Nutritional composition for improving gut microbiota

Abstract

A nutritional composition comprising a digestible carbohydrate, a protein and a lipid wherein the lipid is in the form of lipid globules which are at least partially coated on the surface with phospholipids for use in improving the gut microbiota and reducing opportunistic pathogens in the gut of a human infant.

Description

TECHNICAL FIELD

[0001] The present invention relates to a nutritional composition for infants, in particular an infant formula, a follow-on formula or a young child formula. The present invention further relates to the improvement of the gut microbiota of infants. BACKGROUND

[0002] Human milk is the undisputed gold standard for infant nutrition. However, in some cases breastfeeding is insufficient or unsuccessful for medical reasons, or breast milk is not available due to the choice not to breastfeed. For such cases, infant or follow-on formulas have been developed. Today, commercial infant formulas are commonly used to provide a supplemental or exclusive source of nutrition in the early stages of life. These formulas contain a range of nutrients to meet the nutritional needs of growing infants and typically include fats, carbohydrates, proteins, vitamins, minerals and other nutrients that contribute to optimal growth and development of the infant. Commercial infant formulas are designed to mimic as closely as possible the composition and function of human milk.

[0003] It is known that human milk lipids have a unique physical structure consisting of large lipid globules with a mode diameter of about 4 pm based on volume, with a triglyceride core coated by a tri-layer membrane (milk fat globule membrane, MFGM). Due to the application of industrial processing procedures in order to achieve a stable product, lipid droplets in standard infant formulas typically have a mode diameter of about 0.3-0.5 pm based on volume, and these lipid droplets are not surrounded by MFGM but mainly by milk proteins. Infant formulas have been described with lipid globules having a more similar structure to the lipid globules in human milk (e.g. WO 2010 / 027258 or WO 2010 / 027259).

[0004] US 2022 / 386675 A1 describes a method for non-therapeutically improving the postnatal growth trajectory or physical development of a caesarean section infant by administering a nutritional composition selected from the group comprising infant formulas and follow-on formulas, wherein the lipid globules have a more similar structure to human milk lipid globules.

[0005] It is known in the art that some nutritional ingredients beneficially influence the gut microbiota in addition to prebiotic non-digestible oligosaccharides. For example, it has been described that milk fat globule membrane (MFGM) can beneficially influence the gut microbiota.

[0006] WO 2009 / 082216 describes a composition comprising a phosphosphyngolipid or a degradation product thereof and at least one non-digestible carbohydrate for providing and / or maintaining an optimal gut microbiota.

[0007] Lopez et al. assessed whether specific composition and structure of MFGM and milk polar lipid aggregates (related to their nutritional and health benefits) could be used to tailor functional emulsions inspired by MFGM-coated milk fat globules.

[0008] Berding et al. (2016), doi:10.1097 / MPG.0000000000001200 describe a study in which 2-day-old male piglets (n = 24) were fed a formula (control group (CONT)) or a formula containing polydextrose (1.2 g / 100 g feed), galacto- oligosaccharides (3.5 g / 100 g feed), bovine lactoferrin (0.3 g / 100 g feed), and milk fat globule membrane-10 (2.5 g / 100 g feed) (test group (TEST)) for 30 days. The microbiota of the test group piglets differed from the control group in the ascending colon (p = 0.001) and in the feces (p = 0.05).

[0009] He et al. (2019), doi:10.1038 / s41598-019-47953-4 investigated the fecal microbiome and metabolome of infants fed an experimental formula (EF) supplemented with bovine MFGM and compared to infants fed a standard formula (SF) and a breastfed reference group. The effect of MFGM on the fecal microbiome was moderate; several metabolites (including lactate, succinate, amino acids and their derivatives) were shown to be significantly reduced in the fecal metabolome of infants fed EF compared to infants fed SF.

[0010] Lee et al. (2020), doi:10.1002 / mnfr.202000603 describe a study in which the serum metabolome and fecal microbiota were analyzed using 1H NMR spectroscopy and 16S rRNA gene sequencing, respectively, in a group of Chinese infants given either a standard formula or a formula supplemented with a whey protein fraction enriched in MFGM. Supplementation with MFGM did not induce significant compositional changes in the fecal microbiota, but inhibited microbial diversity and altered microbiota-associated metabolites.

[0011] Despite this, there is still a need in the art for nutritional compositions that can reduce the chance of opportunistic pathogen proliferation, while promoting an increase of beneficial bacteria, thereby improving the gut microbiota and (gut) health.

[0012] Tan et al. (2020), doi:10.6084 / m9.figshare.12689891 describe that gut microbiota modulation plays a role in the treatment and prevention of (gastrointestinal) infections.

[0013] Melli et al. (2015), doi: 10.1016 / j.aller.2015.01.013 describe the link between the gut microbiota and the development of allergies.

[0014] Low et al. (2017), doi: 10.3920 / BM2017.0020 describe the link between an elevated early infant Klebsiella / Bifidobacterium ratio and the development of pediatric allergies in childhood.

[0015] Di Costanzo et al. (2020), doi: 10.3390 / ijms21155275 describe that early gut dysbiosis is associated with the development of food allergies.

[0016] Therefore, improving the gut microbiota of a human subject leads to an improvement of the overall health of the subject, in particular the treatment and / or prevention of gut dysbiosis, infections, and / or allergies. SUMMARY

[0017] The inventors of the present application have surprisingly found that feeding infants with a nutritional composition comprising large, phospholipid-coated lipid spheres improves the gut microbiota of said infants and brings it closer to the gut microbiota observed in breastfed infants. More particularly, the gut microbiota is improved by reducing the relative abundance of opportunistic pathogens in the gut to levels of relative abundance of opportunistic pathogens observed in breastfed infants. The gut microbiota can also be improved by increasing the relative abundance of beneficial bacteria and / or by reducing the ratio between the relative abundance of opportunistic bacteria and the relative abundance of beneficial bacteria.

[0018] Obtaining a gut microbiota similar to that of breastfed infants is considered beneficial. For infants in the first months of life, reducing opportunistic pathogens is particularly advantageous as the gut microbiota is strongly developing at this age. The presence of higher levels of opportunistic pathogens early in life is associated with an impaired gut microbiota and this has been described as a risk factor for infections and non-communicable diseases (NCDs) in children, such as allergies.

[0019] Without wishing to be bound by theory, the inventors believe that reducing the relative abundance of opportunistic pathogens, preferably Enterobacterales, while increasing the relative abundance of beneficial bacteria, preferably Bifidobacteriaceae, has the effect of reducing the risk of developing infections and allergies.

[0020] Therefore, the first aspect of the present application relates to a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0021] a. the lipid spheres have a volume-weighted mode diameter of at least 1.0 pm; and / or

[0022] at least 45 vol.% of the lipid spheres have a diameter between 2 and 12 pm based on the total lipid volume; and

[0023] b. the lipids comprise 0.5 to 20 wt.% phospholipids based on total lipids, and wherein the lipid spheres are at least partially coated on the surface with a layer of phospholipids,

[0024] for use in improving the gut microbiota of a human infant.

[0025] Without wishing to be bound by any theory, the presence of phospholipids (such as MFGM) in the coating of the larger lipid spheres in the nutritional composition results in an improved beneficial effect on the gut microbiota compared to nutritional compositions wherein phospholipids are not present in the coating of the lipid spheres (either not present in the composition or present alone). It is hypothesized that due to the supramolecular lipid structure, i.e. the phospholipid coating and the larger size of the lipid spheres, a larger proportion of the MFGM is able to reach the colon where the gut microbiota resides, and thus results in an improved effect on the gut microbiota. DETAILED DESCRIPTION

[0026] Therefore, the first aspect of the present application relates to a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0027] a. the lipid spheres have a volume-weighted mode diameter of at least 1.0 pm; and / or

[0028] at least 45 vol.% of the lipid spheres have a diameter between 2 and 12 pm based on the total lipid volume; and

[0029] b. the lipids comprise 0.5 to 20 wt.% phospholipids based on total lipids, and wherein the lipid spheres are at least partially coated on the surface with a layer of phospholipids,

[0030] for use in improving the gut microbiota of a human infant.

[0031] For some jurisdictions, the present invention can also be formulated as a method for improving the gut microbiota of a human infant, the method comprising administering a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0032] a. the lipid spheres have a volume-weighted mode diameter of at least 1.0 pm; and / or

[0033] at least 45 vol.% of the lipid spheres have a diameter between 2 and 12 pm based on the total lipid volume; and

[0034] b. the lipids comprise 0.5 to 20 wt.% phospholipids based on total lipids, and wherein the lipid spheres are at least partially coated on the surface with a layer of phospholipids.

[0035] For some jurisdictions, the present invention can also be formulated as the use of digestible carbohydrates, protein and lipids for the manufacture of a nutritional composition for improving the gut microbiota of a human infant, wherein the nutritional composition is selected from the group consisting of infant formula, follow-on formula and baby food formula, and wherein the lipids are in the form of lipid spheres, wherein

[0036] a. the lipid spheres have a mode diameter of at least 1 pm based on volume; and / or

[0037] at least 45 vol.% of the lipid spheres have a diameter between 2 and 12 pm based on the total lipid volume; and

[0038] b. the lipids comprise 0.5 to 20 wt.% phospholipids based on total lipids, and wherein the lipid spheres are at least partially coated on the surface with a layer of phospholipids.

[0039] The present invention can also be formulated as the use of a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, protein and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0040] a. the lipid spheres have a mode diameter of at least 1 pm based on volume; and / or

[0041] at least 45 vol.% of the lipid spheres have a diameter between 2 and 12 pm based on the total lipid volume; and

[0042] b. the lipids comprise 0.5 to 20 wt.% phospholipids based on total lipids, and wherein the lipid spheres are at least partially coated on the surface with a layer of phospholipids,

[0043] Improving the gut microbiota of a human infant.

[0044] In some jurisdictions, the administration of a nutritional composition to an infant is considered non-therapeutic. In those cases, the present application can be expressed as by a method comprising the administration of a nutritional composition, as defined above. For the sake of clarity, the method can also be defined as a non-therapeutic method. By definition, the word "non-therapeutic" excludes any therapeutic effect.

[0045] As used herein, the term "gut microbiota" refers to all microorganisms, including bacteria, archaea, viruses and fungi, found in the digestive tract of a human subject.

[0046] As used herein, "improving the gut microbiota of a human infant" preferably means preventing or treating an impaired gut microbiota of a human infant, or in other words, preventing or treating a dysbiosis of the gut microbiota of a human infant.

[0047] As used herein, the term "dysbiosis" refers to a disruption of the gut microbiome, leading to an imbalance of the gut microbiota.

[0048] Preferably, the gut microbiota is improved by decreasing the relative abundance of opportunistic pathogens in the gut microbiota, and / or by increasing the relative abundance of beneficial bacteria in the gut microbiota, and / or by decreasing the ratio between the relative abundance of opportunistic pathogens and the relative abundance of beneficial bacteria in the gut microbiota. More preferably, the gut microbiota is improved by decreasing the relative abundance of opportunistic pathogens in the gut microbiota.

[0049] Preferably, the opportunistic pathogens are selected from the phyla Proteobacteria and / or Bacillota. More preferably, the opportunistic pathogens are selected from the order Enterobacteriales and the family Clostridiaceae. Most preferably, the opportunistic pathogens are of the order Enterobacteriales.

[0050] Preferably, the Proteobacteria are Gamma Proteobacteria, more preferably Enterobacteriales, even more preferably Enterobacteriaceae, and still even more preferably the Enterobacteriaceae are one or more of the genera Citrobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Providencia, Salmonella, Shigella, and Yersinia. Most preferably, the Enterobacteriaceae are one or more of the genera Enterobacter, Escherichia, Klebsiella, and Shigella. Preferably, the Firmicutes are Clostridiales.

[0051] Preferably, the beneficial bacteria are selected from the family Lactobacillaceae and / or the family Bifidobacteriaceae, more preferably the family Bifidobacteriaceae.

[0052] Preferably, the gut microbiota is improved by decreasing the relative abundance of Enterobacteriales in the gut microbiota, and / or by increasing the relative abundance of Bifidobacteriaceae in the gut microbiota, and / or by decreasing the ratio between the relative abundance of Enterobacteriales and the relative abundance of Bifidobacteriaceae in the gut microbiota.

[0053] In preferred embodiments, the decrease in the relative abundance of opportunistic pathogens in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula, and young child formula, which nutritional composition comprises digestible carbohydrates, proteins, and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0054] a. the lipid spheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipid spheres have a diameter of more than 2 pm based on the total lipid volume; and

[0055] b. the lipids comprise 0-20 wt.% phospholipids based on the total lipids, and wherein the lipid spheres are not coated on the surface with phospholipids.

[0056] More preferably, the decrease in the relative abundance of opportunistic pathogens in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula, and young child formula, which nutritional composition comprises digestible carbohydrates, proteins, and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0057] a. the lipid spheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipid spheres have a diameter larger than 2 pm, based on the total lipid volume; and

[0058] b. the lipid comprises 0-20 wt.% phospholipids, based on total lipids, and wherein the lipid spheres are not coated with phospholipids on the surface.

[0059] In preferred embodiments, an increase in the relative abundance of beneficial bacteria in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, proteins and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0060] a. the lipid spheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipid spheres have a diameter larger than 2 pm, based on the total lipid volume; and

[0061] b. the lipid comprises 0-20 wt.% phospholipids, based on total lipids, and wherein the lipid spheres are not coated with phospholipids on the surface.

[0062] More preferably, an increase in the relative abundance of beneficial bacteria in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, proteins and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0063] a. the lipid spheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipid spheres have a diameter larger than 2 pm, based on the total lipid volume; and

[0064] b. the lipid comprises 0-20 wt.% phospholipids, based on total lipids, and wherein the lipid spheres are not coated with phospholipids on the surface.

[0065] In preferred embodiments, a decrease in the ratio between the relative abundance of opportunistic pathogens and the relative abundance of beneficial bacteria in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula and baby food formula, the nutritional composition comprising digestible carbohydrates, proteins and lipids, wherein the lipids are in the form of lipid spheres, wherein

[0066] a. the lipid spheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipid spheres have a diameter larger than 2 pm, based on the total lipid volume; and

[0067] b. the lipids comprise 0-20 wt.% phospholipids based on total lipids, and wherein the lipospheres are not coated on the surface with phospholipids.

[0068] More preferably, the reduction of the ratio between the relative abundance of opportunistic pathogens and the relative abundance of beneficial bacteria in a human infant is compared to a human infant consuming a nutritional composition selected from the group consisting of infant formula, follow-on formula and young child formula, the nutritional composition comprising digestible carbohydrates, proteins and lipids, wherein the lipids are in the form of lipospheres, wherein

[0069] a. the lipospheres have a volume-weighted mode diameter of 0.3-0.5 pm, and less than 45 vol.% of the lipospheres have a diameter of more than 2 pm based on total liposome volume; and

[0070] b. the lipids comprise 0.5 wt.% to 20 wt.% phospholipids based on total lipids, and wherein the lipospheres are at least partially coated on the surface with a layer of phospholipids.

[0071] The use according to the application preferably reduces the risk of developing an infection in early life, preferably the risk of developing an intestinal infection in early life. In another embodiment, the use according to the application preferably reduces the risk of developing an allergy, more preferably the risk of developing a food allergy.

[0072] In a preferred embodiment, the human infant is at risk of an impaired gut microbiota. Preferably, the human infant at risk of an impaired gut microbiota is selected from the group consisting of an infant born via caesarean section, a preterm infant, an infant born to an overweight or obese mother, an infant born to a mother receiving antibiotics, an infant receiving or having received antibiotics, an infant receiving or having received a proton pump inhibitor, a formula-fed infant, or a combination thereof. More preferably, the human infant at risk of an impaired gut microbiota is selected from the group consisting of an infant born via caesarean section, a preterm infant, and an infant born to a mother receiving intrapartum antibiotics. Most preferably, the human infant at risk of an impaired gut microbiota is an infant born via caesarean section.

[0073] As used herein, "infant born to a mother receiving antibiotics" refers to an infant born to a mother who received antibiotics in the two weeks prior to delivery or during delivery (intrapartum). Preferably, "infant born to a mother receiving antibiotics" refers to an infant born to a mother who received intrapartum antibiotics.

[0074] Preferably, the human infant is aged 0-36 months, more preferably 0-24 months, even more preferably 0-12 months, and most preferably 0-6 months. As used herein, the term "early life" refers to the first 0-36 months of life, preferably the first 0-24 months of life, more preferably the first 0-12 months of life, and most preferably the first 0-6 months of life.

[0075] Lipid globule size

[0076] The lipids are present in the nutritional composition in the form of lipid globules. When the nutritional composition is in liquid form, these lipid globules are emulsified in the aqueous phase. Alternatively, when the nutritional composition is in powder form, the lipid globules are present in the powder and the powder is suitable for reconstitution with water or another food grade aqueous phase. The lipid globules comprise a core and a surface.

[0077] The lipid globules in the nutritional composition preferably have a mode diameter of at least 1.0 pm, more preferably at least 3.0 pm, and most preferably at least 4.0 pm, based on volume. Preferably, the lipid globules have a mode diameter of between 1.0 and 10 pm, more preferably between 2.0 and 8.0 pm, even more preferably between 3.0 and 7.0 pm, and most preferably between 4.0 pm and 6.0 pm, based on volume.

[0078] Alternatively or preferably in addition, the size distribution of the lipid globules is preferably such that at least 45 volume % (vol. %), preferably at least 55 vol. %, even more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 2 and 12 pm. In a more preferred embodiment, at least 45 vol. %, preferably at least 55 vol. %, more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 2 and 10 pm. In a more preferred embodiment, at least 45 vol. %, more preferably at least 55 vol. %, even more preferably at least 65 vol. %, and most preferably at least 75 vol. % of the lipid globules have a diameter between 4 and 10 pm. Preferably, less than 5 vol. % of the lipid globules have a diameter larger than 12 pm.

[0079] The percentage of lipid globules is based on the total lipid volume. The mode diameter relates to the diameter that is present most, based on the total lipid volume, or the peak in a graph that represents diameter on the X-axis and volume (%) on the Y-axis.

[0080] The volume of the lipid globules and their size distribution can be suitably determined using a particle size analyser such as a Mastersizer 2000 (Malvern Instruments, Malvern, UK), for example by the method described by Michalski et al., 2001, Lait 81: 787-796.

[0081] phospholipids

[0082] The lipids in the nutritional composition comprise 0.5 to 20 wt.% of phospholipids based on total lipids, and wherein the lipid globules are at least partially coated on the surface with a layer of phospholipids. Preferably, the nutritional composition comprises 0.6 to 10 wt.%, more preferably 0.7 to 8 wt.%, even more preferably 0.8 to 8 wt.%, even more preferably 1 to 5 wt.% of phospholipids based on total lipids.

[0083] Phospholipids are essentially amphiphilic and include glycerophospholipids and sphingomyelin. By "coated" is meant that the outer surface layer of the lipid globules comprises phospholipids, whereas phospholipids are virtually absent in the core of the lipid globules. A suitable way to determine whether phospholipids are located on the surface of the lipid globules is confocal laser scanning microscopy or transmission electron microscopy; see for example Gallier et al. (A novel infant milk formula concept: Mimicking the human milk fat globule structure, Colloids and Surfaces B: Biointerfaces 136 (2015) 329-339).

[0084] The nutritional composition preferably comprises glycerophospholipids. Examples of glycerophospholipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Preferably, the nutritional composition comprises one or more of PC, PS, PI and PE, more preferably the nutritional composition comprises at least PC.

[0085] The nutritional composition preferably comprises sphingomyelin. Sphingomyelin has a phosphorylcholine or phosphoethanolamine molecule esterified to the 1 -hydroxyl group of ceramide. They are classified as phospholipids and sphingolipids, but not as glycerophospholipids or glycosphingolipids. Preferably, the nutritional composition comprises 0.05 to 10 wt.% sphingomyelin based on total lipids, more preferably 0.1 to 5 wt.%, even more preferably 0.2 to 2 wt.%. Preferably, the nutritional composition comprises at least 5 wt.% sphingomyelin based on total phospholipids, more preferably 5 to 40 wt.% sphingomyelin based on total phospholipids, more preferably 10 to 35 wt.% sphingomyelin based on total phospholipids, even more preferably 15 to 35 wt.% sphingomyelin based on total phospholipids.

[0086] The nutritional composition preferably comprises glycosphingolipids. The term glycosphingolipids herein specifically refers to glycolipids with an amino alcohol sphingosine. The sphingosine backbone is O-linked to a charged head group such as ethanolamine, serine or choline backbone. The backbone is also an amide group linked to a fatty acyl group. Glycosphingolipids are ceramides with one or more sugar residues linked in a beta-glycosidic linkage at the 1 -hydroxyl position and include gangliosides. Preferably, the nutritional composition contains a ganglioside, more preferably at least one ganglioside selected from the group consisting of GM3 and GD3. Preferably, the nutritional composition comprises 0.1 to 10 wt.% glycosphingolipids based on total lipids, more preferably 0.5 to 5 wt.% glycosphingolipids based on total lipids, even more preferably 2 to 4 wt.% glycosphingolipids based on total lipids.

[0087] The nutritional composition preferably comprises cholesterol. The nutritional composition preferably comprises at least 0.005 wt.% cholesterol based on total lipids, more preferably at least 0.02 wt.% cholesterol based on total lipids, more preferably at least 0.05 wt.% cholesterol based on total lipids, even more preferably at least 0.1 wt.% cholesterol based on total lipids. Preferably, the amount of cholesterol in the nutritional composition is no more than 10 wt.% cholesterol based on total lipids, more preferably no more than 5 wt.% cholesterol based on total lipids, even more preferably no more than 1 wt.% cholesterol based on total lipids.

[0088] Preferred sources of phospholipids, glycosphingolipids and / or cholesterol are egg lipids, milk fat, buttermilk fat and buttermilk serum fat (e.g. beta serum fat). Further preferred sources of phospholipids (in particular PC) are soy lecithin and / or sunflower lecithin.

[0089] The nutritional composition preferably comprises phospholipids derived from mammalian milk. Preferably, the nutritional composition comprises phospholipids and glycosphingolipids derived from mammalian milk. Preferably, cholesterol is also obtained from mammalian milk. The nutritional composition preferably comprises phospholipids, glycosphingolipids and / or cholesterol derived from mammalian milk of a cow, a mare, a sheep, a goat, a water buffalo, a horse and a camel, etc. More preferably, the nutritional composition comprises phospholipids, glycosphingolipids and / or cholesterol from cow milk.

[0090] The phospholipids derived from mammalian milk preferably include phospholipids isolated from milk lipid, cream lipid, cream serum lipid, butter serum lipid (beta serum lipid), whey lipid, cheese lipid and / or buttermilk lipid. Buttermilk lipid is typically obtained during the manufacture of buttermilk. Buttermilk serum lipid or beta serum lipid is typically obtained during the manufacture of anhydrous milk fat from butter. Preferably, the phospholipids, glycosphingolipids and / or cholesterol are obtained from milk cream. Suitable commercially available sources of phospholipids from milk are BAEF, SM2, SM3 and SM4 powders from Corman, Salibra from Glanbia, Vivinal MFGM from FrieslandCampina and LacProdan MFGM-10 or PL20 from Arla.

[0091] The use of phospholipids from mammalian milk fat advantageously includes the use of milk fat globule membranes, which are more similar to the situation in human milk. Thus, the use of phospholipids derived from mammalian milk in combination with triglycerides derived from plant lipids enables the manufacture of coated lipid globules with a coating more similar to human milk, while providing an optimal fatty acid profile.

[0092] Preferably, the phospholipids are derived from mammalian milk lipids, more preferably from milk fat globule membranes (MFGM). Preferably, the phospholipids are derived from bovine milk lipids, more preferably from bovine MFGM.

[0093] Preferably, the nutritional composition comprises phospholipids and glycosphingolipids, and more preferably the weight ratio of phospholipids : glycosphingolipids is from 2: 1 to 12: 1, more preferably from 2: 1 to 10: 1 and even more preferably from 2: 1 to 5: 1.

[0094] Methods for obtaining lipid globules with increased size and / or with a phospholipid coating are for example described in WO2010 / 0027258 and WO 2010 / 0027259.

[0095] Lipids

[0096] The nutritional composition according to the present use comprises lipids. The lipids in the present invention comprise one or more selected from the group consisting of triglycerides, polar lipids (such as phospholipids, cholesterol, glycolipids, sphingomyelin), free fatty acids, monoglycerides and diglycerides.

[0097] The lipids preferably represent 30% to 60% of the total calories of the nutritional composition. More preferably, the nutritional composition comprises lipids providing 35% to 55% of the total calories, even more preferably the nutritional composition comprises lipids providing 40% to 50% of the total calories. The lipids are preferably present in an amount of 4 to 6 g / 100 kcal. When in liquid form (e.g. as a ready-to-feed liquid), the nutritional composition preferably comprises 2.1 to 6.5 g lipids / 100 ml, more preferably 3.0 to 4.0 g / 100 ml. Based on dry weight, the nutritional composition preferably comprises 10 to 50 wt.%, more preferably 12.5 to 40 wt.%, even more preferably 19 to 30 wt.% lipids.

[0098] The lipids preferably comprise vegetable lipids. The presence of vegetable lipids advantageously achieves a high content of polyunsaturated fatty acids and / or a more optimal fatty acid profile more similar to human milk fat. Lipids from mammalian milk only (e.g. cow milk) cannot provide the optimal fatty acid profile. The amount of essential fatty acids in mammalian milk is too low.

[0099] Preferably, the nutritional composition comprises at least one, preferably at least two, vegetable lipid source selected from the group consisting of linseed oil (or flaxseed oil), rape seed oil (such as colza oil, canola oil and rapeseed oil), sunflower oil, high oleic sunflower oil, safflower oil, high oleic safflower oil, olive oil, coconut oil, palm oil and palm kernel oil.

[0100] In a preferred embodiment, the nutritional composition comprises 5 to 100 wt.% vegetable lipids based on total lipids, more preferably 10 to 95 wt.%, more preferably 20 to 80 wt.%, even more preferably 25 to 75 wt.%, most preferably 40 to 60 wt.%. It should thus be noted that the nutritional composition can also comprise non-vegetable lipids. Non-vegetable lipids can include mammalian milk fat, mammalian milk-derived lipids as a preferred source of phospholipids, and fish, marine and / or microbial oils as a source of LC-PUFAs.

[0101] Palmitic acid (PA) at the sn-2 position of triglycerides

[0102] The triglycerides preferably are the main fraction of the lipids in the nutritional composition. Triglycerides comprise a glycerol moiety which is attached via ester bonds to three fatty acid residues, which can be the same or different, and are typically selected from saturated and unsaturated fatty acids containing 4 to 26 carbon atoms. Such triglycerides can differ in the fatty acid residues present, and / or can differ in the respective positions of the fatty acid residues relative to the glycerol backbone (e.g. in the sn-1, sn-2 and / or sn-3 position).

[0103] Preferably, the nutritional composition comprises at least 70 wt.% of triglycerides based on total lipids, more preferably at least 80 wt.%, more preferably at least 85 wt.% of triglycerides, even more preferably at least 90 wt.% of triglycerides based on total lipids, even more preferably at least 95 wt.% of triglycerides based on total lipids.

[0104] When the lipid component has an increased amount of palmitic acid (PA) in the sn-2 position in triglycerides based on total PA, a further decrease in the relative abundance of opportunistic pathogens is observed, and thereby an improvement of the human infant gut microbiota is observed. PA relates to palmitic acid and / or acyl chains (C16:0).

[0105] Lipids which can be used to enhance the amount of PA in the sn-2 position in triglycerides based on total PA are commercially available, for example under the trade name Betapol TM Trade names are commercially available from Loders Croklaan company and / or can be prepared in a manner known per se, for example as described in EP 0698078 and / or EP 0758846. Another suitable source is InFat TM In case these lipids are obtained by transesterification or interesterification of plant triglycerides, these sources are considered plant lipids in the context of the present application.

[0106] A preferred source of triglycerides which enhance PA in the sn-2 or beta position in triglycerides is mammalian milk fat, preferably non-human mammalian milk fat, more preferably bovine milk fat. Preferably, the mammalian milk fat, in particular bovine milk fat, is used in the form of anhydrous milk fat, butter oil, butter fat or butter. Preferably, the source of mammalian milk fat is in a homogeneous fat phase, such as butter oil or anhydrous milk fat, and is not in the form of an oil-in-water emulsion, such as cream, as the lipid spheres of the present application can be more easily prepared during the manufacture of the nutritional composition for use according to the present application when the lipid is added to the aqueous phase as a homogeneous fat phase in which the mixture is processed to form an emulsion.

[0107] Preferably, the amount of the source of triglycerides having an increased amount of palmitic acid residues at the sn-2 position of triglycerides comprised in the lipids of the nutritional composition is between 10 and 99.5 wt.% based on total lipids, more preferably between 15 and 85 wt.% based on total lipids, more preferably between 20 and 75 wt.% based on total lipids, more preferably between 25 and 65 wt.% based on total lipids, even more preferably between 30 and 60 wt.% based on total lipids. Preferably, the nutritional composition comprises between 5 and 95 wt.% based on total lipids, more preferably between 20 and 80 wt.% based on total lipids, more preferably between 25 and 75 wt.% based on total lipids, even more preferably between 40 and 60 wt.% based on total lipids of mammalian milk fat.

[0108] In a particularly preferred embodiment, the lipids in the nutritional composition comprise:

[0109] a. 30 to 90 wt.% based on total lipids of plant fat, and

[0110] b. 10 to 70 wt.% based on total lipids of mammalian milk fat.

[0111] More preferably, the lipids in the nutritional composition comprise:

[0112] a. 35 to 75 wt.% based on total lipids of plant fat, and

[0113] b. 25 to 65 wt.% based on total lipids of mammalian milk fat.

[0114] Most preferably, the lipids in the nutritional composition comprise:

[0115] a. 40 to 60 wt.% based on total lipids of plant fat, and

[0116] b. 40 to 60 wt.% based on total lipids of mammalian milk fat.

[0117] The source of lipids in the nutritional composition is preferably chosen such that the amount of palmitic acid (PA) present in the total lipids of the nutritional composition is at least 10 wt.% based on total fatty acids, preferably at least 15 wt.%. Preferably, the amount of PA present in the total lipids based on total fatty acids is below 30 wt.%. More preferably, the amount of PA present in the lipids based on total fatty acids is from 15 to 24 wt.%, even more preferably from 15 to 19 wt.%, even more preferably from 16 to 19 wt.%.

[0118] The lipids in the nutritional composition are preferably selected such that at least 15 wt.%, preferably at least 20 wt.%, more preferably at least 25 wt.%, more preferably at least 30 wt.% of the PA based on total PA is in the sn-2 or beta position in triglycerides. Preferably, the amount of PA in the sn-2 position in triglycerides is no more than 45 wt.%, preferably no more than 40 wt.% based on total PA. Preferably, the amount of PA in the sn-2 position in triglycerides is from 25 wt.% to 40 wt.% based on total PA present in the total lipids.

[0119] In the context of the present application, the weight percentage of fatty acids based on total fatty acids is calculated on the assumption that all fatty acids are free fatty acids, thus not taking into account whether the fatty acids are attached to the glycerol backbone or not.

[0120] Fatty acid composition

[0121] SFA relates to saturated fatty acids and / or acyl chains, MUFA relates to monounsaturated fatty acids and / or acyl chains, PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds; LC-PUFA refers to long chain polyunsaturated fatty acids and / or acyl chains comprising at least 20 carbon atoms in the fatty acyl chain and having 2 or more unsaturated bonds; DHA refers to docosahexaenoic acid and / or acyl chains (22:6, n3); EPA refers to eicosapentaenoic acid and / or acyl chains (20:5 n3); ARA refers to arachidonic acid and / or acyl chains (20:4 n6); DPA refers to docosapentaenoic acid and / or acyl chains (22:5 n3). n3 or omega 3 PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds with the unsaturated bond located at the third carbon atom from the methyl end of the fatty acyl chain; n6 or omega 6 PUFA refers to polyunsaturated fatty acids and / or acyl chains having 2 or more unsaturated bonds with the unsaturated bond located at the sixth carbon atom from the methyl end of the fatty acyl chain.

[0122] The nutritional composition according to the present use preferably comprises LA, which refers to linoleic acid and / or acyl chains (18:2 n6). LA is a precursor of n6 PUFA and n6 LC-PUFA and is an essential fatty acid as it cannot be synthesized by the human body. LA is preferably present in a sufficient amount in order to promote healthy growth and development, but the amount thereof should be as low as possible in order to prevent a negative, competitive influence on the formation of n3 PUFA and an excessively high n6 / n3 ratio. Therefore, the nutritional composition preferably comprises less than 25 wt.%, more preferably less than 20 wt.%, more preferably less than 15 wt.% LA based on total fatty acids. The nutritional composition preferably comprises at least 5 wt.% LA based on total fatty acids, preferably at least 7.5 wt.%, more preferably at least 10 wt.% based on total fatty acids.

[0123] The nutritional composition preferably comprises ALA, which refers to a-linolenic acid and / or acyl chains (18:3 n3). ALA is a precursor of n3 PUFAs and n3 LC-PUFAs and is an essential fatty acid as it cannot be synthesized by the human body. Preferably, ALA is present in an amount sufficient to promote healthy growth and development of the infant. Thus, the nutritional composition preferably comprises at least 0.5 wt.%, more preferably at least 1.0 wt.%, more preferably the nutritional composition comprises at least 1.5 wt.% based on total fatty acids, even more preferably at least 2.0 wt.% of ALA. Preferably, the nutritional composition comprises less than 10 wt.% of ALA based on total fatty acids, more preferably less than 5.0 wt.% of ALA.

[0124] The weight ratio LA / ALA is preferably well balanced in order to ensure optimal n6 / n3 PUFA, n6 / n3 LC PUFA and DHA / ARA ratios in the cell membranes. Thus, the nutritional composition preferably comprises a LA / ALA weight ratio from 2 to 20, more preferably from 3 to 15, more preferably from 5 to 12, more preferably from 5 to 10. Preferably, the n6 PUFA / n3 PUFA weight ratio is from 3 to 20, more preferably from 3 to 15, more preferably from 5 to 12, more preferably from 5 to 10.

[0125] Preferably, the nutritional composition comprises n3 LC-PUFAs, such as EPA, DPA and / or DHA, more preferably DHA. Since the efficiency of the conversion of ALA to DHA in the infant can be low, it is preferred that both ALA and DHA are present in the nutritional composition. Preferably, the nutritional composition comprises at least 0.05 wt.% based on total fatty acids, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.% of DHA. Preferably, the nutritional composition comprises not more than 2.0 wt.% based on total fatty acids, preferably not more than 1.0 wt.% of DHA.

[0126] The nutritional composition preferably comprises ARA. Preferably, the nutritional composition comprises at least 0.05 wt.% based on total fatty acids, preferably at least 0.1 wt.%, more preferably at least 0.2 wt.% of ARA. The group of n6 fatty acids, in particular ARA, counterbalances the group of n3 fatty acids, in particular DHA, so the nutritional composition preferably comprises a relatively low amount of ARA. Preferably, the nutritional composition comprises not more than 2.0 wt.% based on total fatty acids, preferably not more than 1.0 wt.% of ARA. Preferably, the weight ratio between DHA and ARA is between 1 / 4 and 4 / 1, more preferably between 1 / 2 and 2 / 1, more preferably between 0.6 and 1.5.

[0127] Digestible carbohydrates

[0128] The nutritional composition comprises digestible carbohydrates. The digestible carbohydrates preferably provide 30% to 80% of the total calories of the nutritional composition. Preferably, the digestible carbohydrates provide 40% to 60% of the total calories. Based on calories, the nutritional composition preferably comprises 5 to 20 g digestible carbohydrates per 100 kcal, more preferably 7.5 to 15 g. When in liquid form (e.g. as a ready-to-feed liquid), the nutritional composition preferably comprises 3 to 30 g digestible carbohydrates per 100 ml, more preferably 6 to 20, even more preferably 7 to 10 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 20 wt.% to 80 wt.% digestible carbohydrates, more preferably 40 wt.% to 65 wt.%.

[0129] A preferred source of digestible carbohydrates is lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. Lactose advantageously has a low glycemic index. The nutritional composition preferably comprises lactose. The nutritional composition preferably comprises digestible carbohydrates wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.%, most preferably at least 95 wt.% of the digestible carbohydrates is lactose. Based on dry weight, the nutritional composition preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.%.

[0130] Protein

[0131] The nutritional composition comprises protein. The protein preferably provides 5% to 15% of the total calories. Preferably, the nutritional composition comprises protein providing 6% to 12% of the total calories. More preferably, the protein in the nutritional composition is present at less than 3.5 grams per 100 kcal, more preferably the nutritional composition comprises between 1.8 and 2.1 g protein per 100 kcal, even more preferably between 1.85 and 2.0 g protein per 100 kcal. The protein concentration in the nutritional composition is determined by the sum of protein, peptides and free amino acids. Based on dry weight, the nutritional composition preferably comprises less than 12 wt.% protein, more preferably between 9.6 wt.% and 12 wt.%, even more preferably between 10 wt.% and 11 wt.%. Based on ready-to-feed liquid products, the nutritional composition preferably comprises less than 1.5 g protein per 100 ml, more preferably between 1.2 and 1.5 g, even more preferably between 1.25 and 1.35 g.

[0132] The origin of the protein should be chosen in such a way that the minimum requirements for essential amino acid content are met and satisfactory growth is ensured. Thus, protein sources based on cow's milk proteins, such as whey proteins, caseins and mixtures thereof, and proteins based on soy, potato or pea are preferred. In case whey proteins are used, the protein source is preferably based on acid whey or sweet whey, whey protein isolates or mixtures thereof. Preferably, the nutritional composition comprises at least 3 wt.% of casein based on dry weight. Preferably, the casein is intact and / or non-hydrolyzed.

[0133] Non-digestible carbohydrates

[0134] In one embodiment, the nutritional composition preferably comprises non-digestible oligosaccharides. Preferably, the nutritional composition comprises non-digestible oligosaccharides having a degree of polymerization (DP) between 2 and 250, more preferably between 3 and 60. Non-digestible oligosaccharides advantageously further reduce the relative abundance of opportunistic pathogens and improve the gut microbiota of the human infant

[0135] Preferably, the nutritional composition comprises fructooligosaccharides, galactooligosaccharides and / or galacturonooligosaccharides, more preferably fructooligosaccharides and / or galactooligosaccharides, even more preferably galactooligosaccharides, most preferably transgalactooligosaccharides. In a preferred embodiment, the nutritional composition comprises a mixture of galactooligosaccharides and fructooligosaccharides, more preferably transgalactooligosaccharides and fructooligosaccharides. Suitable non-digestible oligosaccharides are for example GOS (FrieslandCampina DOMO), HP or (Orafti).

[0136] Preferably, the nutritional composition comprises 80 mg to 2 g non-digestible oligosaccharides per 100 ml, more preferably 150 mg to 1.5 g, even more preferably 300 mg to 1 g per 100 ml. Based on dry weight, the nutritional composition preferably comprises 0.25 wt.% to 20 wt.%, more preferably 0.5 wt.% to 10 wt.%, even more preferably 1.5 wt.% to 7.5 wt.% of non-digestible oligosaccharides.

[0137] Formulas

[0138] The use according to the present application entails the administration of an infant formula, a follow-on formula or a young child formula. This means that the composition administered is not human milk. It also means that the composition administered is not natural cow's milk or natural milk from another mammal. Alternatively, the term "infant formula" or "follow-on formula" or "young child formula" as used herein means that it relates to an artificially manufactured composition or that it is synthetic. Thus, in one embodiment, the nutritional composition administered is an artificial infant formula or an artificial follow-on formula or an artificial young child formula or a synthetic infant formula or a synthetic follow-on formula or a synthetic young child formula.

[0139] In the present context, infant formula refers to an artificially manufactured nutritional composition intended for infants during the first 0 to about 4 to 6 months of life and intended to be the sole source of nutritional intake. Typically infant formulae are suitable for use as the sole nutritional source. Such formulae are also referred to as starter formulae. Formulae for infants starting at 4 to 6 months of age up to 12 months of age are intended to be a supplementary food to the infant's diet as the infant starts weaning. Such formulae are also referred to as follow-on formulae. Infant formulae and follow-on formulae comply with strict regulations, for example EU Commission Directive 2006 / 141 / EC. In the present context, young child formula refers to an artificially manufactured nutritional composition intended for infants from 12 months to 36 months of age, which is intended to be a supplementary food to the infant's diet. Such formulae are also referred to as growing-up milks.

[0140] The nutritional composition is preferably an infant formula or a follow-on formula. More preferably, the nutritional composition is an infant formula.

[0141] The nutritional composition is preferably an infant formula or a follow-on formula and preferably comprises 3 to 7 g lipids / 100 kcal, preferably 4 to 6 g lipids / 100 kcal, more preferably 4.5 to 5.5 g lipids / 100 kcal, preferably comprises 1.7 to 5 g protein / 100 kcal, preferably 1.8 to 3.5 g protein / 100 kcal, more preferably 1.8 to 2.1 g protein / 100 kcal, more preferably 1.8 to 2.0 g protein / 100 kcal, and preferably comprises 5 to 20 g digestible carbohydrates / 100 kcal, preferably 6 to 16 g digestible carbohydrates / 100 kcal, more preferably 10 to 15 g digestible carbohydrates / 100 kcal.

[0142] Preferably, the nutritional composition is an infant formula or a follow-on formula, which has an energy density of 60 to 75 kcal per 100 ml, more preferably 60 to 70 kcal per 100 ml when in ready-to-feed form. This density ensures an optimal balance between hydration and caloric intake.

[0143] In one embodiment, the nutritional composition is a powder. Suitably, the nutritional composition is in powder form, which can be reconstituted with water or other food grade aqueous liquid to form a ready-to-feed liquid, or in liquid concentrate form, which should be diluted with water to a ready-to-feed liquid. It was found that the lipid spheres maintain their size and coating upon reconstitution.

[0144] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but not to the exclusion of items not specifically recited. Also, the indefinite articles "a" or "an" and "the" and singular forms of nouns, unless the context clearly dictates otherwise, do not exclude the possibility of plural forms. Thus, the indefinite articles "a" or "an" and "the" and singular forms of nouns are typically used in this document to mean "at least one" or "one or more." BRIEF DESCRIPTION OF DRAWINGS

[0145] Figures 1a-1c show the relative abundance of the three Enterobacteriaceae genera in the fecal microbiota of the breastfed reference group, the control group and the test group.

[0146] Figure 2 The relative abundance of the Clostridiales family in the fecal microbiota of the breastfed reference group, the control group and the test group is shown.

[0147] Figures 3a-3b show the relative abundance of the Enterobacteriales order and the Bifidobacteriaceae family in the fecal microbiota of fecal slurries fermented with IMF-A or IMF-1, respectively.

[0148] Figure 4 The ratio between the relative abundance of the Enterobacteriales order versus the Bifidobacteriaceae family in the fecal microbiota of fecal slurries fermented with IMF-A or IMF-1 is shown.

[0149] EXAMPLE

[0150] Example 1

[0151] The clinical study was conducted in 17 research centers in four countries, the Netherlands, Belgium, France and Singapore.

[0152] Subjects and study design

[0153] Healthy term infants with gestational age between 37 and 42 weeks, postnatal age < 35 days, and exclusively formula-fed or exclusively breastfed were eligible for participation. The study was designed as a randomized, double-blind, controlled, prospective, multinational, equicohort trial. After enrollment, formula-fed infants (n = 223) were randomly assigned to receive either the test formula (n = 115) or the control formula (n = 108), stratified by region (Europe / Asia), gender (male / female), and infant age at randomization (< 14 days / > 14 days). Breastfed infants (n = 88) served as a reference group and were eligible if the mother intended to breastfeed exclusively for at least 13 weeks. During the study, infants were exclusively formula-fed or exclusively breastfed. The infant cohort included vaginally born infants and infants born by caesarean section

[0154] Study infant formula

[0155] The two study infant formulas used in this study were complete standard cow milk-based infant formulas, which contained per 100 ml reconstituted formula 66 kcal, 1.3 g protein (intact protein with a casein / whey ratio of 40 / 60), 7.3 g digestible carbohydrates (mainly lactose), 3.4 g fat and 0.8 g short-chain galacto-oligosaccharides (source GOS) and long-chain fructo-oligosaccharides (source Raftilin ), as well as minerals, vitamins trace elements and other micronutrients known in the art and in line with the infant formula directive. The formulas were provided as a powder with instructions to reconstitute with water. About 13.6 g of powder was reconstituted into 100 ml of water to obtain the reconstituted infant formula. The two study formulas only differed in the source and size of the lipids used (Table 1). The fatty acid composition was very similar between the control and test formula, in saturated, monounsaturated and polyunsaturated fatty acids, as well as in n3- and n6-PUFA content.

[0156] Control formula

[0157] The fat component comprised mainly vegetable fat (blend of palm oil, low erucic rapeseed oil, coconut oil, high oleic sunflower oil and sunflower oil) and about 1.5 wt.% LC-PUFA premix (fish oil and microbial oil). No mammalian milk-derived phospholipids were added.

[0158] Test formula

[0159] The fat component consisted of about 50 wt.% vegetable fat (a blend of low erucic rapeseed oil, coconut oil, high oleic sunflower oil and sunflower oil), about 44 wt.% bovine anhydrous milk fat, 1.5 wt.% LC-PUFA containing oil (fish oil and microbial oil), about 3.6 wt.% mammalian milk fat derived from buttermilk enriched in milk phospholipids or milk fat globule membranes (milk phospholipids were about 1.5 wt.% based on total lipids). Lipid droplets in the test formula had a volume-based mode diameter of 5.6 pm and an interface consisting mainly of milk phospholipids, produced as described in WO 2013 / 135739.

[0160] Table 1 Composition of the study formula (per 100 ml)

[0161]

[0162] During the intervention, fecal samples were collected at 3 months of age and the fecal microbiota was determined by 16S rRNA sequencing. DNA was extracted from fecal samples as previously described (Wopereis, H. et al. Intestinal microbiota in infants at high risk for allergy: Effects of prebiotics and role in eczema development. J. Allergy Clin. Immunol. (2017)).

[0163] The V3-V5 region of the 16S rRNA gene was amplified using primers Bact-0341F and Bact-0785R (Klindworth, A. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, e1-e1 (2013)). Sequencing of amplicons was performed on an Illumina MiSeq instrument (Illumina Inc., San Diego, CA, USA) as previously described (Caporaso, J. G. et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012 686, 1621-1624 (2012)). OTU tables were generated from sequencing data using QIIME 1.9.0 (Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335-336 (2010)). Quality control filters were applied as previously described (van den Elsen, L. W. J. et al. Prebiotic oligosaccharides in early life alter gut microbiome development in male mice while supporting influenza vaccination responses. Benef. Microbes 10, 279-291 (2019)).The USEARCH algorithm was used for de novo OTU picking at 97% sequence identity (Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460-2461 (2010)). The SILVA database (release 1.1.9) was used for taxonomic assignment (Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41, D590-D596 (2013)). Singletons and low abundant OTUs with relative abundance <0.002% were excluded from downstream analysis.

[0164] Results

[0165] Figures 1a-1c show the relative abundance of the three Enterobacteriaceae genera in the fecal microbiota of the breastfed reference group, the control group and the test group.

[0166] Figure 2 The relative abundance of the Clostridiaceae family in the fecal microbiota of the breastfed reference group, the control group and the test group is shown.

[0167] It is known that the genera Escherichia, Shigella, Enterobacter, Klebsiella and the Clostridiaceae family encompass opportunistic pathogens. In the figures it can be observed that the relative abundance of these microorganisms is lower in the test group compared to the control group and more similar to the levels of relative abundance observed in the breastfed reference group.

[0168] Example 2

[0169] In vitro studies were carried out to compare the effect of an infant formula according to the application with large lipid globules (IMF-1) with a similar infant formula with smaller lipid globules (IMF-A).

[0170] Study infant formula

[0171] The two research infant formulas used in this example are complete standard cow milk-based infant formulas with the same composition except for the size of the lipid globules. The infant formulas contain 66 kcal, 1.3 g protein (intact protein with a casein / whey ratio of 40 / 60), 7.3 g digestible carbohydrates (mainly lactose), 3.4 g fat and 0.8 g short-chain galacto-oligosaccharides (source GOS) and long-chain fructo-oligosaccharides (source Raftilin ), as well as minerals, vitamins trace elements and other micronutrients known in the art and in line with the infant formula directive, per 100 ml of reconstituted formula.

[0172] The lipid source of both research formulas is identical. The fat component contains vegetable fat (a blend of low erucic rapeseed oil, coconut oil, high oleic sunflower oil, sunflower oil), bovine non- water milk fat, LC-PUFA containing oil (fish oil and microbial oil). In addition, the MFGM-enriched whey protein concentrate also provides additional milk fat including phospholipids (about 1.5 wt.% milk phospholipids based on total lipids). The resulting fatty acid composition of both research formulas is identical in terms of saturated, monounsaturated and polyunsaturated fatty acid as well as n3- and n6-PUFA content. The two research formulas only differ in the size of the lipid globules (Table 2).

[0173] The lipid globules of both IMF-A and IMF-1 have a coating comprising phospholipids. IMF-A is prepared by a standard production process, wherein milk-derived phospholipids are added prior to homogenization. IMF-1 is prepared following the production process as described in WO 2013 / 135739.

[0174] Table 2 Composition of the fat component of IMF-A and IMF-1 (per 100 ml)

[0175]

[0176] Colonic microbiota culture medium for fecal slurry fermentation:

[0177] Yeast extract 1 g / L, ammonium sulphate 2 g / L, K2HPO4 2 g / L, NaHCO3 3.2 g / L, NaCl 4,5 g / L, MgSO4.7H2O 0.5 g / L, Cysteine HCI 0.5 g / L, CaCl2.2H2O 0.4 g / L, Bile salts 25 mg / L, 2 ml metal solution (per L contains: 500 mg EDTA, 200 mg FeSO4.7H2O, 10 mg ZnSO4.7H2O, 3 mg MnCl2.7H2O, 30 mg H3BO3, 20 mg CoCl2.6H2O, 1 mg CuCl2.2H2O, 2 mg NiCl2.6H2O, 3 mg NaMoO4.2H2O, 7.5 mg NaSeO3), and vitamin solution (per L contains: 1 g menadione, 2 g biotin, 2 g pantothenate, 10 g nicotinamide, 0.5 g cobalamin, 4 g thiamine, 5 g p-aminobenzoic acid; filter sterilized), and hemin (10 mg / L).

[0178] Methods

[0179] A healthy one-year old infant fecal sample was obtained. The fecal sample was fermented in the presence of IMF-A or IMF-1.

[0180] The infant fecal sample was thawed under anaerobic conditions and made into approximately 10% (w / v) fecal sample suspension in age-adapted colon microbiota medium (medium pH adjusted to 6.5). The diluted fecal sample was homogenized, allowed to sediment for 5 minutes, and then filtered through a Millex 100 pm vacuum filter. A Biolector Pro plate with pH optodes (BOH2 round well, M2P-labs) was used. Two wells in row C of the plate were filled with 800 pL of fecal solution and 800 pL of each infant formula (IMFA or IMF1), respectively. One feed row of the plate was filled with sterile 3M NaOH.

[0181] Thereafter, the plate was sealed with a vented silicone foil with slits. The plate was incubated in a BioLector Pro (85% moisture, 37°C, 600 rpm, anaerobic (90% N2, 5% CO2, 5% H2)). The experiment was started at a set value of 6.5 pH and continuous pH control was performed. The experiment was stopped after 56 hours of fermentation and the fecal slurry of each well was collected and briefly centrifuged. All manipulations were performed under non-sterile anaerobic conditions. The supernatant was frozen for further analysis while the fecal pellet was used to extract DNA prior to 16S rRNA sequencing.

[0182] The fecal microbiota was determined by the method as described in Example 1.

[0183] Results

[0184] Figures 3a-3b show the relative abundance of Enterobacteriales and Bifidobacteriaceae in the fecal microbiota of fecal slurries fermented with IMF-A or IMF-1, respectively.

[0185] Figure 4 The ratio between the relative abundance of Enterobacteriales versus Bifidobacteriaceae in the fecal microbiota of fecal slurries fermented with IMF-A or IMF-1 is shown.

[0186] Enterobacteriales are known to encompass opportunistic pathogens, while Bifidobacteriaceae are known to encompass beneficial bacteria. It is clear from Figure 3 that IMF-1 reduces the relative abundance of opportunistic pathogens (Figure 3a) while increasing the relative abundance of beneficial bacteria (Figure 3b) compared to IMF-A. Figure 4 The ratio of opportunistic pathogens to beneficial bacteria is lower for IMF-1 compared to IMF-A. The only difference between IMF-A and IMF-1 is the size of the lipid spheres. Therefore, the beneficial effect on the gut microbiota can be attributed to the presence of larger lipid spheres.

Claims

1. A nutritional composition selected from infant formula, follow-up formula, and toddler formula, the nutritional composition comprising digestible carbohydrates, proteins, and lipids, wherein the lipids are in the form of lipid globules, wherein... a. The lipid spheres have a volume-weighted mode diameter of at least 1.0 μm; and / or Based on the total lipid volume, at least 45 vol.% of these lipid spheres have a diameter between 2 and 12 μm; and b. The lipid comprises 0.5 wt.% to 20 wt.% phospholipids based on total lipids, and wherein these lipid globules are at least partially coated with a layer of phospholipids on their surface. For use in improving the gut microbiota of human infants.

2. The nutritional composition for use according to claim 1, wherein the gut microbiota of human infants is improved by treating and / or preventing gut microbiota dysbiosis.

3. The nutritional composition for use according to claim 1 or 2, wherein the gut microbiota is improved by reducing the relative abundance of opportunistic pathogens in the gut microbiota, and / or by increasing the relative abundance of beneficial bacteria in the gut microbiota, and / or by reducing the ratio between the relative abundance of opportunistic pathogens and the relative abundance of beneficial bacteria in the gut microbiota.

4. The nutritional composition for use according to claim 3, wherein these opportunistic pathogens are selected from the order Enterobacteriaceae and the family Clostridium.

5. The nutritional composition for use according to claim 4, wherein the order Enterobacteriaceae is Enterobacteriaceae, preferably one or more of Enterobacter, Escherichia, Klebsiella and Shigella.

6. The nutritional composition for use according to claim 3, wherein the beneficial bacteria are selected from the Lactobacillus family and / or Bifidobacterium family.

7. The nutritional composition for use according to any one of the preceding claims, wherein the use reduces the risk of infection in early life.

8. The nutritional composition for use according to any one of the preceding claims, wherein the use reduces the risk of allergic reactions.

9. The nutritional composition for use according to any one of the preceding claims, wherein the human infant is at risk of impaired gut microbiota.

10. The nutritional composition for use according to claim 9, wherein the human infant at risk of impaired gut microbiota is selected from the group consisting of: infants born by cesarean section, premature infants, infants born to overweight or obese mothers, infants born to mothers receiving antibiotics, infants currently receiving or having received antibiotics, infants currently receiving or having received proton pump inhibitors, formula-fed infants, or combinations thereof.

11. The nutritional composition for use according to any one of the preceding claims, wherein the phospholipids comprise at least 5 wt.% sphingomyelin based on total phospholipids.

12. The nutritional composition for use according to any one of the preceding claims, wherein the phospholipids are mammalian milk-derived phospholipids.

13. The nutritional composition for use according to any one of the preceding claims, wherein the lipid contains at least 10 wt.% palmitic acid based on total fatty acids, and at least 15 wt.% palmitic acid based on total palmitic acid is located at the sn-2 position of the triglyceride.

14. The nutritional composition for use according to any one of the preceding claims, wherein the nutritional composition is for feeding human infants aged 0-36 months.

15. The nutritional composition for use according to any one of claims 3-14, wherein a decrease in the relative abundance of opportunistic pathogens or an increase in the relative abundance of beneficial bacteria in the human infant is compared with that of a human infant consuming a nutritional composition selected from infant formula, follow-up formula, and toddler formula containing digestible carbohydrates, proteins, and lipids, wherein the lipids are in the form of lipid globules, wherein... a. These lipospheres have a volume-weighted mode diameter of 0.3–0.5 μm, and based on the total lipid volume, less than 45 vol.% of these lipospheres have a diameter greater than 2 μm; and b. The lipid comprises 0.5 wt.% to 20 wt.% of phospholipids based on total lipids, and wherein these lipid globules are at least partially coated with a layer of phospholipids on their surface.

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