Patent Application
20240306690
The present invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain belonging to bifidobacteria and a prebiotic mixture of human milk oligosaccharides (HMO) consisting of 2′-fucosyllactose (2FL), difucosyllactose (DFL), 3′-sialyllactose (3SL), 6′-sialyllactose (6SL) and lacto-N-tetraose (LNT), and optionally 3-fucosyllactose (3FL) and applications of the mixtures in human health.
HMOs (human milk oligosaccharides) have become the subject of much interest in recent years due to their roles in numerous biological processes occurring in the human organism. Mammalian milk contains at least 130 of these complex oligosaccharides (Urashima et al, Milk Oligosaccharides, Nova Biomedical Books, New York, 2011, ISBN: 978-1-61122-831-1).
Evidence is accumulating that the resident community of microbes, called the microbiota, in the human digestive tract plays a major role in health and disease. When the composition of the intestinal microbiota is thrown off balance, the human host can suffer consequences. Recent research has implicated intestinal microbiota imbalances in individual disorders as diverse as cancer, obesity, inflammatory bowel disease, psoriasis, asthma, and possibly even autism. Individual non-digestible fibres, including HMOs, are believed to positively modulate the microbiota, and they are of increasing interest for treating one or more of such disorders.
Infancy, especially the first weeks, 3 months, 6 months or 12 months of life is a critical period for the establishment of a balanced gut microbiota.
It is known that the modulation of the gut microbiota during infancy can prospectively have a significant influence in the future health status of the body. For example, the gut microbiome can have an influence on the development of a strong immune system later in life, as well as normal growth, and even on the development of obesity later in life.
The gut microbiome and its evolution during the development of the infant is, however, a fine balance between the presence and prevalence (amount) of many populations of gut bacteria. Some gut bacteria are classified as “generally positive” while others are “generally negative” (or pathogenic) regarding their effect on the overall health of the infant. Certain species of “generally positive” bacteria, such as bifidobacteria, may be under-represented in infants fed conventional infant formula in comparison to breastfed infants. Similarly, some bacterial populations are considered pathogenic and should remain at a low prevalence in the gut microbiota.
Bifidobacterium longum subsp. infantis has been demonstrated to predominate in the gut microbiota of breastfed infants and to benefit the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing short-chain fatty acid production. Reduced abundance of Bifidobacterium species in infants has been correlated to chronic diseases, including asthma and obesity, as well as to lower vaccine response. Researchers have postulated that loss of Bifidobacterium species in the infant gut in populations of developed countries is linked to increased incidence of allergic and autoimmune diseases.
Increasing the abundance of Bifidobacterium species or specifically Bifidobacterium longum subsp. infantis in the gut microbial ecosystem through exogenous administration may be difficult to achieve, especially in formula-fed infants.
Key metabolites produced by Bifidobacterium longum subsp. infantis are lactic acid and acetic acid. Lactic and acetic acid contribute to decreasing stool pH and providing colonization resistance against pathogens in infants (Duar R M, Kyle D and Casaburi G, Colonization Resistance in the Infant Gut: The Role of B. infantis in Reducing pH and Preventing Pathogen Growth; High-Throughput, 2020). Both acids can serve as substrates for other members of the microbiota that produce other short-chain fatty acids (SCFA). SCFA are especially produced by microbial fermentation of dietary fibers in the colon. These colonic fermentations have been known to play a role in energy supply, as trophic factors, and in immune regulation and growing evidence suggests that SCFAs also exert important physiological effects on several organs, including the brain. High abundance of bacteria producing SCFA, particularly butyrate, has been shown to be linked to milder atopic eczema in infants (Nylund L et al., Severity of Atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria, Allergy, 2015).
Due to the loss of Bifidobacterium species in the infant gut and low breast-feeding rates, there is a need to provide infants with both, HMOs and HMO-utilizing bacteria such as B. longum subsp. infantis to support a healthy microbiome for long-term health.
A first aspect of this invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain being bifidobacteria and a prebiotic mixture of human milk oligosaccharides consisting of 2′-fucosyllactose (2FL), 3′-sialyllactose (3SL), difucosyllactose (DFL), 6′-sialyllactose (6SL), lacto-N-tetraose (LNT) and optionally 3-fucosyllactose (3FL).
The present inventors have surprisingly found that a synergistic effect is obtained when this HMO mix is used in combination with Bifidobacterium, in particular the combination of the probiotic, for instance B. longum subsp. infantis, with the HMO mix synergistically promotes a healthier gut environment due to the enhancement of beneficial fermentative metabolites such as short chain fatty acids and the growth stimulation of healthy commensal gut bacteria such as bifidobacteria.
In one aspect the present invention relates to a composition wherein the bifidobacteria is Bifidobacterium animalis subsp. lactis and the prebiotic oligosaccharides mixture consists of 2′-fucosyllactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6′-sialyllactose (6SL), and 3′-sialyllactose (3SL).
In another aspect the present invention relates to a composition wherein the bifidobacteria is a combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis and the prebiotic oligosaccharides mixture consists of 2′-fucosyllactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6′-sialyllactose (6SL), and 3′-sialyllactose (3SL).
In one aspect the present invention relates to a composition for infants or young children comprising at least one probiotic bacterial strain, said probiotic bacterial strain being bifidobacteria, and a prebiotic oligosaccharides mixture consisting of 2′-fucosyllactose (2FL), lactodifucotetraose/difucosyllactose (DFL), lacto-N-tetraose (LNT), 6′-sialyllactose (6SL), and 3′-sialyllactose (3SL) and optionally 3-fucosyllactose (3FL);
wherein the bifidobacteria comprises Bifidobacterium longum subsp. infantis or combination of Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis thereof;
wherein at least one of the bifidobacteria is Bifidobacterium longum subsp. infantis LMG 11588 or a strain having an Average Nucleotide Identity (ANI) of at least 99.9% with this strain.
In one aspect the present invention relates to a nutritional composition selected from the list consisting of an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.
In one aspect the present invention relates to a nutritional composition as defined above for use in: i) preventing and/or treating bacterial infections in an infant or a young child; ii) modulating the microbiota of an infant or a young child; and/or iii) preventing and/or treating allergy of an infant or a young child.
In one aspect the present invention relates to a composition as defined above for use for modulating the microbiota of an infant or a young child and administration of said composition results in abundance of Bifidobacteriaceae and/or Bifidobacterium longum subsp. infantis being increased.
In one aspect the present invention relates to a composition as defined above for modulating the microbiota of an infant or a young child; and/or preventing and/or treating allergy of an infant or a young child by increasing intestinal short-chain fatty acids (SCFA) production in such infant or young child.
In one aspect the present invention relates to a method of modulating the microbiota of an infant or a young child to increase the abundance of Bifidobacteriaceae and/or Bifidobacterium longum subsp. infantis, the method comprising:
As used herein, the following terms have the following meanings.
The term “infant” means a child under the age of 12 months.
The expression “young child” means a child aged between one and three years, also called toddler.
The expressions “a composition for infants or young children” and “a composition to be administered to infants or young children” can be used interchangeably.
In some embodiments, the composition comprising the HMOs mixture according to the invention is a nutritional composition. The expression “nutritional composition” means a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously, and it usually includes a lipid or fat source and a protein source. In a particular embodiment, the nutritional composition is a synthetic nutritional composition.
The expression “infant formula” as used herein refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 91/321/EEC 2006/141/EC of 22 Dec. 2006 on infant formulae and follow-on formulae). It also refers to a nutritional composition intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialties (incl. Food for Special Medical Purpose). The expression “infant formula” encompasses both “starter infant formula” and “follow-up formula” or “follow-on formula”.
A “follow-up formula” or “follow-on formula” is given from the 6th month onwards. It constitutes the principal liquid element in the progressively diversified diet of this category of person.
The expression “baby food” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.
The expression “infant cereal composition” means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.
The term “fortifier” refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula. In one embodiment, the aqueous composition according to the present invention is a milk fortifier. In such embodiment, the aqueous composition of the invention may be packed in single doses.
The term “supplement” means a foodstuff containing specific nutrients and/or probiotics intended to supplement a diet.
In one embodiment, the present invention is a supplement in form of powder or an oil. In such embodiment, the composition of the invention is administered as a standalone composition, or combined with other ingredients such as maltodextrin.
The term “growing-up milk” (or GUM) refers to a milk-based drink generally with added vitamins and minerals, that is intended for young children or children.
HMO (Human Milk Oligosaccharides) are Oligosaccharide structures that naturally occur in human milk. The HMOs that are added to foodstuffs are typically obtained from cow milk, by chemical synthesis or by a biotechnological production process.
The term “SCFA” means short chain fatty acid(s).
The expression “increasing SCFA production” means that the amount of systemic and/or colonic SCFA, is higher in an individual fed with the nutritional composition according to the present invention in comparison with a standard. The SCFA production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography.
The “Average Nucleotide Identity (ANI)” is a measure of nucleotide-level genomic similarity between the coding regions of two genomes. Average Nucleotide Identity can be assessed as describe here: Yoon S H, Ha S M, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017 October; 110(10): 1281-1286. In the present embodiment the strain Bifidobacterium longum subsp. infantis LMG 11588 (also known as ATCC 17930) represents the reference genome to which a microbial genome is compared. An example of a microorganism genome that has at least 99.9% ANI with B. longum subsp. infantis LMG 11588 can be found in PATRIC (https://www.patricbrc.org), genome ID 1678.111 (strain C). In one embodiment of the present invention, the Bifidobacterium longum subsp. infantis strain does not harbour potentially transferable antibiotic resistances.
The HMO and bifidobacteria mixture of this invention can:
The HMO and bifidobacteria mixture can be administered to a human in any suitable form such as, for example, a nutritional composition in a unit dosage form (for example, a tablet, a capsule, a sachet of powder, etc.).
The HMO and bifidobacteria mixture of this invention can also be added to a nutritional composition. For example, it can be added to an infant formula, a food composition, a rehydration solution, or a dietary maintenance or supplement for infants or young children. The nutritional composition can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, or a supplement. In some particular embodiments, the composition of the invention is an infant formula, a fortifier or a supplement that may be intended for the first 4 or 6 months of age. In a preferred embodiment the nutritional composition of the invention is an infant formula. In some other embodiments the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow-on/follow-up formula fortifier.
Macronutrients such as edible fats, carbohydrates and proteins can also be included to such a nutritional composition. Edible fats include, for example, coconut oil, soy oil and monoglycerides and diglycerides. Carbohydrates include, for example, glucose, edible lactose and hydrolysed corn starch. Proteins include, for example, soy protein, whey, and skim milk. Vitamins and minerals (e. g. calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and B complex) can also be included in such a nutritional composition.
The nutritional composition may be prepared in any suitable manner. A composition will now be described by way of example.
For example, a formula such as an infant formula may be prepared by blending together the protein source, the carbohydrate source and the fat source in appropriate proportions. If used, the emulsifiers may be included at this point. The vitamins and minerals may be added at this point but they are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The temperature of the water is conveniently in the range between about 50° C. and about 80° C. to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture.
The HMO mixture of the present invention may be added at this stage, especially if the final product is to have a liquid form. If the final product is to be a powder, they may likewise be added at this stage if desired.
The liquid mixture is then homogenised, for example in two stages.
The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range between about 80° C. and about 150° C. for a duration between about 5 seconds and about 5 minutes, for example. This may be carried out by means of steam injection, an autoclave or a heat exchanger, for example a plate heat exchanger.
Then, the liquid mixture may be cooled to between about 60° C. and about 85° C. for example by flash cooling. The liquid mixture may then be again homogenised, for example in two stages between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenised mixture may then be further cooled to add any heat sensitive components, such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.
If the final product is to be a powder, the homogenised mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. The powder should have a moisture content of less than about 5% by weight. The HMO mixture of the present invention may also or alternatively be added at this stage by dry-mixing along with the probiotic strain(s) in powder.
Preferred features and embodiments of the invention will now be described by way of non-limiting examples.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J. M. and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M. J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D. M. and Dahlberg, J. E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.
Fecal material was collected from 10 approximately 3-month-old donors in Belgium. 4 donors were born by C-section and 6 were vaginally born. One donor was breast-fed, the other 9 donors were infant formula fed in the week before sampling. Fecal suspensions were prepared, mixed with cryoprotectant, aliquoted, flash frozen and then preserved at −80° C. Just before the experiment, fecal samples were defrosted and immediately used in the experiments.
The fecal material was used to perform short-term batch fermentation experiments that mimic the infant colon. These experiments represent a simplified simulation of the continuous Simulator of the Human Microbial Ecosystem (SHIME®). At the start of the short-term colonic incubations, test ingredients were added to a sugar-depleted nutritional medium containing basal nutrients of the colon. Because nutrients of this sugar-depleted nutritional medium will also be fermented by the colonic microbiota, a blank containing only the sugar-depleted nutritional medium (without product) was included for each donor. Finally, fecal inocula of the infant donors were added. Five treatments and one blank were tested per donor, resulting in 60 independent experiments. Treatments were as follows:
The single HMO was 2′-fucosyllactose (2FL). The HMO mix consisted of 5 HMOs in the following ratio in the dry mix: 23% LNT, 9%6SL, 2%3SL, 52%2FL & 14% DFL. Therefore the addition rate of 2FL was the same in the single HMO and HMO mix treatment.
Reactors were incubated for 48 h at 37° C., under shaking and anaerobic conditions. The incubations were performed in fully independent reactors with sufficiently high volume to not only ensure robust microbial fermentation, but also to allow the collection of multiple samples over time. Sample collection enables assessment of metabolite production and thus to understand the complex microbial interactions that are taking place.
An assessment was made amongst others on short chain fatty acids (SCFA) production at the start of the incubation and after 6 h, 24 h and 48 h. The pattern of SCFA production is an assessment of the microbial carbohydrate metabolism.
Changes in microbial composition were analysed at the start and after 24 h and 48 h of incubation. Samples were analyzed with a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing to convert the proportional values obtained with Illumina into absolute cell counts.
Two donors (both vaginally born and infant formula fed) were excluded from the result evaluation as the sequencing results indicated technical or analytical problems.
Fecal material was collected from a 3-month-old baby donor in Belgium. The donor was different from the 10 donors in Example 1. The experiment was similar to Example 1, but all colonic fermentations were carried out in triplicate and the test ingredients differed. Five treatments and one blank were tested, resulting in 18 experiments in total. Treatments were as follows:
B. longum subsp. infantis A is the strain ATCC 15697, which is the type strain of the subspecies and shares less than 99.9% ANI (98.2%) with B. longum subsp. infantis LMG 11588. The strain B. longum subsp. infantis C has more than 99.9% ANI with B. longum subsp. infantis LMG 11588 and is thus closely related with the LMG 11588 strain.
The HMO mix consisted of 5 HMOs in the following ratio in the dry mix: 23% LNT, 9% 6SL, 2%3SL, 52%2FL & 14% DFL.
An assessment was made amongst others on pH and short chain fatty acids (SCFA) production at the start of the incubation and after 6 h, 24 h and 48 h.
Changes in microbial composition were analysed at the start and after 24 h and 48 h of incubation. Samples were analyzed with a combination of 16S-targeted Illumina sequencing and flow cytometry to determine the number of bacterial cells of each bacterial community present, thus allowing to convert the proportional values obtained with Illumina into absolute cell counts.
The sequencing data revealed that the infant donor had no detectable B. longum subsp. infantis-related operational taxonomic unit present in the inoculum, but it could be detected after addition of the two strains. After 48 h colonic simulation (
Fecal material was collected from 6 donors in Belgium at about 3 months of age (infants) and from same donors at about 12 months of age (toddlers). 3 donors were born by C-section and 3 were vaginally born. One donor was breast-fed, the other 5 donors were infant formula fed in the week before the first sampling. The experiment was similar to Example 1. Five treatments and one blank were tested per donor at two ages, resulting in 72 independent experiments. Treatments were as follows:
The HMO mix consisted of 6 HMOs in the following ratio in the dry mix: for the infant study 16% LNT, 8%6SL, 6%3SL, 49%2FL, 7% DFL & 14%3FL; for the toddler study 10% LNT, 5%6SL, 14%3SL, 29%2FL, 4% DFL & 37%3FL.
An assessment was made amongst others on short chain fatty acids (SCFA) production at the start of the incubation and after 6 h, 24 h and 48 h.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21154300.4 | Jan 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/051336 | 1/21/2022 | WO |
