Use of myelin sheath phospholipids and non-digestible carbohydrates to improve the intestinal microbiota

Abstract

The present invention provides a composition comprising a sphingomyelin or a degradation product thereof and at least one non-digestible carbohydrate for providing and / or maintaining an optimal gut microbiota. The composition is particularly suitable as an infant nutrition.

Description

Technical Field

[0001] The present invention pertains to nutritional compositions for restoring or maintaining a healthy balance of the gut microbiota. These compositions are particularly suitable for infants and young children. Background Technology

[0002] Infants are born without a gut microbiota. After birth, the large intestine grows rapidly, and its microbiota also forms and increases rapidly. The gut microbiota of breastfed infants is mainly composed of species belonging to the genus *Bifidobacterium* and other lactic acid-producing bacteria. The microbiota of formula-fed infants is more diverse, typically containing more species belonging to the genera *Bacteroides*, *Clostridium*, and *Enterobacteriaceae*. This difference in microbiota composition is thought to contribute to many beneficial effects on the immune system, gastrointestinal barrier function, and antimicrobial activity, resulting in a decrease in the incidence and duration of gastrointestinal infections, a reduction in allergic diseases such as food allergies, atopic dermatitis, and allergy-induced asthma, and a reduction in constipation in breastfed infants.

[0003] WO 2006 / 091103 discloses a composition comprising probiotics and two non-digestible sugars for combating several diseases. WO 00 / 08948 discloses a mixture of non-digestible oligosaccharides and polysaccharides for improving the gut microbiota.

[0004] WO 2006 / 041316 discloses the use of sphingomyelin degradation products for reducing the amount of Clostridium difficile.

[0005] WO 2007 / 073194 discloses the use of compositions comprising phospholipids, sphingolipids and cholesterol, optionally comprising non-digestible carbohydrates, in the prevention of obesity and / or diabetes. Summary of the Invention

[0006] This invention is based on the unexpected beneficial effects of compositions comprising sphingomyelin and / or its degradation products—particularly ceramides, sphingoid bases, sphingoid phosphates, and / or lysosphingophospholipids—and non-digestible oligosaccharides on the composition and / or activity of the gut microbiota. These combinations have surprisingly demonstrated beneficial effects on enhancing the composition of the gut microbiota. Specifically, a decrease in the Firmicutes / Bacteroidetes ratio, a decrease in the Clostridium difficile / total bacteria ratio, and / or an increase in the probiotic index have been observed.

[0007] The inventors recognized that the composition of the microbiome formed in infancy influences the composition of the microbiome after infancy. Differences in the microbiome formed in infancy are thought to lead to differences in the composition of the gut microbiome in children at least 7 years of age. Therefore, a well-balanced microbiome formed in infancy has healthy outcomes after infancy, into childhood, adolescence, and / or adulthood.

[0008] Therefore, administering a composition containing sphingomyelin and / or its degradation products and non-digestible carbohydrates from birth to infancy and / or early childhood (before 36 months of age) may have a beneficial effect on the prevention of diseases associated with gut microbiota imbalance in the period following the administration of the composition (i.e., the period before 36 months of age, such as childhood, adolescence, and / or adulthood).

[0009] Therefore, a well-balanced microbiome, particularly one with a low ratio of Firmicutes / Bacteroides or Clostridium / Bacteroides, is believed to influence fat storage in adipocytes and / or have preventive and / or therapeutic effects on obesity. Therefore, the compositions of the present invention are particularly suitable for treating and / or preventing obesity. Since adipocytes, particularly visceral adipocytes, form and proliferate during infancy, the compositions of the present invention are particularly suitable for administration to human subjects under 36 months of age to prevent obesity when such subjects are over 36 months of age.

[0010] Furthermore, due to the aforementioned beneficial effects, conjugates of sphingomyelin and / or its degradation products with non-digestible carbohydrates are particularly advantageous for the treatment and / or prevention of diseases related to the composition of the gut microbiota, such as gastrointestinal infections, diarrhea, gastrointestinal inflammation, and / or constipation.

[0011] Since a well-balanced gut microbiota also improves the immune system and / or enhances intestinal maturation and / or gastrointestinal barrier function, the combinations of the present invention can also be used to treat and / or prevent allergic diseases such as allergic reactions, more specifically food allergies, allergic dermatitis, allergy-induced asthma, and systemic and respiratory infections. Detailed Implementation

[0012] This invention relates to a method for normalizing the gut microbiota of a human subject to that present in healthy lean subjects and / or breastfed infants, the method comprising administering the human subject a composition comprising the following components:

[0013] A: Sphingomyelin, preferably sphingomyelin, and / or at least one sphingomyelin degradation product selected from ceramides, lysphingomyelin, sphingosine phosphate, and free sphingosine bases, and

[0014] B: At least one non-digestible carbohydrate selected from fructo-oligosaccharide, galacto-oligosaccharide, gluco-oligosaccharide, arabino-oligosaccharide, mannan-oligosaccharide, xylo-oligosaccharide, fucose, arabinoglacto-oligosaccharide, glucomanno-oligosaccharide, galactomanno-oligosaccharide, sialic acid-containing oligosaccharides, and uronic acid oligosaccharides.

[0015] In other words, this invention relates to the use of a nutritional composition comprising the following ingredients for preparing a composition that normalizes the gut microbiota of human subjects to that present in healthy lean subjects and / or breastfed infants:

[0016] A: Sphingomyelin, preferably sphingomyelin, and / or at least one sphingomyelin degradation product selected from ceramides, lysphingomyelin, sphingosine phosphate, and free sphingosine bases, and

[0017] B: At least one non-digestible carbohydrate selected from fructooligosaccharides, galacto-oligosaccharides, glucose-oligosaccharides, arabino-oligosaccharides, manno-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides, glucomannan-oligosaccharides, galacto-manno-oligosaccharides, oligosaccharides containing sialic acid, and uronic acid oligosaccharides.

[0018] This invention can also be described as a nutritional composition comprising the following ingredients for normalizing the gut microbiota of human subjects to that present in healthy lean subjects and / or breastfed infants:

[0019] A: Sphingomyelin, preferably sphingomyelin, and / or at least one sphingomyelin degradation product selected from ceramides, lysphingomyelin, sphingosine phosphate, and free sphingosine bases, and

[0020] B: At least one non-digestible carbohydrate selected from fructooligosaccharides, galacto-oligosaccharides, glucose-oligosaccharides, arabino-oligosaccharides, manno-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides, glucomannan-oligosaccharides, galacto-manno-oligosaccharides, oligosaccharides containing sialic acid, and uronic acid oligosaccharides.

[0021] Non-digestible carbohydrates

[0022] The compositions of this invention comprise non-digestible carbohydrates. Non-digestible carbohydrates improve the microbiome. Non-digestible carbohydrates stimulate the growth and / or activity of Bifidobacteria and Bacteroides. As shown in Example 1, it has been found that administering non-digestible carbohydrates advantageously reduces the ratio of Firmicutes / Bacteroides, i.e., Clostridium / Bacteroides.

[0023] Preferably, the composition of the present invention comprises a non-digestible carbohydrate with a DP of 2-250, more preferably 2-60. The non-digestible carbohydrate is at least one, preferably at least two, selected from fructooligosaccharides (including inulin), galacto-oligosaccharides (including trans-galacto-oligosaccharides or β-galacto-oligosaccharides), oligodextrin oligosaccharides (including gentio-oligosaccharide, nigero-oligosaccharide, cyclodextrin-oligosaccharide, and polydextrose), arabino-oligosaccharides, manno-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides (including gum arabic), glucomannan oligosaccharides, galacto-manno-oligosaccharides (including partially hydrolyzed guar gum), oligosaccharides containing sialic acid, and uronic acid oligosaccharides (including galacturonic acid oligosaccharides and pectin degradation products). Preferably, the composition of the present invention comprises fructooligosaccharides, galacto-oligosaccharides and / or galacturonic acid oligosaccharides, more preferably galacto-oligosaccharides, and most preferably β-galacto-oligosaccharides.

[0024] Preferably, the composition comprises galactooligosaccharides. Preferably, the galactooligosaccharides are selected from β-galactooligosaccharides, lacto-N-tetrasaccharides (LNT), lacto-N-neo-tetrasaccharides (neo-LNT), fucosylated lactose, fucosylated LNT, and fucosylated neo-LNT. In a particularly preferred embodiment, the composition of the present invention comprises β-galactooligosaccharides. The β-galactooligosaccharides used in the present invention refer to oligosaccharides composed of more than 50%, preferably more than 65%, of galactose units (based on monomer subunits), having a degree of polymerization (DP) of 2-20, wherein at least 50%, more preferably at least 75%, and even more preferably at least 90% of the galactose units are linked together by β-glycosidic bonds, preferably β-1,4-glycosidic bonds. The average DP is preferably 3-6. Glucose units may be located at the reducing end of the galactose unit chain. β-galactooligosaccharides are sometimes also referred to as trans-galactooligosaccharides (TOS). A suitable source of β-galactooligosaccharides is Vivinal. GOS (available from Borculo Domo Ingredients, Zwolle, Netherlands). Other suitable sources include Oligomate (Yakult), Cupoligo (Nissin), and Bi2muno (Classado).

[0025] Preferably, the composition comprises fructooligosaccharides. The fructooligosaccharides used in this invention refer to carbohydrates composed of more than 50%, preferably more than 65%, of fructose units (based on monomer subunits), wherein at least 50%, more preferably at least 75%, and even more preferably at least 90% of the fructose units are linked together by β-glycosidic bonds, preferably β-2,1-glycosidic bonds. Glucose units may be located at the reducing end of the galactose unit chain. Preferably, the DP or average DP of the fructooligosaccharide is 2-250, more preferably 2-100, and even more preferably 10-60. Fructooligosaccharides include levans, hydrolyzed levans, inulin, hydrolyzed inulin, and synthetic fructooligosaccharides. Preferably, the composition comprises short-chain fructooligosaccharides with an average degree of polymerization (DP) of 3-6, more preferably hydrolyzed inulin or synthetic fructooligosaccharides. Preferably, the composition comprises long-chain fructans with an average DP greater than 20, such as Raftilin HP. Preferably, the composition contains both short-chain and long-chain fructans. Suitable oligofructoses for the composition are also readily available, such as Raftiline. HP (Orafti).

[0026] The compositions of the present invention preferably comprise uronic acid oligosaccharides, more preferably galacturonic acid oligosaccharides. As used herein, galacturonic acid oligosaccharides refer to oligosaccharides in which at least 50% of the monosaccharide units are galacturonic acid. The galacturonic acid oligosaccharides used in the present invention are preferably prepared by degradation of pectin, pectic acid, and / or polygalacturonic acid. Preferably, the degraded pectin is prepared by hydrolysis and / or β-elimination of fruit and / or vegetable pectin, more preferably apple, citrus, and / or beet pectin, and even more preferably by hydrolysis and / or β-elimination of apple, citrus, and / or beet pectin degraded by at least one lysin. In a preferred embodiment, at least one terminal galacturonic acid unit of the galacturonic acid oligosaccharide has a double bond. This double bond effectively protects intestinal epithelial cells from attachment by pathogenic bacteria. Preferably, one of the terminal galacturonic acid units comprises a C4-C5 double bond. The galacturonic acid oligosaccharide can be derivatized. The galacturonic acid oligosaccharide can be methoxylated and / or amidated. In one embodiment, the galacturonic acid oligosaccharide is characterized by a degree of methoxylation greater than 20%, preferably greater than 50%, and more preferably greater than 70%.

[0027] In a preferred embodiment, the composition comprises a mixture of inulin and fructooligosaccharides. In another preferred embodiment, the composition comprises a mixture of galacto-oligosaccharides and fructooligosaccharides, wherein the fructooligosaccharides are selected from short-chain fructooligosaccharides and inulin, more preferably inulin. A mixture of at least two different non-digestible carbohydrates provides a greater degree of beneficial stimulation to the gut microbiota than a single non-digestible carbohydrate. Preferably, the weight ratio between the two different non-digestible carbohydrates—preferably galacto-oligosaccharides and fructooligosaccharides—is between 20 and 0.05, more preferably between 20 and 1. Galacto-oligosaccharides are more similar to human milk oligosaccharides. Preferably, the compositions of the present invention comprise galacto-oligosaccharides with a DP of 2-10 and / or fructooligosaccharides with a DP of 2-60. These combinations have been found to synergistically increase Bifidobacteria and Lactobacilli. Preferably, the composition comprises β-galacto-oligosaccharides, fructooligosaccharides, and pectin degradation products. Compared to galacto-oligosaccharides and fructans, the presence of these three non-digestible oligosaccharides further stimulates Bifidobacteria while reducing Bacteroides and Clostridium. The preferred weight ratio of trans-galacto-oligosaccharides:fructooligosaccharides:pectin degradation products is (20-2):1:(1-20), more preferably (12-7):1:(1-3).

[0028] Preferably, each 100 ml of the composition contains 80 mg to 2 g, more preferably 150 mg to 1.50 g, and even more preferably 300 mg to 1 g of non-digestible oligosaccharides. Based on dry weight, the composition preferably contains 0.25-5.5% by weight, more preferably 0.5-4% by weight, and even more preferably 1.5-3% by weight of non-digestible oligosaccharides. Smaller amounts of non-digestible oligosaccharides are less effective at stimulating beneficial bacteria in the microbiome, while excessive amounts can cause side effects such as bloating and abdominal discomfort.

[0029] Sphingomyelin and its degradation products

[0030] The compositions of this invention comprise sphingomyelin and / or one of its degradation products. The term sphingomyelin as used herein refers to a class of sphingolipids composed of a sphingosine base bonded to a fatty acid via an amide bond and to a polar primary group comprising a phosphate ester (phosphate ester, phosphocholine, phosphoserine, phosphoethanolamine, glycerol phosphate, or phosphoinositol). In another embodiment of the invention, the compositions comprise degradation products of sphingomyelin, particularly ceramides, lysphingomyelin, phosphosphosphingosine, and / or free sphingosine bases. The sphingosine base in this invention is preferably a 2-amino-1,3-dihydroxyalkane or alkene with a chain length of 14 atoms or longer and having a (2S,3R)-erythrochemical stereochemistry. The sphingosine base may contain three, rather than two, hydroxyl groups. Its aliphatic chain may be monounsaturated, polyunsaturated (cis or trans conformation), or saturated. The aliphatic chain may be branched. Sphingosine bases include sphingosine, dihydrosphinganine, phytosphingosine, and dihydrophytosphingosine. Preferably, the composition comprises sphingomyelin. Sphingomyelin is a ceramide-phosphocholine compound. Ceramides are composed of sphingosine bases linked to fatty acids via amide bonds.

[0031] Oral administration of sphingomyelin and / or its degradation products, especially lysphingomyelin, ceramides, phosphosphospirin, and / or sphingosine bases, can reduce harmful bacteria in the gut microbiota, such as Firmicutes, particularly Clostridium. The compositions of this invention simultaneously act as stimulants for beneficial bacteria and inhibitors for harmful bacteria, thus more effectively improving the composition of the microbiota and / or maintaining a healthy balance within it.

[0032] Preferably, each 100 ml of the composition contains 10-200 mg, more preferably 20-150 mg, and even more preferably 60-100 mg of sphingomyelin. Based on fat content, the composition preferably contains 0.25-5.5% by weight, more preferably 0.5-4% by weight, and even more preferably 1.5-3% by weight of sphingomyelin. Based on dry weight, the composition preferably contains 0.07-1.5% by weight, more preferably 0.15-1.1% by weight, and even more preferably 0.5-0.8% by weight of sphingomyelin. Lower amounts are less effective, while higher amounts reduce the content of beneficial essential substances and long-chain fatty acids. Suitable sources of sphingomyelin are milk fats, with more suitable sources being buttermilk fat or whey fat, egg yolk, and / or soy lecithin.

[0033] Preferably, the weight ratio of the non-digestible oligosaccharide to sphingomyelin is between 0.4 and 200, more preferably between 1 and 75, and even more preferably between 3 and 15. This ratio in the composition ensures a balance between stimulating beneficial bacteria in the microbiome and reducing harmful bacteria.

[0034] Other lipids

[0035] In this article, LA refers to linoleic acid (18:2 n6); ALA refers to α-linolenic acid (18:3 n3); LC-PUFA refers to acyl chains and / or long-chain polyunsaturated fatty acids with at least 20 carbon atoms and two or more unsaturated bonds in their fatty acyl chains; DHA refers to docosahexaenoic acid (22:6, n3); EPA refers to eicosapentaenoic acid (20:5 n3); ARA refers to arachidonic acid (20:4 n6); and DPA refers to docosapentaenoic acid (22:5 n3).

[0036] LA is preferably present in an amount sufficient to promote healthy growth and development, but in the lowest possible amount to prevent future obesity. Therefore, the composition preferably contains less than 15% by weight of LA in total fatty acids, preferably 5-14.5% by weight of LA, and more preferably 6-12% by weight of LA. ALA is preferably present in an amount sufficient to promote healthy growth and development. Therefore, the composition of the present invention preferably contains at least 1.0% by weight of ALA in total fatty acids. The composition preferably contains at least 1.6% by weight of ALA in total fatty acids, more preferably at least 2.0% by weight of ALA in total fatty acids. The composition preferably contains less than 10% by weight of ALA in total fatty acids, more preferably less than 5.0% by weight of ALA in total fatty acids. The LA / ALA weight ratio should be well balanced to prevent obesity, especially central obesity, while ensuring normal growth and development. Therefore, the weight ratio of LA / ALA in the composition of the present invention is preferably 2-15, more preferably 2-7, more preferably 3-6, even more preferably 4-5.5, and even more preferably 4-5.

[0037] Preferably, the composition of the present invention contains LC-PUFA, because LC-PUFA inhibits subsequent obesity, more preferably central obesity. More preferably, the composition of the present invention contains n-3LC-PUFA, even more preferably EPA, DPA and / or DHA, even more preferably DHA. These n-3LC-PUFAs have been found to alleviate obesity. Since low concentrations of DHA, DPA and / or EPA are already effective, and due to the importance of normal growth and development, the content of n-3LC-PUFA in the composition of the present invention preferably does not exceed 15% by weight of the total fatty acid content, more preferably not more than 10% by weight, even more preferably not more than 5% by weight. The composition of the present invention preferably contains at least 0.2% by weight, more preferably at least 0.5% by weight, and more preferably at least 0.75% by weight of n-3 LC-PUFA as a percentage of the total fatty acid content. Preferably, the weight ratio of n-6LC-PUFA / n-3 LC-PUFA in the composition of the present invention is very low to prevent subsequent obesity. In the compositions of the present invention, the weight ratio of n-6 LC-PUFA to n-3 LC-PUFA is preferably less than 1.5, more preferably less than 1.0, and even more preferably less than 0.6. Another advantage of LC-PUFA is that they promote the attachment of beneficial bacteria, thereby further improving the microbiome and / or maintaining a well-balanced microbiome.

[0038] Preferably, the composition of the present invention comprises cholesterol and cholesterol esters. The simultaneous presence of sphingomyelin and cholesterol and / or cholesterol esters in the composition delays the digestion of sphingomyelin in the small intestine, thereby increasing the amount of sphingomyelin reaching the large intestine, and thus increasing the activity of sphingomyelin and / or its degradation products against harmful bacteria in the gut microbiota. The composition of the present invention preferably comprises at least 0.005% by weight, more preferably at least 0.01% by weight, more preferably at least 0.05% by weight, and even more preferably at least 0.1% by weight of cholesterol and / or cholesterol esters as a percentage of total fat. The amount of cholesterol and / or cholesterol esters preferably does not exceed 10% by weight of total lipids, more preferably not more than 5% by weight, and even more preferably not more than 1% by weight of total lipids. Most preferably, the amount of cholesterol is 0.5-0.7% by weight of total lipids.

[0039] Nutritional composition

[0040] The compositions of the present invention are preferably taken orally. Degradation of sphingomyelin by digestive enzymes is essential for enhancing its resistance to harmful bacteria in the gut microbiota. The compositions of the present invention are preferably nutritional formulations, more preferably infant formulas. Compositions of the present invention containing non-digestible carbohydrates and sphingomyelin and / or their degradation products are not human milk.

[0041] The compositions of this invention can be advantageously used as a complete nutritional supplement for infants. The compositions of this invention preferably contain lipids, proteins, and digestible carbohydrates, and are preferably administered in liquid form. This invention includes dry foods (such as powders) accompanied by instructions for mixing said dry food mixture with a suitable liquid (e.g., water).

[0042] The present invention advantageously provides a composition in which lipids provide 5-50% of the total calories. The present invention advantageously provides a composition in which proteins provide 5-50% of the total calories. The present invention advantageously provides a composition in which digestible carbohydrates provide 15-90% of the total calories. The present invention advantageously provides a composition in which lipids provide 5-50% of the total calories, proteins provide 5-50% of the total calories, and digestible carbohydrates provide 15-90% of the total calories. Preferably, in the compositions of the present invention, lipids provide 35-50% of the total calories, proteins provide 7.5-12.5% ​​of the total calories, and digestible carbohydrates provide 40-55% of the total calories. To calculate the percentage of total calories provided by protein, the total energy provided by protein, peptides, and amino acids needs to be considered. Total calories refer to the sum of calories provided by lipids, digestible carbohydrates, and protein.

[0043] The proteins used in the nutritional formulations are preferably selected from non-human animal proteins (preferably milk proteins), plant proteins (preferably soy proteins and / or rice proteins), free amino acids, and mixtures thereof. The compositions of the present invention preferably contain casein, whey, hydrolyzed casein, and / or hydrolyzed whey protein. The proteins preferably comprise whole proteins, more preferably whole bovine whey protein and / or whole bovine casein. Since the compositions of the present invention are suitable for alleviating allergic reactions in infants, the proteins are preferably selected from hydrolyzed milk proteins.

[0044] The composition of the present invention preferably contains a digestible carbohydrate selected from sucrose, lactose, glucose, fructose, corn syrup solids, starch and maltodextrin, and more preferably contains lactose.

[0045] The compositions of the present invention preferably contain nucleotides and / or nucleosides, more preferably nucleotides. The compositions preferably contain 5′-cytidine, 5′-uridine, 5′-adenosine, 5′-guanosine, and / or 5′-inosine, more preferably 5′-cytidine, 5′-uridine, 5′-adenosine, 5′-guanosine, and 5′-inosine. The compositions preferably contain 5-100 mg, more preferably 5-50 mg, and more preferably 10-50 mg of nucleotides and / or nucleosides per 100 g dry weight. The presence of nucleotides and / or nucleosides advantageously reduces the ratio of Bacteroides, Porphyromonas, and / or Prevotella to Bifidobacteria.

[0046] The nucleotides and / or nucleosides are believed to work synergistically with other components of the compositions of the present invention.

[0047] Abnormal bowel movements, such as hard stools, insufficient stool volume, and diarrhea, are a serious problem in infants. It has been found that bowel problems can be alleviated by administering the compositions of the present invention in liquid form at a molar osmolality of 50-500 mOsm / kg, more preferably 100-400 mOsm / kg. The reduction of abnormal bowel movements enhances the establishment and development of a healthy gut microbiota.

[0048] For the reasons mentioned above, it is important that the liquid food does not have an excessively high calorie density, but still provides enough calories for the subjects. Therefore, the calorie density of the liquid food is preferably 0.1-2.5 kcal / ml, more preferably 0.5-1.5 kcal / ml, and most preferably 0.6-0.8 kcal / ml.

[0049] application

[0050] The compositions of the present invention, comprising sphingomyelin and / or its degradation products (preferably myelin) and non-digestible carbohydrates, are particularly suitable for use in human subjects under 36 months of age to normalize the composition of the gut microbiota. The compositions of the present invention are particularly suitable for use in human subjects under 36 months of age to normalize the composition of the gut microbiota in human subjects older than 36 months of age.

[0051] Normalizing the gut microbiota of human subjects under 36 months of age involves normalizing the composition of their gut microbiota to that found in healthy, breastfed human subjects under 36 months of age. In human subjects under 36 months of age, normalizing the composition of their gut microbiota when said human subjects are over 36 months of age involves normalizing the composition of their gut microbiota to that of healthy subjects of the same age who were breastfed in the past before the age of 36 months.

[0052] The compositions of the present invention comprising sphingomyelin and / or its degradation products (preferably sphingomyelin) and non-digestible carbohydrates are particularly suitable for use in human subjects older than 36 months of age for normalizing the composition of their gut microbiota. Normalizing the gut microbiota of human subjects older than 36 months of age involves normalizing the composition of their gut microbiota to that of healthy human subjects of normal weight. The compositions of the present invention comprising sphingomyelin and / or its degradation products (preferably sphingomyelin) and non-digestible carbohydrates are particularly suitable for use in obese human subjects for normalizing the composition of their gut microbiota. Normalizing the gut microbiota of obese human subjects involves normalizing the composition of their gut microbiota to that of healthy human subjects of normal weight.

[0053] The compositions of the present invention, comprising sphingomyelin and / or its degradation products (preferably myelin) and non-digestible carbohydrates, are particularly suitable for use in human subjects who are currently using or have recently used antibiotics, for normalizing the composition of their gut microbiota. In the context of this invention, the term "recently" means less than two months, for example, less than one month or less than one week, since antibiotic use was discontinued. Normalizing the gut microbiota of human subjects who are currently using or have recently used antibiotics involves normalizing the composition of their gut microbiota to that of the period prior to antibiotic use or their original gut microbiota composition.

[0054] Normalizing the gut microbiota in this invention specifically involves increasing the probiotic index, decreasing the ratio of Clostridium difficile to total bacteria, decreasing the ratio of Clostridium difficile to total bacteria, decreasing the ratio of Clostridium difficile to total bacteria, increasing the ratio of Lactobacillus to total bacteria, increasing the ratio of Bifidobacterium to total bacteria, decreasing the ratio of Firmicutes to Bacteroides, decreasing the ratio of Clostridium difficile to Bacteroides, and decreasing the ratio of Enterobacteriaceae to total bacteria. The increase or decrease refers to the ratios in the human subjects before taking the composition of this invention, or to a control group that did not take this composition. The probiotic index (PI) involves the total number of (Bifidobacteria / total bacteria) + (Lactobacillus / total bacteria) - (Bacteroides / total bacteria) - (Clostridium difficile / total bacteria), see Palframan et al, 2003, Lett Appl Microbiol 37: 281-284. Preferably, the index may also include the ratio of Eubacterium rectale to total bacteria, especially for adults. Eubacterium rectum produces butyrate, which is beneficial to the intestinal barrier in adults.

[0055] The gut microbiota specifically refers to the composition of the microbiota in the colon. The composition of the gut microbiota can be determined at the phylum, class, family, genus and / or species level using specific probes and / or primers known in the art, through methods known in the art, including FISH, real-time PCR and microarrays.

[0056] The compositions of this invention are particularly advantageous for infants born in hospitals, as they are at risk of developing colonies of harmful bacteria from the hospital environment. The compositions of this invention are even more advantageous for infants born via cesarean section, as these infants lack adequate inoculation with beneficial bacteria from their mothers and are at risk of developing colonies of harmful bacteria from the hospital environment.

[0057] Because the compositions of the present invention have the beneficial effects on the microbiota as described above when administered to human subjects under 36 months of age, the compositions of the present invention are preferably used for the prevention and / or treatment of obesity and / or adiposity, more preferably for the prevention and / or treatment of visceral obesity, even more preferably for the prevention and / or treatment of obesity and / or adiposity in human subjects over 36 months of age, and even more preferably for the prevention and / or treatment of visceral obesity. Furthermore, the compositions of the present invention are preferably used to prevent secondary diseases caused by visceral obesity in human subjects over 36 months of age, such as type 2 diabetes, fasting hyperglycemia, insulin resistance, visceral obesity, hyperinsulinemia, hypertension, cardiovascular disease, cerebrovascular disease, atherosclerosis, dyslipidemia, hyperuricemia, fatty liver, osteoarthritis, and sleep apnea. In one embodiment, obesity, adiposity, and visceral obesity refer to the conditions caused by an imbalance in the gut microbiota. Preferably, microbiome imbalance refers to an increased proportion of Firmicutes / Bacteroides, an increased proportion of Clostridium difficile / total bacteria, and / or a decreased probiotic index compared to healthy lean subjects and / or breastfed infants.

[0058] Since the compositions of the present invention have the beneficial effects on the microbiome as described above when administered to human subjects under the age of 36 months, the compositions of the present invention are preferably used for the treatment and / or prevention of gastrointestinal infections, diarrhea and / or gastrointestinal inflammation, and more preferably for the treatment and / or prevention of gastrointestinal infections, diarrhea and / or gastrointestinal inflammation in human subjects over the age of 36 months.

[0059] Since the compositions of the present invention have the beneficial effects on the microbiome as described above when administered to human subjects under 36 months of age, the compositions of the present invention are preferably used to improve the immune system and / or gastrointestinal barrier function, more preferably for improving the immune system and / or gastrointestinal barrier function when the human subjects are over 36 months of age.

[0060] Since the compositions of the present invention have the beneficial effects on the microbiome as described above when administered to human subjects under 36 months of age, the compositions of the present invention are preferably used for the treatment and / or prevention of at least one disease selected from allergic reactions, asthma, allergic dermatitis, eczema, systemic infections, urinary tract infections, otitis media, and respiratory infections, and more preferably for the treatment and / or prevention of at least one disease selected from allergic reactions, asthma, allergic dermatitis, eczema, systemic infections, urinary tract infections, otitis media, and respiratory infections when the human subjects are over 36 months of age.

[0061] The present invention aims to normalize the gut microbiota of human subjects, preferably those aged over 36 months, more preferably over 8 years, more preferably over 15 years, and most preferably over 18 years.

[0062] In this document and its claims, the verb "comprising" and its variations are used in their non-limiting sense to include items following the word, but do not exclude items not specifically mentioned. Furthermore, the use of "a" or "an" to refer to an element does not preclude the possibility of multiple instances of that element, unless the context clearly indicates that there is only one / one such element. Therefore, "a" or "an" generally means "at least one / one".

[0063] Example

[0064] Example 1

[0065] Fermentation studies were conducted using feces collected fresh from 4–5 healthy adults, as described in Appendix I of WO 2004 / 000340. The experiment was performed twice. The feces were diluted 1:5 in McBain and Farlane media, then placed in dialysis tubes under anaerobic conditions and suspended in a flask with buffer. The tubes were first incubated at 37°C for 16 hours to allow acclimatization, and then inoculated at t=0 with the substances listed in the table below.

[0066] The test material was added daily (at t = 0, 24, and 48 h). The buffer solution was refreshed daily. Samples were collected at t = 0 and t = 72 h. The longer timeframes were used because changes in bacterial count are slower compared to changes in the formation of short-chain acids.

[0067] Table 1: Additives to dialysis tubes containing diluted feces

[0068]

[0069] Vivinal GOS (Borculo Domo, The Netherlands) was used as the source of GOS. RaftilineHP (Orafti, Belgium) was used as the source of inulin.

[0070] D-Sphingosine was added to the dialysis tube instead of sphingomyelin (e.g., myelin) because sphingomyelin is degraded into D-sphingosine in the small intestine. Bovine brain (Fluka Sigma Aldrich Chemie) was used as the source of D-sphingosine.

[0071] Dissolve sphingosine in ethanol (10 mg / ml stock solution). Add 30 μl of the 10 mg / ml stock solution to the dialysis tube.

[0072] Samples were analyzed by fluorescence in situ hybridization (FISH) as described in Franks, AH, et al., Appl. Environ. Microbiol., 1998. 64(9): p. 3336-3345. Bacteroides-Prevotella were detected using the Bac303 probe (Manz, W., et al., Microbiology, 1996. 142(Pt5): p. 1097-106). Clostridium were detected using the Chis150 and Clit135 probes (Franks, AH, et al., Appl. Environ. Microbiol., 1998. 64(9): p. 3336-3345). The Erec482 probe was used to detect Eubacterium rectum and Clostridium coccoides (Franks, AH, et al., Appl. Environ. Microbiol., 1998. 64(9): p. 3336-3345). The Bif164 probe was used to detect Bifidobacteria (Langendijk, P., et al., Appl. Environ. Microbiol., 1995. 61(8): p. 3069-3075). The Lab158 probe was used to detect Lactobacillus and Enterococci (Harmsen, HJM, et al., Microbial Ecology in Health and Disease, 1999. Volume 11: p. 3-12). Enterobacteriaceae were determined using the Ec1531 probe (Poulsen, LK, et al., Infect Immun, 1994, 62(11): p.5191-4). Total bacterial count was determined by DAPI staining. The Firmicutes / Bacteroides ratio was set as the percentage of bacteria detected by the Chis150, Clit135, Erec482, and Lab158 probes divided by the percentage of bacteria detected by the Bac303 probe. The bacterial population detected by Erec482 is typically the largest Firmicutes population in the human gut.

[0073] result:

[0074] The Bac303, Chis150, Clit135, Ec1351, and Lab158 probes all showed signals below the FISH detection limit, i.e., less than 10 species per gram of sample. 6The number of cells or relative abundance was less than 1 / 1000 cells (0.1%). For Bac303, samples were stored under barren conditions, but a weak signal was observed in samples with added GOS and inulin. FISH results for Bif164 and Erec482 probes are shown in Table 2.

[0075] Table 2: FISH results of Bif164 and Erec482 probes.

[0076]

[0077] These results indicate that Bifidobacteria increase when non-digestible carbohydrates—more specifically GOS and / or inulin—are fermented or when sphingosine (as a degradation product of sphingomyelin after digestion in the small intestine) is present, and increase further synergistically when both components are present.

[0078] These results indicate that Clostridium and eubacteria—the largest group of Firmicutes—are reduced when non-digestible carbohydrates—more specifically GOS and / or inulin—are fermented or when sphingosine is present. Further improvements were observed when both components were present.

[0079] These results indicate that consuming non-digestible carbohydrates—more specifically GOS and / or inulin—as well as sphingosine leads to the following improvements: increased probiotic index in the coliform microbiota, decreased Clostridium / total bacteria ratio, decreased Bifidobacterium / total bacteria ratio, decreased Firmicutes / Bacteroides ratio, and decreased Clostridium / Bacteroides ratio.

[0080] Example 2:

[0081] Infants aged 9-10 months were given 500ml of formula containing 1.2g of β-galactooligosaccharide and long-chain inulin daily for one month (Group A). ​​The control group received formula without β-galactooligosaccharide and long-chain inulin (Group B). A total of 138 children participated in this study. Fecal microbiota were analyzed by FISH before and after this period.

[0082] Following the intervention, the ratio of Bifidobacteria to total bacteria in group A was significantly higher than that in group B. Furthermore, the ratio of Clostridium perfringens to total bacteria in group A was lower than that in group B. Initially, the ratio of Firmicutes to Bacteroides in group A was 42 and in group B was 39; however, one month after the intervention, the ratio decreased to 32 in group B and 16 in group A.

[0083] Example 3: Infant Nutritional Products

[0084] Infant nutrition products contain fats that provide 48% of total calories, protein that provides 8% of total calories, and digestible carbohydrates that provide 44% of total calories.

[0085] (i) The lipids comprise approximately 1.4% by weight of sphingomyelin and approximately 4% by weight of cholesterol;

[0086] (ii) The digestible carbohydrate comprises 50.9 g lactose per 100 g powder, and each 100 g powder also contains 5.22 g β-galacto-oligosaccharide with a DP of 2-6 and 0.58 g fructooligosaccharide with a DP of 7-60 as non-digestible carbohydrates;

[0087] (iii) Proteins that contain milk proteins, including casein.

[0088] The composition contains vitamins, trace elements, and minerals that meet EU standards. The packaging label of this infant formula states that it is designed to improve gut microbiota, increase the number of bifidobacteria, reduce the number of pathogenic bacteria, and / or maintain a healthy balance of gut microbiota.

Claims

1. Use of a nutritional composition for preparing a composition that normalizes the gut microbiota of a human subject to that present in healthy lean subjects and / or breastfed infants, said nutritional composition comprising: A: Sphingomyelin, preferably sphingomyelin, and / or at least one sphingomyelin degradation product selected from ceramides, lysphingomyelin, sphingosine phosphate, and free sphingosine bases, and B: At least one non-digestible carbohydrate selected from fructooligosaccharides, galacto-oligosaccharides, glucose-oligosaccharides, arabino-oligosaccharides, manno-oligosaccharides, xyloo-oligosaccharides, fucose-oligosaccharides, arabinogalacto-oligosaccharides, glucomannan-oligosaccharides, galacto-manno-oligosaccharides, oligosaccharides containing sialic acid, and uronic acid oligosaccharides.

2. The use of claim 1, wherein the human subject is less than 36 months old.

3. The use of claim 1 or 2, wherein the microbiota remains normal when the age of the human subject exceeds 36 months.

4. The use of any of the preceding claims, wherein the composition of the gut microbiota is normalized in at least one of the following aspects: an increase in the probiotic index, a decrease in the ratio of Clostridium difficile to total bacteria, a decrease in the ratio of Clostridium difficile to total bacteria, a decrease in the ratio of Clostridium difficile to Clostridium, an increase in the ratio of Lactobacillus to total bacteria, an increase in the ratio of Bifidobacterium to total bacteria, a decrease in the ratio of Firmicutes to Bacteroides, a decrease in the ratio of Clostridium difficile to Bacteroides, and a decrease in the ratio of Enterobacteriaceae to total bacteria.

5. The use of any of the preceding claims for the prevention of obesity and / or excess fat, more preferably for human subjects under 36 months of age, and for the prevention of obesity and / or excess fat in human subjects over 36 months of age.

6. The use of claim 5 for the prevention of diseases selected from type 2 diabetes, fasting hyperglycemia, insulin resistance, visceral obesity, hyperinsulinemia, hypertension, cardiovascular disease, cerebrovascular disease, atherosclerosis, dyslipidemia, hyperuricemia, fatty liver, osteoarthritis and sleep apnea.

7. The use of any one of claims 1-4 for the treatment and / or prevention of gastrointestinal infections, diarrhea and / or gastrointestinal inflammation, allergic reactions, asthma, allergic dermatitis, eczema, systemic infections and respiratory infections.

8. Use of any of the preceding claims, wherein the composition comprises at least 0.25% by weight of sphingomyelin—preferably sphingomyelin—and / or at least one sphingomyelin degradation product selected from ceramides, lysphingomyelin, sphingosine phosphate, and free sphingosine bases.

9. The use of any of the preceding claims, wherein at least two non-digestible carbohydrates are selected.

10. Use according to any of the preceding claims, wherein the composition comprises at least 0.25% by weight of non-digestible carbohydrates in the dry weight of the composition.

11. Use according to any of the preceding claims, wherein the composition comprises lipids providing 35-50% of total calories, digestible carbohydrates providing 40-55% of total calories, and protein providing 7.5-12.5% ​​of total calories.

12. Use according to any of the preceding claims, wherein the composition comprises sphingomyelin and / or its degradation products in a weight ratio of 0.4-200 to non-digestible carbohydrates.