Mixtures of HMO and bifidobacteria

Abstract

The present invention relates to a composition for infants or young children comprising at least one probiotic strain and a prebiotic mixture of human milk oligosaccharides, said probiotic strain being Bifidobacteria, the present invention relates to a composition comprising a prebiotic mixture of human milk oligosaccharides consisting of 2 '-fucosyllactose (2FL), milk difucosyltetraose / difucosyllactose (DFL), milk-N-tetraose (LNT), 6'-sialyllactose (6SL), 3 '-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL), which composition is useful for (i) preventing and / or treating a bacterial infection in an infant or a young child; (ii) modulate the microbiota in the infant or young child; and / or iii) preventing and / or treating allergies in infants or young children.

Description

[0001] This application is a divisional application of Chinese patent application 202280010715.8 (International Application No. PCT / EP2022 / 051336), whose application date is January 21, 2022 and whose invention name is “Mixture of HMO and Bifidobacterium”. Technical Field

[0002] The present invention relates to a composition for infants or young children, comprising at least one probiotic strain belonging to the group of bifidobacteria and a prebiotic mixture of human milk oligosaccharides (HMOs), the prebiotic mixture of HMOs consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL) and lacto-N-tetraose (LNT), and optionally 3-fucosyllactose (3FL), and to the use of these mixtures in human health. Background Art

[0003] In recent years, human milk oligosaccharides (HMOs) have attracted considerable attention due to their roles in a variety of biological processes in humans. 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).

[0004] It has been shown that the symbiotic microbial communities in the human digestive tract (called the microbiome) play an important role in health and disease. When the composition of the intestinal microbiome is out of balance, the human host may suffer the consequences. Recent studies have shown that an imbalance in the intestinal microbiome is a cause of a variety of 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 microbiome, and they are increasingly attracting attention for treating one or more of these diseases.

[0005] The infancy of life, especially the first few weeks, 3 months, 6 months or 12 months, is a critical period for establishing a balanced gut microbiota.

[0006] It is known that the regulation of the gut microbiota during infancy can have a significant impact on the body's future health. For example, the gut microbiome can influence the development of a strong immune system, normal growth, and even the development of obesity later in life.

[0007] However, during the development of an infant, the intestinal microbiome and its evolution are a delicate balance between the presence and proliferation (number) of many intestinal bacterial populations. Regarding the effect of intestinal bacteria on the overall health of the infant, some intestinal bacteria are classified as "usually positive", while other intestinal bacteria are "usually negative" (or pathogenic). Compared to breastfed infants, certain types of "overall positive" bacteria (such as Bifidobacterium) may be insufficient in infants fed conventional infant formula. Similarly, some bacterial populations are considered pathogenic and should be kept low in the intestinal microbiome.

[0008] Bifidobacterium longum subsp. infantis has been shown to dominate the gut microbiota of breastfed infants and to benefit the host by accelerating the maturation of immune responses, 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 associated with chronic diseases, including asthma and obesity, and reduced vaccine response. Researchers have hypothesized that the loss of Bifidobacterium species in the infant gut is associated with an increased incidence of allergic and autoimmune diseases in developed populations.

[0009] Increasing the abundance of Bifidobacterium species, or specifically Bifidobacterium longum subsp. infantis , in the gut microbial ecosystem by exogenous administration may be difficult to achieve, especially in infants fed infant formula.

[0010] The key metabolites produced by Bifidobacterium longum subsp. infantis are lactic acid and acetic acid. Lactic acid and acetic acid help lower fecal pH and provide resistance to colonization of 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 microbiome that produce other short-chain fatty acids (SCFAs). Microbial fermentation of dietary fiber in the colon produces SCFAs in particular. These colonic fermentations are known to play a role in energy supply, as nutritional factors and immunomodulation, and increasing evidence suggests that SCFAs also play an important physiological effect on several organs, including the brain. A high abundance of SCFA-producing bacteria, particularly butyrate-producing bacteria, has been shown to be 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)).

[0011] Due to the loss of Bifidobacterium species in the infant gut and low breastfeeding rates, there is a need to provide infants with both HMOs and HMO-utilizing bacteria (such as Bifidobacterium longum subsp. infantis) to support a healthy microbiome for long-term health. Summary of the Invention

[0012] A first aspect of the present invention relates to a composition for infants or young children, comprising at least one probiotic strain and a prebiotic mixture of human milk oligosaccharides, wherein the probiotic strain is a bifidobacterium and the prebiotic mixture of human milk oligosaccharides consists of 2'-fucosyllactose (2FL), 3'-sialyllactose (3SL), difucosyllactose (DFL), 6'-sialyllactose (6SL), lacto-N-tetraose (LNT) and optionally 3-fucosyllactose (3FL).

[0013] The present inventors have surprisingly found that a synergistic effect is obtained when the HMO mixture is used in combination with Bifidobacteria, especially in combination with probiotics, such as Bifidobacterium longum subsp. infantis synergistically promotes a healthier intestinal environment with the HMO mixture due to the enhancement of beneficial fermentation metabolites such as short-chain fatty acids and the stimulation of the growth of healthy commensal intestinal bacteria such as Bifidobacteria.

[0014] 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), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

[0015] 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), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

[0016] In one aspect, the present invention relates to a composition for infants or young children, comprising at least one probiotic strain and a prebiotic oligosaccharide mixture, wherein the probiotic strain is a bifidobacterium, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL);

[0017] wherein the Bifidobacterium comprises Bifidobacterium longum subsp. infantis or a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis;

[0018] wherein at least one of the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an average nucleotide identity (ANI) of at least 99.9% thereto.

[0019] In one aspect, the invention is directed to a nutritional composition selected from the list consisting of an infant formula, a step 1 infant formula, a follow-on formula, or a step 2 infant formula, a baby food, an infant cereal composition, a growing-up milk, a fortifier such as a human milk fortifier, or a supplement.

[0020] In one aspect, the present invention relates to a nutritional composition as defined above for use in: 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 allergies in infants or young children.

[0021] In one aspect, the present invention relates to a composition as defined above for use in modulating the microbiota of an infant or young child, and administration of said composition results in an increased abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis.

[0022] In one aspect, the present invention relates to a composition as defined above for use in modulating the microbiota of an infant or young child; and / or preventing and / or treating allergies in such infant or young child by increasing the production of short-chain fatty acids (SCFAs) in the intestine of such infant or young child.

[0023] In one aspect, the present invention relates to a method of modulating the microbiota of an infant or young child to increase the abundance of Bifidobacteriacea and / or Bifidobacterium longum subsp. infantis, the method comprising:

[0024] - administering a composition as defined to an infant or young child. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2: Mean total short-chain fatty acid concentrations of 8 stool donors after 24 hours. Mean concentrations increased after supplementation with a single HMO (2FL). A greater increase occurred when supplementing with a mixture of HMOs rather than a single HMO. A further incremental increase was achieved when supplementing with Bifidobacterium infantis B. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG 11588.

[0026] Figure 2 Figure 2: Mean proliferation of operational taxonomic units of the family Bifidobacteriaceae from eight stool donors after 24 hours. Mean proliferation increased after supplementation with a single HMO (2FL). A greater increase occurred when supplementing with a mixture of HMOs rather than a single HMO. A further incremental increase was achieved with supplementation with Bifidobacterium infantis B. Bifidobacterium infantis B is Bifidobacterium longum subsp. infantis LMG11588.

[0027] Figure 3 : Complete acidification after 48 hours (average of three replicates). Average concentrations increased after supplementation with the HMO mixture. A continuous increase was achieved when B. infantis A and B. infantis C were supplemented separately in the presence of the HMO mixture. B. infantis A is B. longum subsp. infantis ATCC 15697. B. infantis C is a strain with an ANI greater than 99.9% of that of B. longum subsp. infantis LMG 11588.

[0028] 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. A continuous increase was obtained when B. infantis A and B. infantis C were supplemented separately in the presence of the HMO mixture. B. infantis A is B. longum subsp. infantis ATCC 15697. B. infantis C is a strain with an ANI greater than 99.9% of that of B. longum subsp. infantis LMG 11588.

[0029] Figure 5 : Reproduction of operational taxonomic units of B. infantis after 48 hours (average of three replicates). The greatest increase was achieved when supplementing with B. infantis C in the presence of the HMO mixture. B. infantis A is B. longum subsp. infantis ATCC 15697. B. infantis C is a strain with an ANI greater than 99.9% of that of B. longum subsp. infantis LMG 11588.

[0030] Figure 6 : Mean total short-chain fatty acid concentrations of 6 stool donors at infant (3 months old) and toddler (12 months old) age after 24 hours. When supplemented with Bifidobacterium infantis B without HMOs or Bifidobacterium lactis without HMOs, the mean total short-chain fatty acid concentrations did not increase significantly compared to the blank. The mean concentrations increased when supplemented with the HMO mixture alone. Further increases were obtained when supplemented with Bifidobacterium infantis B with the HMO mixture, and with a combination of Bifidobacterium infantis B and Bifidobacterium lactis with the HMO mixture. Horizontal dashed lines are added to represent the blank content of total short-chain fatty acid concentrations in infants and toddlers. Bifidobacterium infantis B is Bifidobacterium longum infantis subsp. LMG 11588. Bifidobacterium lactis is Bifidobacterium animalis subsp. lactis CNCM 1-3446. DETAILED DESCRIPTION

[0031] As used herein, the following terms have the following meanings.

[0032] The term "infant" refers to a child under the age of 12 months.

[0033] The expression "young children" refers to children between the ages of one and three years old, also known as toddlers.

[0034] The expressions "composition for an infant or a young child" and "composition to be administered to an infant or a young child" are used interchangeably.

[0035] In some embodiments, the composition comprising the HMO mixture according to the present invention is a nutritional composition. The expression "nutritional composition" refers to a composition that provides nutrients to an individual. Such nutritional compositions are typically administered orally or intravenously and typically include a lipid or fat source and a protein source. In specific embodiments, the nutritional composition is a synthetic nutritional composition.

[0036] As used herein, the expression "infant formula" refers to a food intended specifically for the nutrition of infants during the first few months of life and which itself meets the multiple nutritional needs of such persons (in accordance with Article 2(c) of Directive 91 / 321 / EEC and 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formula and follow-on formula). It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and in Special Products for Infants (including foods for special medical purposes). The expression "infant formula" covers both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0037] "Second infant formula" or "follow-on formula" is given from the 6th month onwards. Infant formula constitutes the main liquid element of the gradually diversified diet of such people.

[0038] The expression "baby food" refers to a food material intended specifically for the nutrition of infants or young children during the first year of life.

[0039] The expression "infant cereal composition" refers to a foodstuff which is intended specifically for the nutrition of an infant or young child during the first year of life.

[0040] 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 present invention is a milk fortifier. In such embodiments, the aqueous composition of the present invention can be packaged in single doses.

[0041] The term "supplement" refers to a food product containing specific nutrients and / or probiotics, which is intended to supplement the diet.

[0042] In one embodiment, the present invention is a supplement in the form of a powder or oil. In such an embodiment, the composition of the present invention is administered as a stand-alone composition, or in combination with other ingredients such as maltodextrin.

[0043] The term "growing-up milk" (or GUM) refers to a milk-based beverage, usually with added vitamins and minerals, which is intended for infants or children.

[0044] HMOs (human milk oligosaccharides) are oligosaccharide structures naturally present in human milk. HMOs added to food are usually obtained from cow's milk by chemical synthesis or through biotechnological production methods.

[0045] The term "SCFA" means short-chain fatty acids.

[0046] The expression "increased SCFA production" means that the amount of systemic and / or colonic SCFAs is higher in an individual fed a nutritional composition according to the invention compared to a standard composition. SCFA production can be measured by techniques known to the skilled person, such as by gas-liquid chromatography.

[0047] "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 described herein: Yoon SH, Ha SM, Lim J, Kwon S, Large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017 Oct; 110(10): 1281-1286. In embodiments of the present invention, strain Bifidobacterium longum subsp. infantis LMG 11588 (also known as ATCC 17930) represents a reference genome to which microbial genomes are compared. An example of a microbial genome with at least 99.9% ANI to 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 invention, the B. longum subsp. infantis strain does not have potentially transferable antibiotic resistance.

[0048] The HMO and Bifidobacterium mixture of the present invention can:

[0049] i) increase the intrinsic intestinal abundance of Bifidobacteria, and

[0050] ii) increase intestinal SCFA production and prolong the reduction in fecal pH, and

[0051] iii) Supports the growth of Bifidobacterium longum subsp. infantis.

[0052] The effect of the HMO and Bifidobacterium mixture on Bifidobacteria and SCFAs is greater than would be expected from the effect of either Bifidobacteria or the HMO mixture alone.Thus, the HMO and Bifidobacterium mixture of the present invention has a surprising synergistic effect.

[0053] These effects can make the intestinal environment less susceptible to invasion and overgrowth of harmful bacteria in the infant's intestine. These effects of the HMO and Bifidobacterium mixture can 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.

[0054] In one embodiment, the Bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an average nucleotide identity (ANI) of at least 99.9% thereto.

[0055] In one embodiment, the Bifidobacterium is Bifidobacterium animalis subsp. lactis CNCM 1-3446.

[0056] In one embodiment, the HMO mixture of the present invention consists essentially of:

[0057] i. 34 to 85% by weight, preferably 42 to 71% by weight, of 2FL;

[0058] ii. 10 to 40 wt%, preferably 14 to 26 wt% LNT;

[0059] iii. 4 to 14 wt%, preferably 5 to 10 wt% DFL; and

[0060] iv. 9 to 31 wt%, preferably 10 to 28 wt% of 6SL and 3SL combined.

[0061] In another embodiment, the HMO mixture of the present invention consists essentially of:

[0062] i. 26 to 65 wt%, preferably 32 to 54 wt% 2FL;

[0063] ii. 8 to 30 wt%, preferably 11 to 20 wt% LNT;

[0064] iii. 3% to 11% by weight, preferably 4% to 8% by weight, of DFL;

[0065] iv. 7 to 23 wt%, preferably 8 to 22 wt% of 6SL and 3SL combined; and

[0066] v. 12 to 38 wt%, preferably 17 to 31 wt% 3FL.

[0067] In one embodiment, the HMO mixture of the present invention consists essentially of:

[0068] i. 40% to 80% by weight, preferably 50% to 70% by weight, of 2FL;

[0069] ii. 10 to 40 wt%, preferably 14 to 26 wt% LNT;

[0070] iii. 4 to 14 wt%, preferably 5 to 10 wt% DFL; and

[0071] iv. 9 to 31 wt%, preferably 10 to 28 wt% of 6SL and 3SL combined.

[0072] In another embodiment, the HMO mixture of the present invention consists essentially of:

[0073] i. 20% to 60% by weight, preferably 25% to 55% by weight of 2FL;

[0074] ii. 8 to 30 wt%, preferably 11 to 20 wt% LNT;

[0075] iii. 2 to 12 wt%, preferably 4 to 8 wt% DFL;

[0076] iv. 7 to 23 wt%, preferably 8 to 22 wt% of 6SL and 3SL combined; and

[0077] v. 10 to 40 wt%, preferably 11 to 37 wt% 3FL.

[0078] The HMO and Bifidobacterium mixture may be administered to a human in any suitable form such as, for example, a nutritional composition in unit dosage form (eg, tablets, capsules, powder sachets, etc.).

[0079] The HMO and bifidobacterium mixture of the present 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 agent or supplement for an infant or young child. The nutritional composition can be, for example, an infant formula, a stage 1 infant formula, a follow-on infant formula, or a stage 2 infant formula, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, or a supplement. In some specific embodiments, the composition of the present invention is an infant formula, fortifier, or supplement intended for the first 4 months or 6 months of age. In a preferred embodiment, the nutritional composition of the present 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 infant formula / stage 2 infant formula fortifier).

[0080] Macronutrients such as edible fats, carbohydrates, and proteins may also be included in such nutritional compositions. Edible fats include, for example, coconut oil, soybean oil, and mono- 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 (e.g., calcium, phosphorus, potassium, sodium, chlorine, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, C, and B complex) may also be included in such nutritional compositions.

[0081] The nutritional composition may be prepared in any suitable manner. The composition will now be described by way of example.

[0082] For example, a formula food such as an infant formula can be prepared by blending a protein source, a carbohydrate source, and a fat source in an appropriate ratio. 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. Water (preferably water subjected to reverse osmosis) can then be mixed 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 ingredients. Commercially available liquefiers can be used to form a liquid mixture.

[0083] 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 ingredients can also be added at this stage as needed.

[0084] The liquid mixture is then homogenized, for example in two stages.

[0085] The liquid mixture can then 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 done by steam injection, autoclave, or heat exchanger (e.g., plate heat exchanger).

[0086] The liquid mixture is then cooled, for example, by rapid cooling, to a temperature between about 60°C and about 85°C. The liquid mixture is then homogenized again, for example, in two stages, with the pressure in the first stage being between about 10 MPa and about 30 MPa, and the pressure in the second stage being between about 2 MPa and about 10 MPa. The homogenized mixture can then be further cooled to allow the addition of any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture are then conveniently adjusted.

[0087] If the final product is to be a powder, the homogenized mixture is transferred to a suitable drying apparatus, such as a spray dryer or freeze dryer, and converted to a powder. The moisture content of the powder should be less than about 5% by weight. The HMO mixture of the present invention can also or alternatively be added at this stage by dry mixing with the probiotic strain in powdered form.

[0088] Preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0089] 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 one of ordinary skill in the art and are described in the literature. See, e.g., Sambrook, J., Fritsch, E.F., and Maniatis, T., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., 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.

[0015] The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in the field of DNA synthesis. The present invention relates to the synthesis of oligonucleotides in a practical way, for example in

[0090] Example

[0091] Example 1 - Study of the synbiotic effect of Bifidobacterium longum subsp. infantis LMG 11588 in combination with 2FL or 5HMO mixtures Element effect

[0092] Materials and methods

[0093] Feces were collected from 10 donors in Belgium, approximately 3 months of age. Four donors were born by cesarean section, and six by vaginal delivery. One donor was breastfed, and the other nine had been fed infant formula for the week prior to sampling. Fecal suspensions were prepared, mixed with a cryoprotectant, aliquoted, snap-frozen, and then stored at -80°C. Fecal samples were thawed immediately before the experiment and used immediately.

[0094] Fecal material was used to perform short-term batch fermentation experiments simulating the infant colon. These experiments represent the human microbial ecosystem ( ) was used as a simplified simulation of a continuous simulator. At the beginning of the short-term colonic incubation, the test ingredients were added to a sugar-depleted nutrient medium containing basal nutrients for the colon. Because the nutrients of this sugar-depleted nutrient medium will also be fermented by the colonic microbiota, a blank containing only sugar-depleted nutrient medium (without product) was included for each donor. Finally, a fecal inoculum from an infant donor was added. Five treatments and one blank were tested per donor, resulting in 60 independent experiments. The treatments were as follows:

[0095] 1. Blank (control)

[0096] 2. Single HMO (1.3 g / L)

[0097] 3.5HMO mixture (2.5g / L)

[0098] 4. Bifidobacterium longum subsp. infantis LMG 11588 (1x10 7 CFU / ml)

[0099] 5. Single HMO (1.3g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0100] (1×10 7 CFU / ml)

[0101] 6.5HMO mixture (2.5g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0102] (1×10 7 CFU / ml)

[0103] The single HMO was 2'-fucosyllactose (2FL). The HMO mix consisted of five HMOs in the following proportions in the dry mix: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL, and 14% DFL. Therefore, the addition rate of 2FL was the same for both the single HMO and HMO mix treatments.

[0104] The reactors were incubated at 37°C for 48 hours under shaking and anaerobic conditions. Incubations were performed in fully self-contained reactors with sufficiently high volumes to not only ensure robust microbial fermentation but also allow for the collection of multiple samples over time. Sample collection enabled the assessment of metabolite production and, therefore, understanding of the complex microbial interactions occurring.

[0105] The production of short chain fatty acids (SCFAs) was assessed at the beginning of the incubation and after 6, 24 and 48 hours, etc. The profile of SCFA production is an assessment of microbial carbohydrate metabolism.

[0106] Changes in microbial composition were analyzed at the beginning of incubation and after 24 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 the conversion of ratio values ​​obtained with Illumina to absolute cell counts.

[0107] Two donors (one born by vaginal delivery and one fed infant formula) were excluded from outcome evaluation because sequencing results indicated technical or analytical problems.

[0108] result

[0109] Figure 1 The concentrations of short-chain fatty acids (SCFAs) obtained in the colonic fermentation averaged over 8 stool donors are shown. When only B. longum subsp. infantis LMG 11588 was added, there was no increase in SCFAs compared to the control (no HMO, no B. infantis). With the single HMO 2FL, an increase relative to the control was observed; in the case of the HMO mixture, this increase was even greater. However, when B. longum subsp. infantis was added on top of the single HMO or HMO mixture, an additional increase in SCFA concentration was achieved compared to no addition of B. longum subsp. infantis. The effect of the combination of a single HMO or an HMO mixture with B. 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 clear synergistic (synbiotic) effect. The greatest increase in SCFAs was achieved with the combination of the HMO mixture and B. longum subsp. infantis.

[0110] Figure 2 The cell counts of Bifidobacteriaceae obtained from colonic fermentation averaged from 8 stool donors are shown. 7The level of CFU / ml increased in the variants to which it was added. When only B. longum infantis was added, a small increase in Bifidobacteriaceae was observed compared to the control (no HMO, no B. infantis). With the single HMO 2FL, an increase in Bifidobacteriaceae was observed relative to the control; this increase was even greater with the HMO mixture. However, when B. longum infantis was added on top of a single HMO or HMO mixture, an additional increase in Bifidobacteriaceae was achieved compared to not adding B. longum infantis. For the combination of the HMO mixture and B. longum 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 clear synergistic (synbiotic) effect. Therefore, B. longum infantis LMG 11588 has grown and / or its addition has stimulated the growth of other members of the Bifidobacteriaceae. Therefore, the combination of the HMO mixture and B. longum infantis was most effective in increasing the number of Bifidobacteriaceae. The overall results correlate with the SCFA data shown above.

[0111] Example 2 - Study of a synbiotic combining two different strains of Bifidobacterium longum subsp. infantis with a 5HMO mixture effect

[0112] Materials and methods

[0113] 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:

[0114] 1. Blank (control)

[0115] 2.5HMO mixture (2.5g / L)

[0116] 3. Bifidobacterium longum subspecies infantis A (1E+07 cfu / ml)

[0117] 4. Bifidobacterium longum subspecies infantis C (1E+07 cfu / ml)

[0118] 5. Bifidobacterium longum subspecies infantis A (1E+07 cfu / ml) + 5HMO mixture (2.5 g / L)

[0119] 6. Bifidobacterium longum subspecies infantis C (1E+07 cfu / ml) + 5HMO mixture (2.5 g / L)

[0120] Bifidobacterium longum subsp. infantis A is strain ATCC 15697, which is a typical strain of the subspecies and shares less than 99.9% of the ANI (98.2%) with Bifidobacterium longum subsp. infantis LMG 11588. Strain Bifidobacterium longum subsp. infantis C has an ANI greater than 99.9% with Bifidobacterium longum subsp. infantis LMG 11588 and is therefore closely related to the LMG 11588 strain.

[0121] The HMO mixture consisted of 5 HMOs in the following proportions in the dry mix: 23% LNT, 9% 6SL, 2% 3SL, 52% 2FL, and 14% DFL.

[0122] Among others, pH and short-chain fatty acid (SCFA) production were assessed at the beginning of the incubation and after 6, 24 and 48 hours.

[0123] Changes in microbial composition were analyzed at the beginning of incubation and after 24 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 the conversion of ratio values ​​obtained with Illumina to absolute cell counts.

[0124] result

[0125] Figure 3 and Figure 4 It was shown that the addition of either B. longum subsp. infantis strain alone had no effect on acidification or SCFA production in this donor. The addition of the HMO mixture alone resulted in a significant increase in SCFA. The use of the HMO mixture in combination with either B. longum subsp. infantis strain further increased SCFA production. The best effect was achieved with the combination of the HMO mixture with B. longum subsp. infantis C (a strain with an ANI of over 99.9% with B. longum subsp. infantis LMG 11588). The effect of the combination of the HMO mixture and B. longum subsp. infantis relative to the control (no HMO, no B. infantis) was greater than the sum of the effects of the individual components relative to the control, thus showing a clear synbiotic effect. This synbiotic effect was more pronounced when using B. longum subsp. infantis C than when using B. longum subsp. infantis A.

[0126] Sequencing data showed that no operational taxonomic units of Bifidobacterium longum subsp. infantis were detectable in the infant donor inocula, but were detected after the addition of both strains. Figure 5) after the addition of HMOs, B. longum subsp. infantis remained low for both strains. However, for the combination of B. longum subsp. infantis C and the HMO mixture, a large increase in operational taxonomic units related to B. longum subsp. infantis was detected, indicating a strong growth stimulation of this species. This again illustrates the synergistic effect that can be achieved with the combination of the HMO mixture and B. longum subsp. infantis.

[0127] Example 3 - Study of Bifidobacterium longum subsp. infantis LMG in an infant and toddler colonic model 11588 and Synbiotic effects of combinations of 6HMO mixtures with and without Bifidobacterium lactis

[0128] Materials and methods

[0129] Feces were collected from 6 donors (infants) of about 3 months old in Belgium and from the same donors (toddlers) of about 12 months old. 3 donors were born by caesarean section and 3 donors were born by vaginal delivery. One donor was breastfed and the other 5 donors were fed infant formula one week before the first sampling. The experiment was similar to Example 1. Five treatments and one blank were tested for each donor at two ages, resulting in 72 independent experiments. The treatments were as follows:

[0130] 1. Blank (control)

[0131] 2. Bifidobacterium longum subsp. infantis LMG 11588 (1x10 8 CFU / ml)

[0132] 3. Bifidobacterium animalis subsp. lactis CNCM I-3446 (1x10 8 CFU / ml)

[0133] 4.6HMO mixture (2.5g / L)

[0134] 5.6HMO mixture (2.5g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0135] (1×10 8 CFU / ml)

[0136] 6.6HMO mixture (2.5g / L) + Bifidobacterium longum subsp. infantis LMG 11588

[0137] (1×10 8 CFU / ml)+Bifidobacterium animalis subsp. lactis CNCM I-3446

[0138] (1×10 8 CFU / ml)

[0139] The HMO mixture consisted of six HMOs in the following proportions in the dry mix: 16% LNT, 8% 6SL, 6% 3SL, 49% 2FL, 7% DFL, and 14% 3FL for the infant study; and 10% LNT, 5% 6SL, 14% 3SL, 29% 2FL, 4% DFL, and 37% 3FL for the toddler study.

[0140] Short chain fatty acid (SCFA) production was assessed at the beginning of the incubation and after 6, 24 and 48 hours, among others.

[0141] result

[0142] Figure 6 The concentrations of short-chain fatty acids (SCFAs) obtained from the average colonic fermentation of 6 stool donors of two different ages over 24 hours are shown. Compared to the blank control, for both models, infants and toddlers, no meaningful increase in SCFAs occurred when only Bifidobacterium longum subsp. infantis LMG 11588 or Bifidobacterium animalis subsp. lactis CNCM 1-3446 was added. With 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 effect of the combination 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 clear synergistic (synbiotic) effect. The synbiotic effect of the HMO mixture with Bifidobacterium longum infantis was maintained when Bifidobacterium animalis subsp. lactis was also added; the addition of Bifidobacterium animalis subsp. lactis resulted in a slight further increase in SCFA concentrations. The greatest increase in SCFAs in both the infant and toddler models was achieved with the 6HMO mixture in combination with the following species: Bifidobacterium longum infantis subsp. and Bifidobacterium animalis subsp. lactis.

Claims

1. A composition for infants or young children, comprising at least one probiotic strain and a prebiotic oligosaccharide mixture, wherein the probiotic strain is a bifidobacterium, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucosyllactose / 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 or a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infanttis; At least one of the bifidobacteria is Bifidobacterium longum subsp. infantis LMG 11588.

2. The composition according to claim 1, comprising at least one probiotic strain and a prebiotic oligosaccharide mixture, wherein the probiotic strain is bifidobacterium, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and 3-fucosyllactose (3FL); wherein the bifidobacterium comprises Bifidobacterium longum subsp. infantis or a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infanttis; At least one of the bifidobacteria is Bifidobacterium longum subsp. infantis LMG 11588.

3. 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), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

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

5. The composition according to any one of claims 1 to 4, wherein the at least one Bifidobacterium is Bifidobacterium animalis subsp. lactis CNCM 1-3446.

6. A composition according to any one of the preceding claims, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42 to 71 wt% 2FL; ii. 10 wt% to 40 wt% LNT; iii. 4 to 14 wt% DFL; and iv. 9 to 31 wt% of 6SL and 3SL combined.

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

8. The composition of any one of claims 1 to 5, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 26 to 65 wt% 2FL; ii. 8 to 30 wt% LNT; iii. 3 to 11 wt% DFL; iv. 7 wt% to 23 wt% of 6SL and 3SL combined; and v. 12 wt% to 38 wt% 3FL.

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

10. The composition of any one of claims 1-5, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50 to 70 wt% 2FL; ii. 14 to 26 wt% LNT; iii. 4 to 14 wt% DFL; and iv. 9 to 31 wt% of 6SL and 3SL combined.

11. The composition of claim 10, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50 to 70 wt% 2FL; ii. 14 to 26 wt% LNT; iii. 5 to 10 wt% DFL; and iv. 10 to 28 wt% of 6SL and 3SL combined.

12. The composition of any one of claims 1-5, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 20 to 60 wt% 2FL; ii. 8 to 30 wt% LNT; iii. 2 to 12 wt% DFL; iv. 7 wt% to 23 wt% of 6SL and 3SL combined; and v. 10 wt% to 40 wt% 3FL.

13. The composition of claim 12, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 25 to 55 wt% 2FL; ii. 11 to 20 wt% LNT; iii. 4 to 8 wt% DFL; iv. 8 to 22 wt% of 6SL and 3SL combined; and v. 11 wt% to 37 wt% 3FL.

14. The composition according to any one of claims 1 to 5, which is a nutritional composition selected from the list consisting of an infant formula, a step 1 infant formula, a follow-on formula or a step 2 infant formula, a baby food, an infant cereal composition, a growing-up milk, a fortifier, or a supplement.

15. The composition of claim 14, wherein the fortifier is a human milk fortifier.

16. Use of the composition of any one of claims 1 to 15 in the preparation of a medicament for: i) preventing and / or treating bacterial infections in infants or young children; ii) regulating the microbiota of infants or young children; and / or iii) preventing and / or treating allergic reactions in infants or young children.

17. The use according to claim 16, wherein the medicament is for modulating the microbiota of an infant or young child, and administration of the medicament results in an increase in the abundance of Bifidobacteriaceae and / or Bifidobacterium longum subsp. infantis.

18. The use according to claim 16, wherein the medicament is for regulating the microbiota of an infant or young child; and / or preventing and / or treating allergic reactions in such infant or young child by increasing the production of short-chain fatty acids (SCFA) in the intestine of such infant or young child.

19. The method according to any one of claims 16 to 18, wherein the bifidobacterium is Bifidobacterium longum subsp. infantis LMG 11588 and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

20. The method according to any one of claims 16 to 18, wherein the bifidobacterium is a combination of Bifidobacterium animalis subsp. Lactis CNCM 1-3446 and Bifidobacterium longum subsp. infantis LMG11588, and the prebiotic oligosaccharide mixture consists of 2'-fucosyllactose (2FL), lactodifucosyllactose / difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

21. The use according to any one of claims 16 to 18, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42 to 71 wt% 2FL; ii. 10 wt% to 40 wt% LNT; iii. 4 to 14 wt% DFL; and iv. 9 to 31 wt% of 6SL and 3SL combined.

22. The use according to claim 21, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 42 to 71 wt% 2FL; ii. 14 to 26 wt% LNT; iii. 5 to 10 wt% DFL; and iv. 10 to 28 wt% of 6SL and 3SL combined.

23. The use according to any one of claims 16 to 18, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 26 to 65 wt% 2FL; ii. 8 to 30 wt% LNT; iii. 3 to 11 wt% DFL; iv. 7 wt% to 23 wt% of 6SL and 3SL combined; and v. 12 wt% to 38 wt% 3FL.

24. The use according to claim 23, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 32 wt% to 54 wt% 2FL; ii. 11 to 20 wt% LNT; iii. 4 to 8 wt% DFL; iv. 8 to 22 wt% of 6SL and 3SL combined; and v. 17 wt% to 31 wt% 3FL.

25. The use according to any one of claims 16 to 18, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50 to 70 wt% 2FL; ii. 14 to 26 wt% LNT; iii. 4 to 14 wt% DFL; and iv. 9 to 31 wt% of 6SL and 3SL combined.

26. The use according to claim 25, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 50 to 70 wt% 2FL; ii. 14 to 26 wt% LNT; iii. 5 to 10 wt% DFL; and iv. 10 to 28 wt% of 6SL and 3SL combined.

27. The use according to any one of claims 16 to 18, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 20 to 60 wt% 2FL; ii. 8 to 30 wt% LNT; iii. 2 to 12 wt% DFL; iv. 7 wt% to 23 wt% of 6SL and 3SL combined; and v. 10 wt% to 40 wt% 3FL.

28. The use according to claim 27, wherein the prebiotic oligosaccharide mixture consists essentially of: i. 25 to 55 wt% 2FL; ii. 11 to 20 wt% LNT; iii. 4 to 8 wt% DFL; iv. 8 to 22 wt% of 6SL and 3SL combined; and v. 11 wt% to 37 wt% 3FL.

29. The use according to any one of claims 16-18, wherein the composition is a nutritional composition selected from the list consisting of an infant formula, a step 1 infant formula, a follow-on formula or a step 2 infant formula, a baby food, an infant cereal composition, a growing-up milk, a fortifier, or a supplement.

30. The use of claim 29, wherein the fortifier is a human milk fortifier.

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