constitutes the main liquid element in the gradually diversified diet of such persons.

[0037] The expression "baby food" refers to a foodstuff intended specifically for the nutrition of infants or young children during the first year of life.

[0038] The expression "infant cereal composition" refers to a foodstuff intended specifically for the nutrition of infants or young children during the first year of life.

[0039] The term "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula. In one embodiment, the aqueous composition according to the invention is a milk fortifier. In such embodiments, the aqueous composition of the invention can be packaged in single doses.

[0040] The term "supplement" refers to a foodstuff containing specific nutrients and / or probiotics and intended to supplement the diet.

[0041] In one embodiment, the invention is a supplement in powder or oil form. In such an embodiment, the composition of the invention is administered as a separate composition or in combination with other ingredients such as maltodextrin.

[0042] The term "growing-up milk" (or GUM) refers to a milk-based beverage, usually fortified with vitamins and minerals, which is intended for consumption by infants or children.

[0043] HMO (human milk oligosaccharide) is an oligosaccharide structure that occurs naturally in human milk. HMO added to foodstuffs is usually obtained from cow's milk by chemical synthesis or by biotechnological production methods.

[0044] The term "SCFA" means short-chain fatty acid.

[0045] The expression "increased SCFA production" means that the amount of systemic and / or colonic SCFA is higher in an individual fed a nutritional composition according to the invention compared to an individual fed a standard composition. SCFA production can be measured by techniques known to the person skilled in the art such as by gas-liquid chromatography.

[0046] "Average nucleotide identity (ANI)" is a measure of the genomic similarity at the nucleotide level between the coding regions of two genomes. Average nucleotide identity can be evaluated as described herein: 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 (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 an embodiment of the invention, the strain Bifidobacterium longum subsp. infantis LMG 11588 (also known as ATCC 17930) represents the reference genome against which microbial genomes are compared. Examples of microbial genomes having at least 99.9% ANI with Bifidobacterium 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 invention, Bifidobacterium longum subsp. infantis strains do not have potentially transferable antibiotic resistance.

[0047] The HMO and Bifidobacterium mixture of the present invention can:

[0048] i) increase the indigenous intestinal abundance of Bifidobacterium, and

[0049] ii) increase intestinal SCFA production and prolong fecal pH reduction, and

[0050] iii) support the growth of Bifidobacterium longum subsp. infantis.

[0051] The effect of the HMO and Bifidobacterium mixture on Bifidobacterium and SCFAs is greater than what would be expected from the effect of Bifidobacterium alone or the HMO mixture alone. Thus, the HMO and Bifidobacterium mixture of the present invention has a surprising synergistic effect.

[0052] These effects can render the intestinal environment less susceptible to invasion and overgrowth of harmful bacteria in the infant intestine. These effects of the HMO and Bifidobacterium mixture can prevent and / or treat conditions such as allergy, bacterial infection, inflammatory bowel disease, irritable bowel syndrome, obesity, and other conditions associated with impaired inflammation and barrier function.

[0053] In one embodiment, the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having at least 99.9% average nucleotide identity (ANI) with this strain.

[0054] In one embodiment, the Bifidobacterium is Bifidobacterium animalis subsp. lactis CNCM I-3446.

[0055] In one embodiment, the HMO mixture of the present invention consists essentially of:

[0056] i. 34% to 85% by weight, preferably 42% to 71% by weight of 2FL;

[0057] ii. 10% to 40% by weight, preferably 14% to 26% by weight of LNT;

[0058] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL; and

[0059] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL.

[0060] In another embodiment, the HMO mixture of the present invention consists essentially of:

[0061] i. 26% to 65% by weight, preferably 32% to 54% by weight of 2FL;

[0062] ii. 8% to 30% by weight, preferably 11% to 20% by weight of LNT;

[0063] iii. 3% to 11% by weight, preferably 4% to 8% by weight of DFL;

[0064] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL; and

[0065] v. 12% to 38% by weight, preferably 17% to 31% by weight of 3FL.

[0066] In one embodiment, the HMO mixture of the present invention consists essentially of:

[0067] i. 40% to 80% by weight, preferably 50% to 70% by weight of 2FL;

[0068] ii. 10% to 40% by weight, preferably 14% to 26% by weight of LNT;

[0069] iii. 4% to 14% by weight, preferably 5% to 10% by weight of DFL; and

[0070] iv. 9% to 31% by weight, preferably 10% to 28% by weight of a combination of 6SL and 3SL.

[0071] In another embodiment, the HMO mixture of the present invention consists essentially of:

[0072] i. 20% to 60% by weight, preferably 25% to 55% by weight of 2FL;

[0073] ii. 8% to 30% by weight, preferably 11% to 20% by weight of LNT;

[0074] iii. 2% to 12% by weight, preferably 4% to 8% by weight of DFL;

[0075] iv. 7% to 23% by weight, preferably 8% to 22% by weight of a combination of 6SL and 3SL; and

[0076] v. 10% to 40% by weight, preferably 11% to 37% by weight of 3FL.

[0077] The HMO and Bifidobacterium mixture can be administered to a human in any suitable form such as, for example, a nutritional composition in unit dosage form (e.g., tablets, capsules, powder sachets, etc.).

[0078] The HMO and Bifidobacterium mixture of the present invention can also be added to a nutritional composition. For example, it can be added to infant formula, food compositions, rehydration solutions, or dietary maintenance agents or supplements for infants or toddlers. The nutritional composition can be, for example, infant formula, stage 1 infant formula, follow-on formula or stage 2 infant formula, baby food, infant cereal composition, fortifiers such as human milk fortifier or supplements. In some specific embodiments, the composition of the present invention is an infant formula, fortifier or supplement intended for infants up to 4 months or 6 months of age. In a preferred embodiment, the nutritional composition of the present invention is 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., human milk fortifier) or a formula food fortifier (such as an infant formula fortifier or a follow-on formula / stage 2 infant formula fortifier).

[0079] Macronutrients such as edible fats, carbohydrates, and proteins can also be included in such nutritional compositions. Edible fats include, for example, coconut oil, soybean oil, and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose, and hydrolyzed corn starch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (such as 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 nutritional compositions.

[0080] The nutritional composition can be prepared in any suitable manner. The composition will now be described by way of example.

[0081] For example, a formula food such as infant formula can be prepared by blending a protein source, a carbohydrate source, and a fat source in appropriate proportions. If used, an emulsifier can be added at this time. Vitamins and minerals can be added at this time, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc. can be dissolved in the fat source. Then water (preferably water that has undergone reverse osmosis) can be mixed in to form a liquid mixture. The water temperature is suitably in the range of about 50°C to about 80°C to help disperse the components. A commercially available liquefier can be used to form the liquid mixture.

[0082] Especially if the final product is in liquid form, the HMO mixture of the present invention can be added at this stage. If the final product is a powder, these components can be added at this stage as needed.

[0083] Then, for example, the liquid mixture is homogenized in two stages.

[0084] Then, the liquid mixture can be heat-treated to reduce the bacterial load, for example, by rapidly heating the liquid mixture to a temperature in the range of about 80 °C to about 150 °C for a duration between about 5 seconds and about 5 minutes. This can be carried out by steam injection, autoclave or heat exchanger (e.g., plate heat exchanger).

[0085] Then, the liquid mixture is cooled to between about 60 °C and about 85 °C, for example, by rapid cooling. Then the liquid mixture is again homogenized, for example, in two stages, where the pressure in the first stage is between about 10 MPa and about 30 MPa, and the pressure in the second stage is between about 2 MPa and about 10 MPa. Then the homogenized mixture can be further cooled to add any heat-sensitive components, such as vitamins and minerals. At this point, the pH and solids content of the homogenized mixture are conveniently adjusted.

[0086] If the final product is to be a powder, the homogenized mixture is transferred to a suitable drying device, such as a spray dryer or freeze dryer and converted into a powder. The moisture content of the powder should be less than about 5 wt%. The HMO mixture of the present invention can also or alternatively be added at this stage by dry mixing with probiotic strains in powder form.

[0087] The preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0088] Unless otherwise indicated, the practice of the present invention will employ conventional chemical, biochemical, molecular biological, microbiological, and immunological techniques, which are all within the capabilities of those 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, Second Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al., (1995 and periodic supplements), Current Protocols in Molecular Biology, Chapters 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; and Lilley, D.M., and Dahlberg, J.E., 1992, Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. These general texts are hereby incorporated by reference.

[0089] Example

[0090] Example 1 - Study of the synbiotic effect of the combination of Bifidobacterium longum subsp. infantis LMG 11588 with a mixture of 2FL or 5HMO Effect

[0091] Materials and methods

[0092] Fecal samples were collected from 10 donors in Belgium, approximately 3 months old. Four donors were delivered by cesarean section and six donors were delivered vaginally. One donor was breastfed and the other nine donors were fed infant formula in the week before sampling. Fecal suspensions were prepared, mixed with cryoprotectant, aliquoted, flash-frozen, and then stored at -80 °C. Immediately before the experiment, the fecal samples were thawed and used in the experiment right away.

[0093] The fecal material was used for short-term batch fermentation experiments simulating the infant colon. These experiments represent a simplified simulation of a continuous simulator of the human microbial ecosystem. At the start of the short-term colon incubation, the test components were added to a sugar-depleted nutrient medium containing colon basal nutrients. Since the nutrients in this sugar-depleted nutrient medium would also be fermented by the colonic microbiota, a blank containing only the sugar-depleted nutrient medium (without products) was included for each donor. Finally, the fecal inoculum from the infant donors was added. Five treatments and one blank were tested for each donor, resulting in 60 independent experiments. The treatments were as follows:

[0094] 1. Blank (control)

[0095] 2. Single HMO (1.3 g / L)

[0096] 3. 5-HMO mixture (2.5 g / L)

[0097] 4. Bifidobacterium longum subsp. infantis LMG 11588 (1x10 7 CFU / ml)

[0098] 5. Single HMO (1.3 g / L) + Bifidobacterium longum subsp. infantis LMG 11588 (1×10 7 CFU / ml)

[0099] 6. 5-HMO mixture (2.5 g / L) + Bifidobacterium longum subsp. infantis LMG 11588 (1×10 7 CFU / ml)

[0100] The single HMO was 2'-fucosyllactose (2FL). The HMO mixture consisted of 5 HMOs, and the proportions in the dry mixture were as follows: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL, and 14% DFL. Thus, the addition rate of 2FL was the same in the single HMO and HMO mixture treatments.

[0101] The reactor was incubated for 48 hours at 37 °C, with shaking and under anaerobic conditions. The incubation was carried out in a completely independent reactor with a volume high enough to ensure not only robust microbial fermentation but also allow collection of multiple samples over time. Sample collection enabled assessment of metabolite production and thus understanding of the complex microbial interactions taking place.

[0102] The production of short-chain fatty acids (SCFAs) was evaluated at the start of incubation and at 6 hours, 24 hours, and 48 hours later, etc. The pattern of SCFA production is an assessment of microbial carbohydrate metabolism.

[0103] Changes in the microbial composition were analyzed at the start of incubation and at 24 hours and 48 hours of incubation. Samples were analyzed using a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing conversion of the proportional values obtained with Illumina into absolute cell counts.

[0104] Two donors (vaginal delivery donor and formula-fed donor) were excluded from the result evaluation because the sequencing results indicated technical or analytical problems.

[0105] Results

[0106] Figure 1 Shown are the short-chain fatty acid (SCFA) concentrations obtained in colonic fermentations averaged over 8 fecal donors. When only Bifidobacterium longum subsp. infantis LMG 11588 was added, there was no increase in SCFAs compared to the control (no HMO, no Bifidobacterium infantis). With the use of the single HMO 2FL, an increase relative to the control was observed; in the case of using an HMO mixture, this increase was even greater. However, when Bifidobacterium longum subsp. infantis was added on top of the single HMO or HMO mixture, an additional increase in SCFA concentration was achieved compared to not adding Bifidobacterium longum subsp. infantis. The combined effect of the single HMO or HMO mixture with Bifidobacterium longum subsp. infantis relative to the control was greater than the sum of the effects of the individual components relative to the control, thus showing a distinct synergistic (synbiotic) effect. The greatest increase in SCFAs could be obtained with the combination of the HMO mixture and Bifidobacterium longum subsp. infantis.

[0107] Figure 2 Shown are the cell counts of the Bifidobacteriaceae obtained in colonic fermentations averaged over 8 fecal donors. Bifidobacterium longum subsp. infantis LMG 11588 at 1x10 7The levels of CFU / ml were added to the variants to which it was added. When only Bifidobacterium longum subsp. infantis was added, a small increase in Bifidobacteriaceae was observed compared to the control (no HMO, no Bifidobacterium infantis). Using the single HMO 2FL, an increase in Bifidobacteriaceae was observable relative to the control; in the case of using an HMO mixture, this increase was even greater. However, when Bifidobacterium longum subsp. infantis was added on top of the single HMO or the HMO mixture, an additional increase in Bifidobacteriaceae was achieved compared to not adding Bifidobacterium longum subsp. infantis. For the combination of the HMO mixture and Bifidobacterium longum subsp. infantis, the increase in Bifidobacteriaceae relative to the control was greater than the individual effect of either component relative to the control, thus showing a distinct synergistic (synbiotic) effect. Therefore, Bifidobacterium longum subsp. infantis LMG 11588 has grown and / or its addition has stimulated the growth of other members of Bifidobacteriaceae. Therefore, the combination of the HMO mixture and Bifidobacterium longum subsp. infantis most effectively increases the number of Bifidobacteriaceae. The overall results are related to the SCFA data shown above.

[0108] Example 2 - Study of the synbiotic effect of the combination of two different strains of Bifidobacterium longum subsp. infantis with a mixture of 5HMO Effect

[0109] Materials and methods

[0110] Feces were collected from a 3-month-old infant donor in Belgium. This donor was different from the 10 donors in Example 1. This experiment was similar to Example 1, but all colonic fermentations were performed in triplicate and the test components were different. Five treatments and one blank were tested, resulting in a total of 18 experiments. The treatments were as follows:

[0111] 1. Blank (control)

[0112] 2. 5HMO mixture (2.5 g / L)

[0113] 3. Bifidobacterium longum subsp. infantis A (1E+07 cfu / ml)

[0114] 4. Bifidobacterium longum subsp. infantis C (1E+07 cfu / ml)

[0115] 5. Bifidobacterium longum subsp. infantis A (1E+07 cfu / ml) + 5HMO mixture (2.5 g / L)

[0116] 6. Bifidobacterium longum subsp. infantis C (1E+07 cfu / ml) + 5HMO mixture (2.5 g / L)

[0117] Bifidobacterium 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 Bifidobacterium longum subsp. infantis LMG 11588. The strain Bifidobacterium longum subsp. infantis C has more than 99.9% ANI with Bifidobacterium longum subsp. infantis LMG 11588 and is thus closely related to the LMG 11588 strain.

[0118] The HMO mixture consists of 5 HMOs, and the proportions in the dry mixture are as follows: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL, and 14% DFL.

[0119] The pH and short-chain fatty acid (SCFA) production were evaluated at the start of incubation and after 6 hours, 24 hours, and 48 hours, etc.

[0120] The changes in the microbial composition were analyzed at the start of incubation and after 24 hours and 48 hours of incubation. The samples were analyzed using a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing the proportion values obtained with Illumina to be converted to absolute cell counts.

[0121] Results

[0122] Figure 3 and Figure 4 showed that the addition of either Bifidobacterium longum subsp. infantis strain alone had no effect on acidification or SCFA production in this donor. The addition of the HMO mixture alone led to a significant increase in SCFAs. The combination of the HMO mixture with either Bifidobacterium longum subsp. infantis strain further increased SCFA production. The best effect was achieved with the combination of the HMO mixture and Bifidobacterium longum subsp. infantis C (a strain with more than 99.9% ANI with Bifidobacterium longum subsp. infantis LMG 11588). The effect of the combination of the HMO mixture and Bifidobacterium longum subsp. infantis relative to the control (no HMO, no Bifidobacterium infantis) was greater than the sum of the effects of the single components relative to the control, thus showing a distinct synbiotic effect. This synbiotic effect was more significant when using Bifidobacterium longum subsp. infantis C compared to using Bifidobacterium longum subsp. infantis A.

[0123] The sequencing data showed that no detectable Bifidobacterium longum subsp. infantis-related operational taxonomic units were present in the inoculum in the infant donor, but they could be detected after the addition of the two strains. 48-hour colon simulation ( Figure 5)Afterwards, when HMO was not added, for both strains, Bifidobacterium longum subsp. infantis remained at a low level. However, for the combination of Bifidobacterium longum subsp. infantis C and the HMO mixture, a large increase in the Bifidobacterium longum subsp. infantis - related operational taxonomic units was detected, indicating a strong growth stimulation of this bacterial species. This again shows the synergistic effect that can be obtained with the combination of the HMO mixture and Bifidobacterium longum subsp. infantis.

[0124] Example 3 - Study of the synbiotic effect of the combination of Bifidobacterium longum subsp. infantis LMG 11588 with a mixture of 6HMO in the presence and absence of Bifidobacterium lactis in an infant and toddler colon model Effect

[0125] Materials and methods

[0126] Fecal samples were collected from 6 donors (infants) approximately 3 months old and from the same donors (toddlers) approximately 12 months old in Belgium. 3 donors were born by cesarean section and 3 donors were born vaginally. One donor was breastfed and the other 5 donors were formula - fed with infant formula one week before the first sampling. This experiment was similar to Example 1. Five treatments and one blank were tested for each donor at both ages, resulting in 72 independent experiments. The treatments were as follows:

[0127] 1. Blank (control)

[0128] 2. Bifidobacterium longum subsp. infantis LMG 11588 (1x10 8 CFU / ml)

[0129] 3. Bifidobacterium animalis subsp. lactis CNCM I - 3446 (1x10 8 CFU / ml)

[0130] 4. 6 - HMO mixture (2.5 g / L)

[0131] 5. 6 - HMO mixture (2.5 g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0132] (1×10 8 CFU / ml)

[0133] 6. 6 - HMO mixture (2.5 g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0134] (1×10 8 CFU / ml) + Bifidobacterium animalis subsp. lactis CNCM I - 3446

[0135] (1×10 8 CFU / ml)

[0136] The HMO mixture consists of 6 HMOs and their proportions in the dry mixture are as follows: for the infant study, 16% LNT, 8% 6SL, 6% 3SL, 49% 2FL, 7% DFL, and 14% 3FL; for the toddler study, 10% LNT, 5% 6SL, 14% 3SL, 29% 2FL, 4% DFL, and 37% 3FL.

[0137] The production of short-chain fatty acids (SCFAs) was evaluated at the start of incubation and after 6 h, 24 h, and 48 h, etc.

[0138] Results

[0139] Figure 6 Shown are the short-chain fatty acid (SCFA) concentrations obtained in the colonic fermentation averaged over 6 fecal donors of two different ages at 24 h. Compared to the blank control, for both models, i.e., infants and toddlers, no significant increase in SCFAs occurred when only Bifidobacterium longum subsp. infantis LMG 11588 or Bifidobacterium animalis subsp. lactis CNCM I-3446 was added. Using the 6HMO mixture, an increase in SCFA concentration relative to the blank control was observed; the effect was stronger in the toddler model than in the infant model. However, when Bifidobacterium longum subsp. infantis was additionally added to the 6HMO mixture, an additional increase in SCFA concentration was achieved compared to not adding Bifidobacterium longum subsp. infantis. The additional effect of Bifidobacterium longum subsp. infantis was stronger in the infant model than in the toddler model. In both models, the combined effect of the HMO mixture and Bifidobacterium longum subsp. infantis relative to the control was greater than the sum of the effects of the individual components relative to the blank control, thus showing a distinct synergistic (synbiotic) effect. When Bifidobacterium animalis subsp. lactis was additionally added, the synbiotic effect of the HMO mixture and Bifidobacterium longum subsp. infantis was maintained; the addition of Bifidobacterium animalis subsp. lactis led to a slight further increase in SCFA concentration. The greatest increase in SCFAs in both the infant and toddler models was obtained with the combination of the 6HMO mixture with the following strains: the combination with Bifidobacterium longum subsp. infantis and the combination with Bifidobacterium animalis subsp. lactis.

Claims

1. A composition for infants or toddlers, the composition comprising at least one probiotic strain and a prebiotic oligosaccharide mixture, the probiotic strain being Bifidobacterium, the prebiotic oligosaccharide mixture consisting of 2'-fucosyllactose (2FL), lactodifucotetraose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL); wherein the Bifidobacterium comprises Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis ), or a combination of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis ), CNCM I-3446 and Bifidobacterium longum subsp. infantis ( longum subsp. infantis ); wherein at least one of the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588.

2. The composition according to claim 1, wherein the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucotetraose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

3. The composition according to claim 1, wherein the Bifidobacterium is a combination of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. Lactis ), CNCM I-3446 and Bifidobacterium longum subsp. infantis LMG11588, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucotetraose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

4. The composition according to any one of claims 1-3, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42% to 71% by weight of 2FL; ii. 10% to 40% by weight of LNT; iii. 4% to 14% by weight of DFL; and iv. 9% to 31% by weight of a combination of 6SL and 3SL.

5. The composition according to claim 4, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42% to 71% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 5% to 10% by weight of DFL; and iv. 10% to 28% by weight of a combination of 6SL and 3SL.

6. The composition according to any one of claims 1 - 3, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 26% to 65% by weight of 2FL; ii. 8% to 30% by weight of LNT; iii. 3% to 11% by weight of DFL; iv. 7% to 23% by weight of a combination of 6SL and 3SL; and v. 12% to 38% by weight of 3FL.

7. The composition according to claim 6, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 32% to 54% by weight of 2FL; ii. 11% to 20% by weight of LNT; iii. 4% to 8% by weight of DFL; iv. 8% to 22% by weight of a combination of 6SL and 3SL; and v. 17% to 31% by weight of 3FL.

8. The composition according to any one of claims 1 - 3, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50% to 70% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 4% to 14% by weight of DFL; and iv. 9% to 31% by weight of a combination of 6SL and 3SL.

9. The composition according to claim 8, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50% to 70% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 5% to 10% by weight of DFL; and iv. 10% to 28% by weight of a combination of 6SL and 3SL.

10. The composition according to any one of claims 1 - 3, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 20% to 60% by weight of 2FL; ii. 8% to 30% by weight of LNT; iii. 2% to 12% by weight of DFL; iv. 7% to 23% by weight of a combination of 6SL and 3SL; and v. 10% to 40% by weight of 3FL.

11. The composition according to claim 10, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 25% to 55% by weight of 2FL; ii. 11% to 20% by weight of LNT; iii. 4% to 8% by weight of DFL; iv. 8% to 22% by weight of a combination of 6SL and 3SL; and v. 11% to 37% by weight of 3FL.

12. The composition according to any one of claims 1 - 3, wherein the composition is a nutritional composition selected from the list consisting of: infant formula, stage 1 infant formula, follow - on formula or stage 2 infant formula, baby food, infant cereal composition, growing milk, fortifier, or supplement.

13. The composition according to claim 12, wherein the fortifier is a human milk fortifier.

14. Use of the composition according to any one of claims 1 - 13 in the preparation of a medicament for: i) preventing and / or treating bacterial infections in infants or young children; ii) modulating the microbiota of infants or young children; and / or iii) preventing and / or treating allergic reactions in infants or young children.

15. The use according to claim 14, wherein the medicament is for modulating the microbiota of infants or young children, and administering the medicament results in an increase in the abundance of Bifidobacteriaceae ( Bifidobacteriaceae ) and / or Bifidobacterium longum subsp. infantis.

16. The use according to claim 14, wherein the medicament is for modulating the microbiota of infants or young children; and / or preventing and / or treating allergic reactions in such infants or young children by increasing the production of gut short - chain fatty acids (SCFAs) in the infants or young children.

17. The use according to any one of claims 14 - 16, wherein the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto - N - tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

18. The use according to any one of claims 14 - 16, wherein the Bifidobacterium is a combination of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. Lactis ) CNCM I - 3446 and Bifidobacterium longum subsp. infantis LMG 11588, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto - N - tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

19. The use according to any one of claims 14 - 16, wherein the prebiotic oligosaccharide mixture consists essentially of the following substances: i. 42% to 71% by weight of 2FL; ii. 10% to 40% by weight of LNT; iii. 4% to 14% by weight of DFL; and iv. 9% to 31% by weight of the combined 6SL and 3SL.

20. Use according to claim 19, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42% to 71% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 5% to 10% by weight of DFL; and iv. 10% to 28% by weight of the combined 6SL and 3SL.

21. Use according to any one of claims 14 - 16, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 26% to 65% by weight of 2FL; ii. 8% to 30% by weight of LNT; iii. 3% to 11% by weight of DFL; iv. 7% to 23% by weight of the combined 6SL and 3SL; and v. 12% to 38% by weight of 3FL.

22. Use according to claim 21, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 32% to 54% by weight of 2FL; ii. 11% to 20% by weight of LNT; iii. 4% to 8% by weight of DFL; iv. 8% to 22% by weight of the combined 6SL and 3SL; and v. 17% to 31% by weight of 3FL.

23. Use according to any one of claims 14 - 16, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50% to 70% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 4% to 14% by weight of DFL; and iv. 9% to 31% by weight of the combined 6SL and 3SL.

24. Use according to claim 23, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50% to 70% by weight of 2FL; ii. 14% to 26% by weight of LNT; iii. 5% to 10% by weight of DFL; and iv. 10% to 28% by weight of the combined 6SL and 3SL.

25. Use according to any one of claims 14 - 16, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 20% to 60% by weight of 2FL; ii. 8% to 30% by weight of LNT; iii. 2% to 12% by weight of DFL; iv. 7% to 23% by weight of the combined 6SL and 3SL; and v. 10% to 40% by weight of 3FL.

26. Use according to claim 25, wherein the prebiotic oligosaccharide mixture consists essentially of the following substances: i. 25% to 55% by weight of 2FL; ii. 11% to 20% by weight of LNT; iii. 4% to 8% by weight of DFL; iv. 8% to 22% by weight of the combined 6SL and 3SL; and v. 11% to 37% by weight of 3FL.

27. Use according to any one of claims 14 - 16, wherein the composition is a nutritional composition selected from the list consisting of: infant formula, stage 1 infant formula, follow - on formula or stage 2 infant formula, baby food, infant cereal composition, growing-up milk, fortifier, or supplement.

28. Use according to claim 27, wherein the fortifier is a human milk fortifier.

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