Nutritional composition containing 3-hydroxybutyrate to improve the gastrointestinal barrier

Abstract

The present invention relates to a nutritional composition comprising 3-hydroxybutyric acid for use in improving the gastrointestinal barrier. In particular, the present invention relates to improving the gastrointestinal barrier in an individual, preferably an infant or a child.

Description

Technical Field

[0001] The present invention relates to a nutritional composition containing 3-hydroxybutyric acid for use in improving the gastrointestinal barrier. In particular, the present invention relates to improving the gastrointestinal barrier in an individual, preferably an infant or a child.

Background Art

[0002] The gastrointestinal barrier plays an important role as a functional barrier against macromolecules and pathogenic organisms while enabling the absorption of nutrients such as minerals and vitamins. The intestinal epithelial barrier is the first line that separates the body from the external environment including the microbiota, dietary components, and environmental toxins. A properly functioning intestinal epithelial cell barrier is extremely important for maintaining the overall quality of life and regulating a wide range of functions including immunity and hormonal balance, and as a result, can affect a variety of health and clinical outcomes. Considering the extensive impact of the intestinal epithelial barrier on overall health, there is a need for compositions that can provide health benefits such as optimizing an individual's health and overall quality of life by improving, enhancing, or protecting intestinal barrier function.

[0003] This need is particularly prominent in infants and young children. During postnatal growth, the newly born intestine undergoes a maturation process that ends with the establishment of a functional barrier. This phenomenon is called intestinal closure and seems to be affected by diet. Therefore, several studies using infants (JPGN, 1995, 21: 383-6) and animal models (Pediatr Res, 1990, 28: 31-7) have shown that barrier maturation is faster in neonates fed breast milk than in neonates fed formula milk. This can explain the higher morbidity of allergies and infections in formula-fed infants compared to breast-fed infants.

[0004] Breast milk is recommended for all infants. However, in some cases, breastfeeding may be insufficient or unsuccessful for medical reasons, or mothers may choose not to breastfeed. Infant formulas have been developed for these situations. Fortifiers have also been developed to enhance breast milk or infant formula with specific components. In such cases, it is even preferable to provide means to improve the gastrointestinal barrier of infants and toddlers through nutritional interventions such as complete nutrition or nutritional supplements.

[0005] Certain groups of infants and young children have a particular need for compositions that can provide health benefits, such as improving intestinal barrier function. Such infants and young children include, for example, premature infants, low birth weight infants, and / or infants or young children with slow growth. In fact, the intestinal barrier is highly permeable and easily damaged, and its structure and function are less mature in such infants than in healthy full-term infants. This can then lead to other problems, such as infections or allergies. For such infants, supplementing pharmaceutical management with nutritional compositions that can improve intestinal barrier function is particularly advantageous. Neonatal and juvenile animals, such as pet animals, may also need improvement in their intestinal barrier for the same reasons.

[0006] The intestinal barrier can also be impaired in older children and adults, such as those suffering from and / or severe gastrointestinal diseases (e.g., inflammatory bowel disease, chronic diarrhea) and / or receiving parenteral nutrition. The same is true for animals, such as pets, that suffer from these conditions.

[0007] Therefore, improved, and in particular more efficient and / or reliable, nutritional compositions for improving the intestinal barrier are considered advantageous.

[0008] The effects of nutrients on improving the gastrointestinal barrier have been studied in the past. Here, we will cite the case of polyamines, which have been the subject of many studies. For example, AFBekebrede, J. Keijer, WJJ Gerrits and VCJ de Boer; The Molecular and Physiological Effects of Protein-Derived Polyamines in the Intestine; Nutrients, 2020, 12, pp 197ff reviews the effects of polyamines (i.e., spermine, spermidine, putrescine, and cadaverine) on biochemical, cellular, and physiological processes focusing on the colon. It is stated that polyamines support intestinal physiology by supporting barrier function, such as inducing intestinal maturation and increasing lifespan.

[0009] 3-hydroxybutyrate has been previously reported to have potentially beneficial effects on intestinal cell maturation (Q. Wang, Y. Zhou, P. Rychahou, TW-M. Fan, ANLane, HL Weiss and BMEvers, Ketogenesis contributes to intestinal cell differentiation, Cell Death and Differentiation (2017) 24, 458-468). However, this literature does not mention intestinal barrier function, structure, protection, and repair.

[0010] Other components such as probiotics and prebiotics, including human breast milk oligosaccharides (HMOs), have been identified as having a positive effect on the intestinal barrier.

[0011] Human breast milk oligosaccharides are indigestible oligosaccharides and therefore cannot be metabolized by enzymes produced by infants or young children. However, these oligosaccharides encounter bacteria in the microbiome of the infant or young child's gastrointestinal tract and are metabolized by those bacteria. The metabolism of HMOs in the infant gastrointestinal tract has been previously studied. Bacteria metabolize HMOs by two different classes of mechanisms, depending on the bacterial genus / species. Bifidobacterium longum subsp. infantis internalizes HMOs in their native form without digesting them into smaller fragments, and then metabolizes HMOs by an intracellular mechanism. Through this mechanism, B. infantis releases important metabolites such as acetic acid and lactic acid into the infant's gastrointestinal tract. Such metabolites are beneficial for the growth of other bacteria in the microbiome, such as Bifidobacterium bifidum. Bifidobacterium bifidum has a very different method of metabolizing HMOs. This bacterial species actually releases enzymes that digest HMOs via extracellular mechanisms, thus cleaving HMOs into small fragments in the infant's gastrointestinal tract, which in turn make such fragments consuming across the diverse bacterial species in the microbiota. For example, Sela et al.; Nursing our microbiota: molecular linkages between bifidoacteria and milk oligosaccharides; Trends Microbiol, 2010, 18(7):298-307 describes in detail the diverse mechanisms used by microbiota bacteria to metabolize HMOs. In particular, Figures 3A and 3B illustrate such mechanisms.

[0012] It would be particularly advantageous to provide improved nutritional compositions, especially more efficient and / or reliable ones, for infants and young children with dysfunctional microbiomes, such as formula-fed infants and premature infants, infants born by cesarean section, and infants and young children who have received or are receiving antibiotic treatment. For example, Chemikova et al.; The premature infant gut microbiome during the first 6 weeks of life differs based on gastrointestinal maturity at birth, Pediatric Research, 2018, 84:71-79, shows how the microbiome of premature infants differs from that of full-term infants (see, for example, Table 1 on page 72). Furthermore, Korpela et al.; Early life colonization of the human gut: microbes matter everywhere; Current Opinion on Microbiology, 2018, 44:70-78 provides a meta-analysis of many studies evaluating infant microbiomes, extracting the range of quantities of five major genera present in the microbiomes of infants between birth and 2 years of age, full-term infants (breastfed and formula-fed), infants born by cesarean section, and infants treated with antibiotics. This publication reveals that the microbiomes of infants born by cesarean section and infants treated with antibiotics are significantly different from those of full-term infants born by birth.

[0013] Several publications have also highlighted significant differences in the gut microbiota between breastfed and formula-fed infants. See, for example, Lee et al.; Comparison of the gut microbiota profile in breast-fed and formula-fed Korean infants using pyrosequencing; Nutrition Research and Practice, 2015, 9(3):242-248.

[0014] The typical microbiome of breastfed infants is known to be particularly efficient at metabolizing HMOs. Further improvement of the effects of nutritional compositions on the health of all infants and young children, especially those with dysfunctional microbiomes that cannot metabolize HMOs in an optimal manner, would be beneficial. There is a clear need to develop appropriate methods for improving the gastrointestinal barrier in infants and young children, particularly those with dysfunctional microbiomes.

[0015] Further optimization of the effects of nutritional compositions on the gastrointestinal barrier in all individuals, particularly infants and young children, would be beneficial. There is a clear need to develop appropriate methods for improving the gastrointestinal barrier in individuals, especially infants and young children.

[0016] Furthermore, there is a need to provide such health benefits in a manner particularly suitable for young or vulnerable populations (such as those with impaired health, infants, and young children) without classical pharmaceutical interventions.

[0017] There is a need to provide such health benefits to these subjects in a manner that does not induce side effects and / or is easily delivered and is well acceptable to parents or healthcare professionals.

[0018] Furthermore, there is a need to supply such benefits in a manner that keeps the cost of supply reasonable and most of them affordable.

[0019] Therefore, it is clear that there is a need to develop alternative methods other than classical pharmaceutical interventions such as the use of drugs, at least in light of the associated risks of side effects.

[0020] [Overview of the prefecture] This invention relates to a nutritional composition containing 3-hydroxybutyrate for use in improving the gastrointestinal barrier. This effect is demonstrated by the results of in vitro studies in an intestinal epithelial cell model.

[0021] 3-Hydroxybutyric acid (HMO) is a breakdown product of HMO, specifically 2-fucosyl lactose (2'FL). It is advantageous to use 3-Hydroxybutyric acid as the active ingredient in place of the HMO from which it originates. 3-Hydroxybutyric acid is formed in the gastrointestinal tract by the metabolism of HMO, particularly 2-fucosyl lactose (2'FL), by the gut microbiota. However, the rate of 3-Hydroxybutyric acid formation from HMO through this biochemical process is influenced by many parameters. Directly adding 3-Hydroxybutyric acid to a nutritional composition allows for more precise administration of the amount of 3-Hydroxybutyric acid delivered to the target gastrointestinal tract. In particular, the metabolism of HMO in the gastrointestinal tract is highly dependent on the gut microbiota present in the subject; therefore, the effects of HMO mediated by HMO metabolites are highly dependent on the composition of the microbiota as described above. Thus, direct administration of metabolites effective in improving the intestinal barrier is advantageous in that it can be effective even in subjects with abnormal or non-abnormal but functionally impaired gut microbiota.

[0022] The inventors have found an improved nutritional composition that is advantageous in that individuals ingesting the composition do not need to have a microbiome capable of metabolizing HMOs in order to benefit from the effect of HMOs on the intestinal barrier. In other words, individuals with a dysfunctional microbiome can also fully benefit.

[0023] Such a composition is particularly suitable for a subject having a dysfunctional microbiota, especially an infant and a young child, preferably an infant who is being breastfed and does not have a microbiota similar to that of a full-term infant born by vaginal delivery, and thus has a risk of metabolizing HMO in a suboptimal manner. Such infants and young children are, for example, formula-fed infants, infants born by cesarean section, premature infants, and infants who have received antibiotic administration.

[0024] Therefore, an object of the present invention relates to a nutritional composition that improves the gastrointestinal barrier, such as improvement of barrier protection, improvement of barrier structure, improvement of barrier function, and / or barrier repair. Such aspects of the gastrointestinal barrier are all interrelated. Appropriate gastrointestinal structure and function are maintained by appropriate barrier protection and restored by appropriate barrier repair. Therefore, any of these aspects, considered together or individually, contribute to a sustained reduction in disease susceptibility.

[0025] Therefore, one aspect of the present invention relates to a nutritional composition containing 3-hydroxybutyric acid for use in improving the gastrointestinal barrier in a subject, preferably an infant (i.e., a child under 12 months of age) or a young child (e.g., a child between 1 and 8 years of age).

[0026] In a preferred embodiment of the present invention, such improvement to the gastrointestinal barrier is improved barrier protection, barrier structure, and barrier function, preferably barrier protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] [Figure 1A]Demonstrates the effectiveness of HMO fermentation products in providing preventive epithelial barrier protection. The co-cultures are treated with HMO and then epithelial barrier dysfunction is induced by cytokine-mediated inflammation. Inflammation is induced in all groups except the control -ve (intact barrier). Before inducing inflammation, the protection rate is calculated by analyzing the evolution of transepithelial electrical resistance (TEER) compared to the control -ve (100% protection) and control +ve (0% protection). Error bars represent LSD5% / 2. A) Feeding for 2 days. B) Feeding for 21 days. [Figure 1B] Demonstrates the effectiveness of HMO fermentation products in providing preventive epithelial barrier protection. The co-cultures are treated with HMO and then epithelial barrier dysfunction is induced by cytokine-mediated inflammation. Inflammation is induced in all groups except the control -ve (intact barrier). Before inducing inflammation, the protection rate is calculated by analyzing the evolution of transepithelial electrical resistance (TEER) compared to the control -ve (100% protection) and control +ve (0% protection). Error bars represent LSD5% / 2. A) Feeding for 2 days. B) Feeding for 21 days. [Figure 2A] Demonstrates the effectiveness of HMO fermentation products in inducing resistance to inflammatory epithelial barrier dysfunction. The co-cultures are treated with HMO and then inflammation is induced in all groups except the control -ve (intact barrier) where epithelial barrier dysfunction is induced by cytokine-mediated inflammation. The lag time is calculated as the time it takes for the transepithelial electrical resistance (TEER) to decrease below that of the control -ve (intact barrier) after induction of inflammation. Error bars represent LSD5% / 2. A) Feeding for 2 days. B) Feeding for 21 days. [Figure 2B] Demonstrates the effectiveness of HMO fermentation products in inducing resistance to inflammatory epithelial barrier dysfunction. The co-cultures are treated with HMO and then inflammation is induced in all groups except the control -ve (intact barrier) where epithelial barrier dysfunction is induced by cytokine-mediated inflammation. The lag time is calculated as the time it takes for the transepithelial electrical resistance (TEER) to decrease below that of the control -ve (intact barrier) after induction of inflammation. Error bars represent LSD5% / 2. A) Feeding for 2 days. B) Feeding for 21 days. [Figure 3A]This study demonstrates the effectiveness of HMO fermentation products in limiting susceptibility to inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control group (intact barrier). The graph shows the median transepithelial electrical resistance (TEER) during inflammation induction. Error bars represent LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 3B] This study demonstrates the effectiveness of HMO fermentation products in limiting susceptibility to inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control group (intact barrier). The graph shows the median transepithelial electrical resistance (TEER) during inflammation induction. Error bars represent LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 4A] This study demonstrates the effectiveness of HMO fermentation products in reducing the severity of symptoms of inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control group (intact barrier). The graph shows the final transepithelial electrical resistance (TEER) at the end of inflammation induction. Error bars represent LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 4B] This study demonstrates the effectiveness of HMO fermentation products in reducing the severity of symptoms of inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control group (intact barrier). The graph shows the final transepithelial electrical resistance (TEER) at the end of inflammation induction. Error bars represent LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 5A]This study demonstrates the efficacy of HMO fermentation products in reducing the severity of symptoms of inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control-ve (intact barrier). After inflammation induction, epithelial barrier integrity was calculated by analyzing the permeability of FITC-labeled dextran (FD4) from the apical membrane compartment to the basement membrane compartment, compared to the control-+ve (value set to 100, representing barrier dysfunction). Bars represent the mean, and intervals represent mean + / - LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 5B] This study demonstrates the efficacy of HMO fermentation products in reducing the severity of symptoms of inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with HMO, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control-ve (intact barrier). After inflammation induction, epithelial barrier integrity was calculated by analyzing the permeability of FITC-labeled dextran (FD4) from the apical membrane compartment to the basement membrane compartment, compared to the control-+ve (value set to 100, representing barrier dysfunction). Bars represent the mean, and intervals represent mean + / - LSD 5% / 2. A) 2 days of feeding. B) 21 days of feeding. [Figure 6] This study demonstrates the efficacy of 3-hydroxybutyrate in reducing inflammation-induced epithelial barrier dysfunction. Co-cultures were treated with 3-hydroxybutyrate, and then epithelial barrier dysfunction was induced by cytokine-mediated inflammation. Inflammation was induced in all groups except the control group (ve, representing intact barrier). After inflammation induction, epithelial barrier integrity was calculated by analyzing the permeability of FITC-labeled dextran (FD4) from the apical membrane compartment to the basal membrane compartment, compared to the control group (+ve, value set to 100, representing barrier dysfunction). Bars represent the mean, and intervals represent the mean + / - standard error (SE). P-values ​​were obtained by a two-sided, two-sample Student's t-test assuming unequal variances.

[0028] The present invention will be described in more detail below. [Modes for carrying out the invention]

[0029] definition Before discussing the present invention in further detail, the following terms and general common technical knowledge are first defined.

[0030] The term "3-hydroxybutyric acid" refers to the compound with CAS registry number CAS 300-85-6.

[0031] The term "infant" refers to a child under 12 months of age (<12 months). The term "child" also refers to a toddler, meaning a child between 1 and 8 years old (≥1 year to <3 years). The term "child" also refers to a child between 3 and 8 years old (≥3 years to <8 years).

[0032] "An infant or toddler born by cesarean section" means an infant or toddler who was delivered by cesarean section. This term means that the infant or toddler was not delivered vaginally.

[0033] "An infant or toddler born by childbirth" refers to an infant or toddler who was delivered vaginally and not by cesarean section.

[0034] "Premature" or "immature" refers to an infant or toddler born before full term. Generally, it refers to an infant or toddler born before 37 weeks of gestation.

[0035] A "low birth weight infant" refers to a newborn weighing less than 2500g (5.5 pounds) due to either premature birth or restricted fetal growth. Therefore, An infant or toddler whose birth weight is between 1500 and 2500 g (commonly referred to as "low birth weight" or LBW). An infant or toddler whose birth weight is between 1000 and 1500 g (referred to as "very low birth weight" or VLBW). This category includes infants or toddlers whose birth weight is less than 1000g (referred to as "extremely low birth weight" or ELBW).

[0036] "SGA infants" refer to infants whose birth weight is below the 10th percentile for infants of the same gestational age.

[0037] The term "nutritional composition" means a composition that provides nutrition to a subject. This nutritional composition is usually taken orally and typically contains a lipid source or fat source and a protein source.

[0038] In certain embodiments, the nutritional composition of the present invention is a hypoallergenic nutritional composition. The expression "hypoallergenic nutritional composition" means a nutritional composition that is unlikely to cause an allergic reaction.

[0039] In certain embodiments, the compositions of the present invention are "synthetic nutritional compositions." The term "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means, which may be chemically identical to a mixture naturally occurring in mammalian milk (i.e., a synthetic composition is not breast milk).

[0040] As used herein, the expression “infant formula” refers to a food intended for specific nutritional supplementation purposes for infants during the first month of life, which, by itself, satisfies the nutritional requirements for infants in this category (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 for infant formulas and follow-on formulas). This also refers to nutritional compositions intended for infants, as defined in Codex Alimentarius (Codex STAN72-1981), and special infant foods (including foods for special medical purposes). The expression “infant formula” encompasses both “infant starter formulas” and “follow-up formulas” or “follow-on formulas.”

[0041] A "follow-up formula" or "follow-on formula" is given from 6 months of age onward. Such formulas constitute the main liquid component in the increasingly diverse diet of infants in this category.

[0042] The term "infant food" refers to food intended for specific nutritional purposes during the first year of life for infants or toddlers.

[0043] The expression "infant grain composition" refers to a food product intended for specific nutritional purposes during the first year of life for infants or toddlers.

[0044] The term "growing-up milk" (or GUM) refers to a milk beverage intended for infants or children, generally fortified with vitamins and minerals.

[0045] The term "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula.

[0046] The term "weaning period" refers to the period in an infant's or toddler's diet during which breast milk is replaced by other foods.

[0047] The expressions "days / weeks / months / years of survival" and "age in days / weeks / months / years since birth" can be used interchangeably.

[0048] The phrase "improvement of the intestinal barrier" may encompass one or more of the following: Improvement of barrier repair, including (but not limited to) restoration of the integrity of the gastrointestinal barrier, such as repair of damaged barriers, reduction of permeability when inflammation is induced in the gastrointestinal mucosa, and repair of the mucosa. Improvement of barrier structures, including (but not limited to) the gastrointestinal barrier, the integrity of the gastrointestinal barrier, the tight junction structure, and the integrity of the intestinal epithelium. Improvement of barrier function, e.g., improved gastrointestinal barrier resistance; reduction of gastrointestinal barrier permeability, e.g., reduction of pathogens that travel from the intestines through the intestinal barrier; e.g., reduction of symbiotic bacteria that travel from the intestines through the intestinal barrier; reduction of allergens that travel from the intestines through the intestinal barrier; reduction of toxic compounds that travel from the intestines through the intestinal barrier; and reduction of disease susceptibility. Improved barrier protection, including (but not limited to) prevention of barrier dysfunction, prevention of leakage from the barrier, protection of tight junctions, and protection of the integrity of the intestinal epithelium.

[0049] "Breast milk" should be understood as breast milk or the mother's colostrum.

[0050] Oligosaccharides are polymers that contain a small number (typically 3 to 10) monosaccharides.

[0051] The terms "HMO" or "HMO(plural)" or "of HMO" refer to human breast milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, suggesting that HMOs may exhibit important functions not directly related to their calorie value. In particular, they have been shown to play an essential role in the early development of infants, such as the maturation of the immune system. Many types of HMOs are found in human breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine, with a wide variety of bonds between them, resulting in a great many different oligosaccharides in human breast milk, with over 130 such structures identified to date. Almost all of these oligosaccharides have a lactose residue at the reducing end, and sialic acid and / or fucose (if present) occupy the non-reducing terminal position. HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosylated oligosaccharides).

[0052] "Fucosylated oligosaccharides" are oligosaccharides that contain fucose residues. Such oligosaccharides are neutral in nature. Some examples include 2-FL (2'-fucosyl lactose), 3-FL (3-fucosyl lactose), difucosyl lactose, lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyl lacto-N-hexaose, fucosyl lacto-N-neohexaose, difucosyl lacto-N-hexaose I, difucosyl lacto-N-neohexaose II, and any combination thereof. While not bound by theory, it is thought that the fucosyl epitopes of fucosylated oligosaccharides may act as decoys on mucosal surfaces. Through competitive effects, such epitopes may prevent and / or limit the action of pathogens (of viral or bacterial origin) or their secreted components (e.g., toxins) that cause infection, particularly by avoiding their binding to natural ligands. Therefore, although not bound by theory, it is thought that they reduce the risk of infection / inflammation, especially LRT / ear infections and / or inflammation. Furthermore, it is thought that fucosylated oligosaccharides enhance the growth and metabolic activity of certain symbiotic microorganisms, reduce inflammatory responses, create an unfavorable environment for pathogens, and thus lead to colonization resistance.

[0053] The expressions "fucosylated oligosaccharides containing 2'-fucosyl epitopes" and "2'-fucosylated oligosaccharides" encompass fucosylated oligosaccharides that possess certain morphological homology due to their 2'-fucosyl epitope content, and thereby may exhibit certain functional homology. While not theoretically bound, the 2'-fucosyl epitopes of these fucosylated oligosaccharides are thought to be particularly specific to pathogens (or their secretions) involved in LRT and / or ear infections.

[0054] The term "N-acetylated oligosaccharide" encompasses both "N-acetyl-lactosamine" and "oligosaccharides containing N-acetyl-lactosamine." These are neutral oligosaccharides that have an N-acetyl-lactosamine residue. Appropriate examples include LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose) and any combination thereof. Other examples include lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, isolact-N-octaose, para-lacto-N-octaose, and lacto-N-decaose.

[0055] The phrases "at least one fucosylated oligosaccharide" and "at least one N-acetylated oligosaccharide" mean "at least one type of fucosylated oligosaccharide" and "at least one type of N-acetylated oligosaccharide."

[0056] "HMO precursors" are important compounds that intervene in the production of HMOs, such as sialic acid and / or fucose.

[0057] "Sialized oligosaccharides" are oligosaccharides containing charged sialic acid, i.e., oligosaccharides that have a sialic acid residue. They have acidic properties. Some examples are 3-SL (3'-sialyl lactose) and 6-SL (6'-sialyl lactose).

[0058] The nutritional compositions of the present invention may be in solid form (e.g., powder) or liquid form. The amounts of various components (e.g., oligosaccharides) may be expressed in g / 100g of the composition on a dry weight basis if it is in solid form, e.g., powder, or as a concentration in g / L of the composition if it refers to liquid form (the latter also encompasses liquid compositions that can be obtained from powder after reconstitution in a liquid such as milk or water, e.g., reconstituted infant formula or follow-up / follow-up formula or growing-up milk or infant cereal products or any other formulations designed for infant nutrition).

[0059] The term "prebiotics" refers to indigestible carbohydrates that have a beneficial effect on the host by selectively stimulating the growth and / or activity of healthy bacteria in the human colon (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125:1401-12).

[0060] The term "probiotics" refers to preparations or components of microbial cells that have a beneficial effect on the health or quality of life of the host. (Salminen S, Ouwehand A, Benno Y. et al. "Probiotics: how should they be defined." Trends Food Sci. Technol. 1999:10 107-10). Microbial cells, in a broad sense, include bacteria or yeast.

[0061] The term "cfu" should be understood as a colony-forming unit.

[0062] All percentages (%) are based on weight unless otherwise specified.

[0063] In addition, in the context of the present invention, the terms “comprising” or “comprises” do not exclude other possible elements. The compositions of the present invention, including many embodiments described herein, may consist of, or essentially consist of, any additional or optional components, constituents, or limitations described herein, in addition to the essential components and limitations described herein, or as necessary.

[0064] No reference to prior art documents in this specification should be construed as an acknowledgment that such prior art is well known or that it forms part of a general understanding common in the art.

[0065] The present invention is described in further detail below. Note that the various aspects, features, examples and embodiments described herein are interchangeable and / or can be combined.

[0066] Nutritional composition As disclosed in the Examples section, 3-hydroxybutyric acid has been found to be effective in improving the gastrointestinal barrier. Accordingly, one aspect of the present invention relates to a nutritional composition comprising 3-hydroxybutyric acid for use in improving the gastrointestinal barrier in subjects. In a preferred embodiment, the subjects are infants (children under 12 months of age) or toddlers (1 to under 3 years of age). In another preferred embodiment, the nutritional composition is growing-up milk, and the subjects are children (3 to under 8 years of age).

[0067] In one embodiment, such improvement of the gastrointestinal barrier includes improvement of the barrier structure, improvement of the barrier function, improvement of barrier protection, and / or improvement of barrier repair.

[0068] In another embodiment, such improvement of the gastrointestinal barrier includes improvement of barrier function, improvement of barrier structure, and / or improvement of barrier protection.

[0069] In further embodiments, such improvement of the gastrointestinal barrier is an improvement in barrier protection.

[0070] In yet another embodiment, such use is for the purpose of preventing barrier dysfunction, preventing barrier leakage, protecting tight junction structures, and protecting the integrity of the intestinal epithelium. In yet another embodiment, such prevention of barrier leakage is to prevent pathogens, allergens, and / or toxic compounds from penetrating from the gastrointestinal tract through the intestinal barrier into the body.

[0071] When the gastrointestinal barrier lacks adequate protection, i.e., during an inflammatory challenge, it loses its optimal structure and function; therefore, the gastrointestinal barrier provides improved structure and function.

[0072] Therefore, in one embodiment, such use is for improving the strength, integrity, tight bonding structure, and integrity of the gastrointestinal barrier.

[0073] In another embodiment, such use is for improving gastrointestinal barrier resistance and reducing gastrointestinal barrier permeability. Preferably, such use is for reducing pathogens that travel from the intestines through the intestinal barrier, such as reducing commensal bacteria that travel from the intestines through the intestinal barrier, reducing allergens that travel from the intestines through the intestinal barrier, reducing toxic compounds that travel from the intestines through the intestinal barrier, and reducing disease susceptibility.

[0074] The effects / benefits outlined above are preferably obtained in the small intestine.

[0075] In further embodiments, the nutritional composition is formulated for administration to an infant. In another embodiment, such infant is selected from the group consisting of premature infants, infants born under gestation, and low birth weight infants, preferably the infant is premature. The nutritional composition according to the present invention is considered particularly useful for these types of infants because their barriers are less mature and more permeable than those of healthy full-term infants, and therefore the described benefits are more important to these infants than to "normal" infants.

[0076] In some embodiments, 3-hydroxybutyric acid may be present in amounts of 0.01 mg / L to 10 g / L of the composition, for example, 0.1 mg / L to 1 g / L, or 0.5 mg / L to 500 mg / L. In a particular embodiment, 3-hydroxybutyric acid is present in an amount of 1 mg / L of the composition. Such concentrations are typically used when the nutritional composition is in the form of a complete nutrition, containing a variety of nutrients such as proteins, fats, and carbohydrates. In another particular embodiment, 3-hydroxybutyric acid is present in amounts of 0.3 mg / L to 300 g / L of the composition, for example, 3 mg / L to 30 g / L, or 15 mg / L to 15 g / L. In a particular embodiment, 3-hydroxybutyric acid is present in an amount of 33 mg / L of the composition. Such concentrations are typically used when the nutritional composition is in the form of a nutritional supplement or fortifier.

[0077] In some embodiments, 3-hydroxybutyric acid may be present in amounts of 0.007 mg / 100g to 7 g / 100g of the composition on a dry weight basis, for example, 0.07 mg / 100g to 0.7 g / 100g or 0.3 mg / 100g to 0.4 g / 100g of the composition on a dry weight basis. In certain embodiments, 3-hydroxybutyric acid is present in an amount of 1 mg per 100 g of the composition on a dry weight basis. Such concentrations are typically used when the nutritional composition is a complete nutritional form containing a variety of nutrients such as proteins, fats, and carbohydrates.

[0078] In another specific embodiment, the amount of 3-hydroxybutyric acid is 0.003 mg to 3 g per serving, for example, 0.03 mg to 0.3 g or 0.15 mg to 0.15 g per serving. In a particular embodiment, the amount of 3-hydroxybutyric acid is 0.3 mg per serving. Such concentrations are preferably used when the nutritional composition is in the form of a nutritional supplement or fortifier.

[0079] In certain embodiments, 3-hydroxybutyrate is provided in the nutritional composition of the present invention in such an amount that, through normal intake of the nutritional composition, a total daily dose of 0.006 mg to 6 g, for example, 0.06 mg to 0.6 g or 0.3 mg to 0.3 g is provided to the target of intake of the composition, preferably an infant or toddler, respectively, a child who takes it. A minimum amount of 3-hydroxybutyrate is considered necessary to have the desired effect in a measurable manner.

[0080] The nutritional composition according to the present invention typically contains a carbohydrate source. Any carbohydrate can be used, such as lactose, sucrose, saccharose, maltodextrin, starch, or mixtures thereof, but one preferred carbohydrate source is lactose.

[0081] The nutritional composition according to the present invention may also contain oligosaccharides. Preferably, the composition contains oligosaccharides (i.e., other than the human breast milk oligosaccharides described above) and / or at least fiber and / or at least their precursors. Other oligosaccharides and / or fiber and / or their precursors may be selected from a list including galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, human breast milk oligosaccharides (as defined in the Definitions section), bovine milk oligosaccharides and any combination thereof. These components may typically be present in amounts of 0 to 10% by weight of the composition.

[0082] Suitable commercially available products that can be used to prepare the composition of the present invention include combinations of FOS and inulin, such as the product sold by BENEO under the trademark Orafti, or polydextrose sold by Tate & Lyle under the trademark STA-LITE®.

[0083] The nutritional composition according to the present invention may also optionally contain at least one precursor of an oligosaccharide. One or more precursors of oligosaccharides may be present. For example, precursors of human breast milk oligosaccharides are sialic acid, fucose, or a mixture thereof. In some embodiments, the composition contains sialic acid.

[0084] In certain examples, the nutritional composition contains 0-3 g / L of oligosaccharide precursors, or 0-2 g / L, 0-1 g / L, 0-0.7 g / L, 0-0.5 g / L, 0-0.3 g / L, or 0-0.2 g / L of oligosaccharide precursors. The composition according to the present invention may contain 0-2.1 g of oligosaccharide precursors (or more) per 100 g of composition on a dry weight basis, for example, 0-1.5 g, 0-0.8 g, or 0-0.15 g of oligosaccharide precursors (or more) per 100 g of composition on a dry weight basis.

[0085] The nutritional composition of the present invention may further contain at least one probiotic (or probiotic strain), such as a probiotic bacterial strain.

[0086] The most commonly used probiotic microorganisms are primarily bacteria and yeasts of the following genera: Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium, and Saccharomyces.

[0087] In some specific embodiments, the probiotics are probiotic strains. In some specific embodiments, this is in particular Bifidobacteria and / or Lactobacilli.

[0088] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM I-2116, Lactobacillus johnsonii CNCM I-1225, all available from Valio Oy (Finland) under the LGG trademark; Streptococcus salivarius DSM 13084, sold by BLIS Technologies Limited (New Zealand) under the designation KI2; and especially Bifidobacterium lactis CNCM, sold by Christian Hansen company (Denmark) under the trademark Bb 12. Examples include Bifidobacterium longum ATCC BAA-999, sold under the trademark BB536 by Morinaga Milk Industry Co., Ltd. (Japan), Bifidobacterium breve, sold under the trademark Bb-03 by Danisco, Bifidobacterium breve, sold under the trademark M-16V by Morinaga, Bifidobacterium infantis, sold under the trademark Bifantis by Procter & Gambie Co., and Bifidobacterium breve, sold under the trademark R0070 by Institut Rosell (Lallemand).

[0089] The nutritional composition according to the present invention may contain, on a dry weight basis, 10E3 to 10E12 cfu of probiotic strain per gram of composition, more preferably 10E7 to 10E12 cfu, for example, 10E8 to 10E10 cfu of probiotic strain.

[0090] In one embodiment, the probiotics are live bacteria. In another embodiment, the probiotics are non-replicating or inactivated. In some other embodiments, both viable and inactivated probiotics may be present. Probiotic components and metabolites may also be added.

[0091] In one embodiment, the nutritional composition of the present invention is a complete nutritional composition (satisfying all or most of the nutritional requirements of the subject). In another embodiment, the nutritional composition is a supplement or fortifier intended to supplement, for example, human breast milk, or an infant formula or follow-on / follow-up formula.

[0092] In some specific embodiments, the compositions of the present invention are infant formulas, fortifiers, or supplements that may be intended for infants aged 4, 6, or 12 months. In preferred embodiments, the nutritional compositions of the present invention are infant formulas. In fact, it is believed that the nutritional interventions of the present invention may be most effective when administered in the early stages of life (e.g., at 1 month, 4 months, 6 months, or 12 months of age).

[0093] The nutritional composition according to the present invention may be, for example, an infant formula, an infant starter formula, a follow-on formula or follow-up formula, a growing-up milk, baby food, an infant cereal composition, or a fortifier such as a human breast milk fortifier, a supplement, a healthcare product, a medical food, an enteral nutrition composition, or a food for animals.

[0094] In some specific embodiments, the composition of the present invention is an infant formula, fortifier, or supplement that may be intended for infants in their first four or six months of age. In preferred embodiments, the nutritional composition of the present invention is an infant formula.

[0095] In some embodiments, the nutritional composition of the present invention is a fortifier. The fortifier may be a formula fortifier, such as a breast milk fortifier (e.g., a human breast milk fortifier) ​​or an infant formula fortifier or a follow-on / follow-up formula fortifier.

[0096] If the nutritional composition is a supplement, it can be provided in the form of a unit dose. In such cases, it is particularly useful to specify the amount of 3-hydroxybutyrate in relation to the period or daily dose to be administered to infants or young children as described above.

[0097] The nutritional composition of the present invention can be in solid (e.g., powder), liquid, or gelatinous form.

[0098] In certain embodiments, the nutritional composition is a supplement in powder form, provided in the form of a sachet, tablet, capsule, or lozenge, or in liquid form such as a liquid distributed as drops in breast milk or the nutritional composition, or directly to the mouth of an infant or young child.

[0099] In another embodiment, the supplement may further contain carriers, protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flowing agents, taste masking agents, bulking agents, gelling agents, and gel-forming agents. Such supplements may also contain conventional pharmaceutical additives and auxiliaries, additives, and diluents, including, but not limited to, water, gelatin of any origin, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, and fillers. If the supplement is in powder form, it may contain a carrier. However, it is preferable that the supplement does not contain a carrier. If the supplement is in the form of a syrup, the HMO is preferably dissolved or suspended in water acidified with citrate.

[0100] Furthermore, supplements may contain vitamins, mineral trace elements, and other micronutrients in accordance with the recommendations of government agencies such as the USRDA.

[0101] The nutritional composition of the present invention may be in solid (e.g., powder), liquid, or gelatinous form. In certain embodiments, the nutritional composition is a supplement containing 3-hydroxybutyric acid, which is in powder form and provided in sachets, or in the form of a syrup, preferably with a total solid concentration of 5-75 g / 100 mL (5-75% (w / v)). If the supplement is in powder form, it may contain a carrier. However, it is preferable that the supplement does not contain a carrier. If the supplement is in the form of a syrup, the 3-hydroxybutyric acid is preferably dissolved or suspended in water acidified with citrate.

[0102] The nutritional compositions of the present invention typically contain a protein source. The protein amount can be 1.6 to 3 g / 100 kcal. In some embodiments, particularly when the composition is intended for premature infants, the protein amount can be 2.4 to 4 g / 100 kcal, or more than 3.6 g / 100 kcal. In some other embodiments, the protein amount may be less than 2.0 g / 100 kcal, for example, 1.8 to 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0103] As long as the minimum requirements regarding essential amino acid content are met and satisfactory growth is guaranteed, the type of protein is not considered to be of high importance in this invention. Therefore, protein sources based on whey, casein, and mixtures thereof may be used, not just soy-based protein sources. With respect to whey protein, the protein source may be based on acidic whey, sweet whey, or mixtures thereof, and may contain α-lactalbumin and β-lactoglobulin in any desired proportion.

[0104] In some embodiments, the primary protein source is whey (i.e., more than 50%, for example, more than 60% or more than 70% of the protein is derived from whey protein).

[0105] The protein may be an intact protein, a hydrolyzed protein, or a mixture of an intact protein and a hydrolyzed protein. The term "intact" means that the main part of the protein is intact, i.e., its molecular structure has not changed, for example, at least 80% of the protein has not changed, for example, at least 85% of the protein has not changed, preferably at least 90% of the protein has not changed, and more preferably at least 95% of the protein has not changed, for example, at least 98% of the protein has not changed. In certain embodiments, the protein is not changed at all.

[0106] In the context of this invention, the term "hydrolyzed" means that a protein has been hydrolyzed, or broken down into its constituent amino acids. The protein may be fully hydrolyzed or partially hydrolyzed. For example, it may be desirable to supply partially hydrolyzed protein (degree of hydrolysis 2-20%) to infants or young children who are considered to be at risk of developing milk allergies. When hydrolyzed protein is required, the hydrolysis process may be carried out as desired, as is known in the art. For example, hydrolyzed whey protein can be prepared by enzymatically hydrolyzing a whey fraction in one or more steps. It has been found that when the whey fraction used as a starting material is substantially lactose-free, the lysine blackage that the protein undergoes during the hydrolysis process is significantly reduced. This can reduce the degree of lysine blackage from about 15% by weight of total lysine to less than 10% by weight of lysine, and for example, about 7% by weight of lysine significantly improves the nutritional value of the protein source.

[0107] In one embodiment of the present invention, at least 70% of the protein is hydrolyzed, preferably at least 80% of the protein is hydrolyzed, for example at least 85% of the protein is hydrolyzed, more preferably at least 90% of the protein is hydrolyzed, for example at least 95% of the protein is hydrolyzed, and particularly at least 98% of the protein is hydrolyzed. In a particular embodiment, 100% of the protein is hydrolyzed.

[0108] In a particular embodiment, the protein of the nutritional composition is hydrolyzed, completely hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein can be 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, greater than 20, greater than 40, greater than 60, greater than 80, or greater than 90.

[0109] Alternatively, protein components can be replaced with mixtures or synthetic amino acids, for example, for premature or low-birth-weight infants.

[0110] In certain embodiments, the nutritional composition according to the present invention is a hypoallergenic composition. In other specific embodiments, the composition according to the present invention is a hypoallergenic nutritional composition.

[0111] The nutritional composition according to the present invention generally contains a lipid source. This is particularly appropriate when the nutritional composition of the present invention is an infant formula. In this case, the lipid source may be any lipid or fat, preferably a source suitable for use in an infant formula. Some suitable fat sources include palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids linoleic acid and alpha-linolenic acid may also be added, as may small amounts of oil such as fish oil or microbial oil rich in preformed arachidonic acid and docosahexaenoic acid. The fat source may have an n-6 fatty acid to n-3 fatty acid ratio of about 5:1 to about 15:1, for example, about 8:1 to about 10:1.

[0112] The nutritional compositions of the present invention may also contain all vitamins and minerals that are understood to be essential in daily diets and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins, and other nutrients that may be optionally included in the compositions of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amount of certain minerals and other vitamins will vary depending on the target population.

[0113] If necessary, the nutritional composition of the present invention may also contain emulsifiers and stabilizers, such as soy, lecithin, and citrate esters of mono and diglycerides.

[0114] The nutritional composition of the present invention may also contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, and nucleosides.

[0115] The nutritional composition of the present invention may also contain carotenoids (including multiple carotenoids). In some specific embodiments of the present invention, the nutritional composition of the present invention does not contain any carotenoids.

[0116] The nutritional compositions according to the present invention can be prepared by any suitable method. The compositions will now be described as examples.

[0117] For example, nutritional compositions such as infant formulas can be prepared by blending protein sources, carbohydrate sources, and fat sources in appropriate proportions. Emulsifiers, if used, can be added at this stage. Vitamins and minerals may be added at this stage, but are usually added later to avoid thermal decomposition. Any lipophilic vitamins and emulsifiers can be dissolved in the fat source before blending. Water, preferably reverse-osmotic water, can then be mixed to form a liquid mixture. The water temperature should be, as appropriate, in the range of about 50°C to about 80°C to aid in the dispersion of the components. A commercially available liquefaction device can be used to form the liquid mixture.

[0118] In particular, if the final product is in liquid form, fucosylated oligosaccharides (including multiple types) and N-acetylated oligosaccharides (including multiple types) may be added at this stage. If the final product is in powder form, any oligosaccharide may be added at this stage if desired.

[0119] Next, the liquid mixture is homogenized, for example, in two steps.

[0120] The liquid mixture can then be subjected to a heat treatment to reduce bacterial load, for example, by rapidly heating the liquid mixture to a temperature in the range of approximately 80°C to 150°C for approximately 5 seconds to approximately 5 minutes. This heating can be carried out by steam injection, by autoclave, or by heat exchanger, such as a plate heat exchanger.

[0121] Next, the liquid mixture can be cooled to approximately 60°C to 85°C, for example, by flash cooling. Then, the liquid mixture can be homogenized again, for example, in two stages: in the first stage to approximately 10 MPa to 30 MPa, and in the second stage to approximately 2 MPa to 10 MPa. The homogenized mixture can then be further cooled, and any heat-sensitive components, such as vitamins and minerals, can be added. The pH and solid content of the homogenized mixture are usually adjusted at this point.

[0122] If the final product is a powder, the homogenized mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer to convert it into a powder. The moisture content of the powder should be less than approximately 5% by weight. Fucosylated oligosaccharides (or multiple) and N-acetylated oligosaccharides (or multiple) may also be added at this stage, or alternatively, together with probiotic strains (or multiple) (if used), by dry mixing or by blending them in the form of crystalline syrup, and the mixture is spray-dried or freeze-dried.

[0123] If a liquid composition is preferred, the homogenized mixture may be sterilized and then aseptically filled into a suitable container, or it may be filled into a container first and then retorted.

[0124] In another embodiment, the composition of the present invention may be a supplement. The supplement may be in the form of, for example, a tablet, capsule, lozenge, or liquid. The dietary supplement may further contain protective hydrophilic colloids (such as gum, protein, or modified starch), binders, film-forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (such as oils, fats, waxes, or lecithin), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flowing agents, taste masking agents, bulking agents, gelling agents, and gel-forming agents. Such supplements may also contain conventional pharmaceutical additives and auxiliaries, additives, and diluents, including, but not limited to, water, gelatin of any origin, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavoring agents, preservatives, stabilizers, emulsifiers, buffers, lubricants, colorants, wetting agents, and fillers.

[0125] Furthermore, supplements may contain vitamins, minerals, trace elements, and other micronutrients, in accordance with the recommendations of government agencies such as the USRDA, in addition to organic or inorganic carrier materials suitable for oral or parenteral administration.

[0126] The nutritional compositions of the present invention are for use in any type of human subject, preferably infants or toddlers. Infants or toddlers may be full-term or premature. In certain embodiments, the nutritional compositions of the present invention are for use in premature infants, low birth weight infants, and / or underweight infants (SGA) or toddlers. In certain embodiments, the nutritional compositions of the present invention are for use in premature infants, low birth weight infants, and / or underweight infants (SGA).

[0127] The nutritional composition of the present invention may also be used in infants or young children born by cesarean section or delivered vaginally.

[0128] In some embodiments, the compositions according to the present invention may be used before and / or during weaning.

[0129] Nutritional compositions can be administered (or given or fed) at the appropriate age and duration as needed.

[0130] The nutritional composition can be given, for example, immediately after the birth of an infant. The composition of the present invention can also be given to an infant during the first week, or the second week, or the third week, or the first month, or the second month, or the third month, or the fourth month, or the sixth month, or the eighth month, or the tenth month, or the first year, or the second year or older. In some particularly advantageous embodiments of the present invention, the nutritional composition is given (or administered) to the infant within the first four, six, or twelve months after birth. In some other embodiments, the nutritional composition of the present invention is administered at a few days after birth (e.g., day 1, day 2, day 3, day 5, day 10, day 15, day 20, etc.), or at several weeks (e.g., week 1, week 2, week 3, week 4, week 5, week 6, week 7, week 8, week 9, week 10, etc.), or at several months (e.g., month 1, month 2, month 3, month 4, month 5, month 6, month 7, month 8, month 9, month 10, etc.). This may be particularly true if the infant is precocious, but is not necessarily so.

[0131] In one embodiment, the composition of the present invention is given to an infant or toddler as a composition to supplement breast milk. In some embodiments, the infant or toddler receives breast milk for at least the first two weeks, the first one month, two months, four months, or six months. In one embodiment, the nutritional composition of the present invention is given to the infant or toddler after breastfeeding for such a period, or together with breastfeeding for such a period. In another embodiment, the composition is given to the infant or toddler alone or as a primary nutritional composition for at least one period, for example, after one month, two months, or four months after birth, for at least one month, two months, four months, or six months.

[0132] In one embodiment, the nutritional composition of the present invention is a complete nutritional composition (satisfying all or most of the nutritional requirements of the subject). In another embodiment, the nutritional composition is a supplement or fortifier intended to supplement, for example, human breast milk or an infant formula or follow-up formula.

[0133] All patent and non-patent documents cited in this application are incorporated into this application exactly as they appear in the documents.

[0134] The present invention will be described in more detail by the following non-limiting embodiments. [Examples]

[0135] Example 1 Table 1 below shows an example of the composition of a nutritional composition according to the present invention (e.g., an infant formula). This composition is provided for illustrative purposes only.

[0136] Table 1: Composition of the infant formula in Example 1 [Table 1]

[0137] Example 2: Correlation between HMO metabolites and the effects of HMO on intestinal barrier function. Purpose of the exam To compare the effects of microbiome fermentation products (i.e., metabolites) of 2'FL, 2'FL+LNnT, and combinations of six HMOs (2'FL, 3'SL, 6'SL, LNT, LNnT, and DiFL) in terms of their protective and strengthening capabilities against the epithelial barrier before and after inflammation induction. Inflammation induction will be performed in a cell line co-culture model of intestinal epithelial cells, including Caco-2 cells and HT29-MTX cells, containing the respective amounts of individual metabolites identified in the microbiome fermentation products of each HMO or HMO mixture.

[0138] method The microbial fermentation products of 2'FL, 2'FL+LNnT, and combinations of six HMOs (2'FL, 3'SL, 6'SL, LNT, LNnT, and DiFL) were obtained by culturing the fecal microbiota of 3-month-old breastfed infants in a continuous human gastrointestinal tract model called the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), obtained from ProDigest (Gent, Belgium). The microbiota of the same infant was inoculated into four separate SHIME culture vessels. After 2 weeks of microbiota stabilization, either 2'FL, a combination of 2'FL and LNnT (2:1 ratio), or a combination of six HMOs (2'FL, 3'SL, 6'SL, LNT, LNnT, and DiFL) was supplied daily to the SHIME vessels of the microbiota at a concentration of 5 g / L for 21 days. The ratio of each HMO under the six HMO blends was set as follows: 2'FL:3'SL:6'SL:LNnT:LNT:DiFL = 0.55:0.07:0.09:0.05:0.18:0.06. The fermentation culture medium on each microbiome container was collected before the initial feed, and then 2 and 21 days after the initial feed. All fermentation culture media were then centrifuged, and the supernatant containing HMO metabolites was collected.

[0139] For the epithelial barrier assay, human cell lines Caco-2 and HT29-MTX were obtained from the American Type Culture Collection (ATCC) and the European Collection of Authenticated Cell Cultures (ECACC), respectively. Caco-2 cells and HT29-MTX cells were maintained separately in culture flasks in Dulbecco's Minimum Essential Medium (DMEM) supplemented with GlutaMAX (Invitrogen), 1% Minimum Essential Medium, 100 μg / mL streptomycin, 100 μg / mL penicillin, and thermo-inactivated fetal bovine serum (FBS: 15% for Caco-2 and 10% for HT29-MTX) at 37°C and humidified 10% CO2.

[0140] For co-culture testing of the epithelial barrier, each cell line was grown in its own flask until it reached a 90% confluent monolayer. The cell lines were then trypsinized with 1× trypsin. The co-cultures of Caco-2 and HT29-MTX were then divided into 6×10⁻⁶ samples. 4 / cm 2 And, 1.12cm 2 Seeds were seeded on a Transwell® polycarbonate semipermeable membrane (0.4 μm) and grown for 21 days in DMEM supplemented with GlutaMAX®, 1% minimal essential medium, 100 μg / mL streptomycin, 100 μg / mL penicillin, and 10% thermally inactivated FBS.

[0141] On the day of the experiment, the culture medium was replaced with fresh medium at least 4 hours before processing. First, the co-culture was pre-treated with fermented medium recovered from the SHIME experiment. Specifically, fermented medium from the infant microbiome supplied with 2'FL, 2'FL-LNnT, or a combination of six HMOs (HMO6) (2'FL, 3'SL, 6'SL, LNT, LNnT, and DiFL) was added to the apical compartment of the transwell at a concentration of 20% v / v. Unfermented cultured SHIME medium (without infant microbiome) was used as a control. After 36 hours of pre-treatment, epithelial barrier dysfunction was induced for a further 48 hours by adding TNF-α (2.5 ng / mL) and IFN-γ (10 ng / mL) to the basolateral compartment of the transwell. Transepithelial electrical resistance was continuously measured using a Cellzscope® instrument during the experiment. At the end of the experiment, the permeation of FITC-labeled dextran (4000 Da) from the apical membrane compartment to the basal membrane compartment within 2 hours was quantified.

[0142] Metabolome analysis was performed on microbial fermentation products of 2'FL, 2'FL+LNnT, and six HMO combinations (2'FL, 3'SL, 6'SL, LNT, LNnT, and DiFL) obtained using SHIME. Metabolome analysis was performed using the Metabolon® platform (Metabolon Inc., Morrisville, NC, USA). Briefly, samples were extracted and evenly divided for analysis on LC / MS / MS and polar LC platforms. For metabolite identification and quantification by integration of peak areas, ions were matched to an in-house reference library using proprietary software. All datasets were provided electronically to Nestle for analysis.

[0143] Statistical analysis was performed on metabolome data to investigate the correlation between the level of effectiveness of each microbial fermentation product in reading out each barrier and the concentration of each metabolite in such microbial fermentation products. The statistical analysis was performed as follows:

[0144] During the epithelial barrier test, differences between treatments were evaluated using one-way ANOVA, followed by multiple comparisons using Fisher's minimum significance method with a statistical significance level of α=5% (LSD5%).

[0145] A three-stage analysis was performed for metabolome correlation analysis.

[0146] Regardless of the treatment, all samples were initially divided into two groups: pre-feeding samples (day 0) and post-feeding samples (days 2 and 21). Metabolites that significantly changed between day 0 and day 21 were evaluated using one-way ANOVA, followed by multiple comparisons using Fisher's minimum significance method at LSD 5%.

[0147] Next, significant metabolites were clustered based on the treatment. Metabolites that clustered more with HMO treatment compared to lactose were selected and used to investigate their correlation with the epithelial barrier test.

[0148] The correlation between metabolite levels and epithelial barrier data was examined using the Pearson correlation coefficient.

[0149] result Figures 1A and 1B show that HMO fermentation products have efficacy in providing preventative epithelial barrier protection, whereas fermentation of lactose, the main carbohydrate in human breast milk and infant formula, does not have such efficacy.

[0150] Figures 2A and 2B demonstrate the effectiveness of HMO fermentation products in inducing resistance to pro-inflammatory epithelial barrier dysfunction.

[0151] Figures 3A and 3B demonstrate the effectiveness of HMO fermentation products in limiting susceptibility to pro-inflammatory epithelial barrier dysfunction.

[0152] Figures 4A and 4B demonstrate the effectiveness of HMO fermentation products in reducing the severity of symptoms of pro-inflammatory epithelial barrier dysfunction.

[0153] Figures 5A and 5B demonstrate the effectiveness of HMO fermentation products in reducing the severity of symptoms of pro-inflammatory epithelial barrier dysfunction.

[0154] Table 3 shows the correlation between the amount of 3-hydroxybutyrate in HMO fermentation products and different readouts of epithelial barrier integrity. The table shows that 3-hydroxybutyrate has a significantly positive correlation with all readouts of barrier integrity.

[0155] [Table 2]

[0156] conclusion These data indicate that 3-hydroxybutyrate is effective in improving and protecting the intestinal barrier.

[0157] Example 3: Direct effects of 3-hydroxybutyric acid Purpose of the exam 3-hydroxybutyrate was tested for its protective and strengthening effects on the epithelial barrier after inflammation induction in a co-culture model of intestinal epithelial cell lines, including Caco-2 cells and HT29-MTX cells.

[0158] Materials and methods Human cell lines Caco-2 and HT29-MTX were obtained from the American Type Culture Collection (ATCC) and the European Collection of Authenticated Cell Cultures (ECACC), respectively. Caco-2 cells and HT29-MTX cells were maintained separately in culture flasks in Dulbecco's Minimum Essential Medium (DMEM) supplemented with GlutaMAX (Invitrogen), 1% Minimum Essential Medium, 100 μg / mL streptomycin, 100 μg / mL penicillin, and thermo-inactivated fetal bovine serum (FBS: 15% for Caco-2 and 10% for HT29-MTX) at 37°C and humidified 10% CO2.

[0159] For co-culture testing of the epithelial barrier, each cell line was grown in its own flask until it reached a 90% confluent monolayer. The cell lines were then trypsinized with 1× trypsin. The co-cultures of Caco-2 and HT29-MTX were then divided into 6×10⁻⁶ samples. 4 / cm 2 And, 1.12cm 2 Seeds were seeded on a Transwell polycarbonate semipermeable membrane (0.4 μm) and grown for 21 days in DMEM supplemented with GlutaMAX, 1% minimal essential medium, 100 μg / mL streptomycin, 100 μg / mL penicillin, and 10% thermally inactivated FBS.

[0160] On the day of the experiment, the culture medium was replaced with fresh medium at least 4 hours before processing. The co-culture was first pre-treated with 3-hydroxybutyrate. Specifically, a stock solution was prepared by diluting 3-hydroxybutyrate (Sigma 166898; CAS 300-85-6) with DMSO to a final concentration of 1 mM. On the day of the experiment, this stock solution was further diluted to a concentration of 100 μM with fresh medium. The diluted 3-hydroxybutyrate was then added to the apical membrane compartment of the Transwell at a final concentration of 10 μM (e.g., 50 μl in a 500 μL apical membrane compartment volume). As a control, the same amount of DMSO as the stock 3-hydroxybutyrate was diluted with fresh culture medium, and the diluted DMSO was added to the apical membrane compartment of the Transwell at a concentration of 20% v / v. Unfermented cultured SHIME medium (without infant microbiota) was used as a control. After 36 hours of pretreatment, epithelial barrier dysfunction was induced for a further 48 hours by adding TNF-α (2.5 ng / mL) and IFN-γ (10 ng / mL) to the basement membrane compartment of the transwell. At the end of the experiment, the permeation of FITC-labeled dextran (4000 Da) from the apical membrane compartment to the basement membrane compartment within 2 hours was quantified. FD4 permeation data were analyzed using a two-sided, two-sample Student's t-test assuming unequal variances.

[0161] result Figure 6 shows the effectiveness of 3-hydroxybutyrate in protecting the barrier by counteracting inflammation-induced increased epithelial barrier permeability.

[0162] conclusion The data presented confirms the results of correlation studies and provides evidence of the effectiveness of 3-hydroxybutyrate in improving and protecting the intestinal barrier.

Claims

1. A nutritional composition containing 3-hydroxybutyric acid for use in improving the gastrointestinal barrier in a subject, The nutritional composition is an infant formula, an infant starter formula, a follow-on formula or follow-up formula, a growing-up milk, baby food, an infant cereal composition, a human breast milk fortifier, or a supplement.

2. The nutritional composition according to claim 1, wherein the target is an infant (a child under 12 months of age) or a toddler (1 year to under 3 years of age).

3. The nutritional composition according to claim 1, wherein the nutritional composition is growing-up milk, and the target is a child aged 3 to under 8 years.

4. The nutritional composition according to any one of claims 1 to 3, wherein the improvement of the gastrointestinal barrier is an improvement in the barrier structure, an improvement in the barrier function, an improvement in barrier protection and / or an improvement in barrier repair.

5. The nutritional composition according to any one of claims 1 to 4, wherein the improvement of the gastrointestinal barrier is an improvement in barrier function, an improvement in barrier structure, and / or an improvement in barrier protection.

6. The nutritional composition according to any one of claims 1 to 5, wherein the improvement of the gastrointestinal barrier is an improvement in barrier protection.

7. The nutritional composition according to any one of claims 1 to 6, wherein the use is for the purpose of preventing barrier dysfunction, preventing barrier leakage, protecting tight junction structures, and improving the integrity of the intestinal epithelial lining.

8. The nutritional composition according to claim 7, wherein the prevention of barrier leakage is the prevention of pathogens, allergens and / or toxic compounds moving from the gastrointestinal tract through the intestinal barrier into the body.

9. The nutritional composition according to any one of claims 1 to 8, wherein the subject has a functionally impaired microbiome.

10. The nutritional composition according to claim 9, wherein the dysfunctional microbiota has less than 80% Actinobacteriae bacteria, and the percentage is defined by the total number of bacteria in the microbiota.

11. The nutritional composition according to claim 9 or 10, wherein the dysfunctional microbiota has less than 20% Bifidobacterium bifidum, and the percentage is defined by the number based on the total number of bacteria in the microbiota.

12. The nutritional composition according to any one of claims 1 to 2 and 4 to 11, wherein the subject is an infant selected from the group consisting of premature infants, infants born under gestation, and infants with low birth weight.

13. The nutritional composition according to any one of claims 1 to 12, wherein 3-hydroxybutyric acid is present in the nutritional composition in an amount of 0.01 mg / L to 10 g / L or 0.3 mg / L to 300 g / L on a dry weight basis.

14. The nutritional composition according to any one of claims 1 to 13, wherein 3-hydroxybutyric acid is present in an amount of 0.003 mg to 3 g per serving.

15. The nutritional composition according to any one of claims 1 to 14, wherein 3-hydroxybutyric acid is provided in an amount such that a total daily dose of 0.006 mg to 6 g per day is provided to the subject ingesting the nutritional composition through normal intake of the nutritional composition.

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