Human milk oligosaccharides for improving gut microbiome and methods for determining state of maturation of gut

Abstract

The present invention provides methods for inducing the enteric microbiome trajectory of a formula-fed infant to converge with a reference enteric microbiome trajectory obtained from a human milk-fed infant, comprising administering to the formula-fed infant an effective amount of a mixture of human milk oligosaccharides (HMOs). The invention also provides methods for determining the gut maturity status of a formula-fed infant comprising providing a reference gut microbiome trajectory obtained from a human milk-fed infant.

Description

Technical Field

[0001] The present invention relates to methods for inducing convergence of an infant's gut microbiome trajectory with a reference gut microbiome trajectory and maintaining or sustaining convergence with a reference gut microbiome trajectory. The present invention also relates to methods for determining the intestinal maturation state of an infant and / or toddler. Background Art

[0002] Breast milk generally provides the reference nutrition for all infants, and exclusive breastfeeding is usually recommended for the first six months. Feeding pattern (exclusive breastfeeding or partial breastfeeding vs. no breastfeeding in early infancy) has a recognized effect on the composition and function of the gut microbiome. For example, the longer the duration of exclusive breastfeeding, the less gut microbiota dysbiosis associated with diarrhea. In addition, differences in the gut microbiota between breastfed and non-breastfed infants may persist after 6 months of age (see, e.g., Ho, NT et al., 2018. Nature communications, 9(1), pp. 1-13).

[0003] The health benefits observed in breastfed infants can include protection against infection and possibly reducing the development of overweight and diabetes later in life. This suggests that early life microbiome maturation altered by nutrition may affect later health. Many observational cohort studies and randomized controlled trials have shown that the composition of infant formula consumed has a considerable effect on the microbiome (see, for example, Dogra, SK et al., 2021. Microorganisms, 9(10), p. 2110).

[0004] For example, studies have shown that adding two specific human milk oligosaccharides, 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), to infant formula results in a gut microbiome composition closer to that of breastfed infants. However, at 12 months, the gut microbiome composition of the breastfed group was still significantly different from that of the formula group (see, e.g., Berger, B. et al., 2020. Mbio, 11(2), pp. e03196-19). Summary of the invention

[0005] The present inventors have shown that the addition of a mixture of human milk oligosaccharides (HMOs) to infant formula, follow-on formula and growing-up milk can induce the gut microbiome trajectory of formula-fed infants to converge and maintain or sustain convergence with a reference gut microbiome trajectory obtained from a human milk-fed group. When a mixture of HMOs was added to infant formula, follow-on formula and growing-up milk, the gut microbiome trajectories converged earlier and the number of formula-fed infants and toddlers who were outliers was significantly reduced.

[0006] In one aspect, the present invention provides a mixture of human milk oligosaccharides (HMOs) for inducing a gut microbiome trajectory of an infant to converge with a reference gut microbiome trajectory and to maintain or sustain convergence with a reference gut microbiome trajectory.

[0007] In another aspect, the present invention provides a method for inducing convergence of an infant's gut microbiome trajectory with a reference gut microbiome trajectory and maintaining or sustaining convergence with a reference gut microbiome trajectory, the method comprising administering to the infant an effective amount of a mixture of human milk oligosaccharides (HMOs).

[0008] In another aspect, the present invention provides the use of a mixture of human milk oligosaccharides (HMOs) to induce a gut microbiome trajectory of an infant to converge with a reference gut microbiome trajectory and to maintain or sustain convergence with a reference gut microbiome trajectory.

[0009] The mixture of HMOs may comprise any suitable HMO, which may be administered in any suitable form, in any suitable amount and for any suitable duration. Suitably, the mixture of HMOs may comprise at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide and at least one sialylated oligosaccharide. Suitably, the mixture of HMOs comprises or consists of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL). Suitably, the mixture of HMOs is administered in the form of infant formula and / or growing-up milk. Suitably, the mixture of HMOs comprises or consists of: (i) 2'FL in an amount of about 55 wt% to about 60 wt%; (ii) diFL in an amount of about 5 wt% to about 7 wt%; (iii) LNT in an amount of about 18 wt% to about 20 wt%; (iv) 3'-SL in an amount of about 6 wt% to about 8 wt%; and (v) 6'-SL in an amount of about 9 wt% to about 11 wt%, based on the total weight of the HMOs. Suitably, the mixture of HMOs is administered to infants until at least about 6 months of age, until at least about 9 months of age, until at least about 12 months of age, or until at least about 15 months of age.

[0010] In a preferred embodiment, the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk. Suitably, the mixture of HMOs is administered in the form of a newborn formula comprising a total amount of HMOs of about 0.5 g / L to about 5.0 g / L, about 1.0 g / L to about 3.0 g / L or about 1.5 g / L to about 2.5 g / L; a follow-on formula comprising a total amount of HMOs of about 0.1 g / L to about 2.0 g / L, about 0.2 g / L to about 1.0 g / L, about 0.3 g / L to about 0.8 g / L, about 0.35 g / L to about 0.65 g / L; and / or a growing-up milk comprising a total amount of HMOs of about 0.1 g / L to about 1.0 g / L, about 0.2 g / L to about 0.8 g / L, about 0.28 g / L to about 0.52 g / L or about 0.3 g / L to about 0.5 g / L.

[0011] Suitably, the mixture of HMOs is administered in the form of a newborn infant formula comprising: (i) 2'FL in an amount of about 0.5 g / L to about 3.0 g / L; (ii) diFL in an amount of about 0.05 g / L to about 0.3 g / L; (iii) LNT in an amount of about 0.1 g / L to about 1.0 g / L; (iv) 3'-SL in an amount of about 0.05 g / L to about 0.3 g / L; and (v) 6'-SL in an amount of about 0.05 g / L to about 0.5 g / L. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula comprising: (i) 2'FL in an amount of about 0.70 g / L to about 1.05 g / L or about 1.16 g / L to about 1.74 g / L; (ii) diFL in an amount of about 0.05 g / L to about 0.11 g / L or about 0.12 g / L to about 0.18 g / L; (iii) LNT in an amount of about 0.23 g / L to about 0.36 g / L or about 0.39 g / L to about 0.58 g / L; (iv) 3'-SL in an amount of about 0.09 g / L to about 0.13 g / L or about 0.14 g / L to about 0.21 g / L; and (v) 6'-SL in an amount of about 0.12 g / L to about 0.17 g / L or about 0.19 g / L to about 0.28 g / L. Suitably, the mixture of HMOs is administered in the form of an follow-on formula comprising: (i) 2'FL in an amount of about 0.19 g / L to about 0.34 g / L; (ii) diFL in an amount of about 0.03 g / L to about 0.05 g / L; (iii) LNT in an amount of about 0.06 g / L to about 0.11 g / L; (iv) 3'-SL in an amount of about 0.04 g / L to about 0.09 g / L; and (v) 6'-SL in an amount of about 0.03 g / L to about 0.06 g / L. Suitably, the mixture of HMOs is administered in the form of a growing-up milk comprising: (i) 2'FL in an amount of about 0.15 g / L to about 0.28 g / L; (ii) diFL in an amount of about 0.01 g / L to about 0.04 g / L; (iii) LNT in an amount of about 0.05 g / L to about 0.09 g / L; (iv) 3'-SL in an amount of about 0.03 g / L to about 0.08 g / L; and (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L.

[0012] In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of HMOs of about 1.5 g / L. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula comprising: (i) 2'FL in an amount of about 0.87 g / L; (ii) diFL in an amount of about 0.10 g / L; (iii) LNT in an amount of about 0.29 g / L; (iv) 3'-SL in an amount of about 0.11 g / L; and (v) 6'-SL in an amount of about 0.14 g / L. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of HMOs of about 2.5 g / L. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula comprising: (i) 2'FL in an amount of about 1.45 g / L; (ii) diFL in an amount of about 0.14 g / L; (iii) LNT in an amount of about 0.48 g / L; (iv) 3'-SL in an amount of about 0.18 g / L; and (v) 6'-SL in an amount of about 0.24 g / L. In some embodiments, the mixture of HMOs is administered in the form of a follow-on infant formula comprising a total amount of HMOs of about 0.5 g / L. In some embodiments, the mixture of HMOs is administered in the form of an follow-on formula comprising: (i) 2'FL in an amount of about 0.26 g / L; (ii) diFL in an amount of about 0.04 g / L; (iii) LNT in an amount of about 0.09 g / L; (iv) 3'-SL in an amount of about 0.06 g / L; and (v) 6'-SL in an amount of about 0.05 g / L. In some embodiments, the mixture of HMOs is administered in the form of a growing-up milk comprising a total amount of HMOs of about 0.4 g / L. In some embodiments, the mixture of HMOs is administered in the form of a growing-up milk comprising: (i) 2'FL in an amount of about 0.21 g / L; (ii) diFL in an amount of about 0.03 g / L; (iii) LNT in an amount of about 0.07 g / L; (iv) 3'-SL in an amount of about 0.06 g / L; and (v) 6'-SL in an amount of about 0.04 g / L.

[0013] Newborn infant formula milk powder, older infant formula milk powder and growing-up milk can each comprise any other suitable component. Suitably, newborn infant formula milk powder, older infant formula milk powder and growing-up milk each comprise the protein of the amount of about 60kcal / 100mL to about 80kcal / 100mL, the carbohydrate of the amount of about 1.5g / 100kcal to about 2.5g / 100kcal, the lipid of the amount of about 8g / 100kcal to about 15g / 100kcal and about 3g / 100kcal to about 8g / 100kcal.

[0014] At about 12 months of age or earlier, about 11 months of age or earlier, about 10 months of age or earlier, or about 9 months of age or earlier, the infant's gut microbiome trajectory may converge with a reference gut microbiome trajectory and remain or maintain convergence with a reference gut microbiome trajectory. At about 6 months of age or later, about 7 months of age or later, about 8 months of age or later, or about 9 months of age or later, the infant's gut microbiome trajectory may converge with a reference gut microbiome trajectory and remain or maintain convergence with a reference gut microbiome trajectory. At about 6 months to about 12 months of age, about 7 months to about 11 months of age, about 8 months to 10 months of age, or about 9 months of age, the infant's gut microbiome trajectory may converge with a reference gut microbiome trajectory and remain or maintain convergence with a reference gut microbiome trajectory.

[0015] The gut microbiome trajectory of the infant and the reference gut microbiome trajectory can be obtained by any suitable method. The reference gut microbiome trajectory can be obtained from a human milk feeding group (infants and / or toddlers). Suitably, the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory. Suitably, the gut microbiome trajectory is a gut microbiome age trajectory. Suitably, the gut microbiome age trajectory is obtained using genus level data, species level data and / or functional data from the gut microbiome data.

[0016] The infant may be any infant whose gut microbiome composition differs from a reference gut microbiome trajectory. The infant may be a formula-fed infant. Suitably, the infant is full term. Suitably, the infant is delivered by caesarean section.

[0017] Inducing the infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory and maintaining or sustaining convergence with the reference gut microbiome trajectory can be associated with health benefits. For example, inducing the infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory can promote intestinal maturation. Inducing the infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory can promote maturation of the infant's gut microbiome, gut metabolism, gut barrier function, and / or gut immune function.

[0018] In one aspect, the present invention provides a method for determining the state of intestinal maturation of a formula-fed infant or toddler, wherein the method comprises:

[0019] (a) provide gut microbiome data from a population of human milk-fed infants and / or toddlers;

[0020] (b) training a regression model based on gut microbiome data; and

[0021] (c) providing gut microbiome data from a formula-fed infant or toddler and determining whether the formula-fed infant or toddler is an outlier in a trained regression model,

[0022] wherein if the formula-fed infant or toddler is not an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant or toddler is normal, and / or wherein if the formula-fed infant or toddler is an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant or toddler is abnormal.

[0023] In another aspect, the present invention provides a method for determining the intestinal maturation state of a formula-fed infant or toddler, wherein the method comprises:

[0024] (a) provide gut microbiome data from a population of human milk-fed infants and / or toddlers;

[0025] (b) training a regression model based on gut microbiome data to provide gut microbiome trajectories;

[0026] as well as

[0027] (c) provide gut microbiome data from formula-fed infants or toddlers and determine whether the formula-fed infants or toddlers meet the gut microbiome trajectory,

[0028] wherein if the formula-fed infant or toddler meets the gut microbiome trajectory, then the intestinal maturation state of the formula-fed infant or toddler is normal, and / or wherein if the formula-fed infant or toddler does not meet the gut microbiome trajectory, then the intestinal maturation state of the formula-fed infant or toddler is abnormal.

[0029] Any suitable statistical method may be used to determine whether a formula-fed infant or toddler is an outlier in a trained regression model and / or conforms to a gut microbiome trajectory. Suitably, a formula-fed infant or toddler is an outlier and / or does not conform to a trajectory based on a standard error (SE), confidence interval, prediction interval, and / or standard deviation in a trained regression model or gut microbiome trajectory. Suitably, a formula-fed infant or toddler is an outlier and / or does not conform to the trajectory if the gut microbiome data of the formula-fed infant or toddler differs from a trained regression line or gut microbiome trajectory by -2 SE or less or 2 SE or more, -2.5 SE or less or 2.5 SE or more, or -3 SE or less or 3 SE or more, if the gut microbiome data of the formula-fed infant or toddler falls outside the 90%, 95% or 99% confidence interval in the trained regression model or gut microbiome trajectory, if the gut microbiome data of the formula-fed infant or toddler falls outside the 90%, 95% or 99% prediction interval in the trained regression model or gut microbiome trajectory, and / or if the formula-fed infant or toddler has a Z-score of -2 or less or 2 or greater, -2.5 or less or 2.5 or greater, or -3 or less or 3 or greater in the trained regression model or gut microbiome trajectory.

[0030] In another aspect, the present invention provides a data processing system comprising means for performing the method according to the present invention for determining the state of intestinal maturation of a formula-fed infant or toddler.

[0031] In another aspect, the present invention provides a processor configured to perform a method for determining the state of intestinal maturation of a formula-fed infant or toddler according to the present invention.

[0032] In another aspect, the invention provides a computer readable medium comprising instructions which, when executed by a computer, cause the computer to perform a method for determining the intestinal maturity state of a formula-fed infant or toddler according to the invention.

[0033] In another aspect, the invention provides a computer program comprising instructions which, when executed by a computer, cause the computer to perform a method according to the invention for determining the state of intestinal maturation of a formula-fed infant or toddler.

[0034] In another aspect, the invention provides a computer readable data carrier having stored thereon a computer program according to the invention.

[0035] In another aspect, the invention provides a data carrier signal carrying the computer program according to the invention.

[0036] In another aspect, the invention provides the use of one or more reference gut microbiome trajectories for determining the state of intestinal maturation in a formula-fed infant or toddler following administration of a mixture of HMOs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 – Schematic diagram of HMO trial

[0038] Healthy term infants were randomly assigned to standard bovine milk-based infant formula (control group, CG); the same formula with 1.5 g / L HMO (experimental group 1, TG1); or the same formula with 2.5 g / L HMO (experimental group 2, TG2); or human milk feeding group (reference, HMG). Fecal samples were collected at enrollment, 3 months, 6 months, 12 months, and 15 months of age.

[0039] Figure 2 – Modeling the age of the gut microbiome

[0040] The overall process of modeling microbiome age is shown.

[0041] Figure 3 – Example of gut microbiome trajectories by model

[0042] LOESS fits the microbiome age trajectory for each feeding group using an age predictor trained on data from the vaginally delivered HMG group (infants and / or toddlers) using the following: (A) genus-level data (10 features selected, R 2 =0.862); (B) MGS species-level data (20 selected features, R 2 =0.881); (C) MGS species-level data (25 selected features, R 2 =0.844); (D) CAZyme data (30 selected features, R 2 =0.658); (E) Shannon index by gene. The shaded area indicates the 95% confidence interval.

[0043] Figure 4 – Examples of gut microbiome trajectories by delivery mode

[0044] LOESS fits the microbiome age trajectory for each feeding group using an age predictor trained on data from the HMG group (infants and / or toddlers) delivered vaginally using CAZyme data (30 features selected) for: (A) formula-fed group (infants and / or toddlers) delivered vaginally; (B) formula-fed group (infants and / or toddlers) delivered by cesarean section. Shaded areas indicate 95% confidence intervals.

[0045] Figure 5 – Example outliers in genus-level data

[0046] MAZ values ​​were calculated based on an age predictor that used genus-level data (10 features selected, optimized with RMSE, R 2 =0.862) was trained based on data from the HMG group (infants and / or toddlers) who were delivered vaginally. DETAILED DESCRIPTION

[0047] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will appreciate that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0048] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "comprising" and "consisting of" are synonymous with "including" or "containing" and are inclusive or open-ended and do not exclude additional unrecited members, elements, or method steps. The terms "comprising" and "consisting of" also include the term "consisting of".

[0049] Numerical ranges include the numbers that define the range. As used herein, the term "about" means approximately, in the vicinity, roughly, or around. When the term "about" is used in conjunction with a numerical value or range, it modifies the value or range by extending the boundaries above and below the numerical value shown. Generally speaking, the terms "about" and "approximately" are used herein to modify numerical values ​​above and below the stated value by 10%.

[0050] The concentration of a component in a composition described herein may refer to the concentration of the composition after reconstitution, for example, with water.

[0051] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0052] The methods and systems disclosed herein may be used by physicians, healthcare professionals, laboratory technicians, infant and / or toddler care providers, and the like.

[0053] Human Milk Oligosaccharide (HMO) Mix

[0054] The present invention provides a mixture of human milk oligosaccharides (HMOs) for inducing a gut microbiome trajectory of an infant to converge with a reference gut microbiome trajectory and to maintain or sustain convergence with a reference gut microbiome trajectory.

[0055] Human milk oligosaccharides (HMO)

[0056] Any suitable mixture of HMOs may be used in the present invention.

[0057] As used herein, "human milk oligosaccharides" or "HMOs" (also known as human milk polysaccharides) are short polymers of monosaccharides that can be found in high concentrations in human breast milk. Many different types of HMOs are found in human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid, fucose and / or N-acetylglucosamine, with a wide variety of bonds between them, thus explaining the large number of different oligosaccharides in human milk. Most HMOs have a lactose moiety at their reducing end, while the terminal positions of the non-reducing end are occupied by sialic acid and / or fucose (if any). HMOs can be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucosylated oligosaccharides).

[0058] The mixture of HMOs may comprise two or more individual HMOs, three or more individual HMOs, four or more individual HMOs, or five or more individual HMOs. In some embodiments, the mixture of HMOs comprises five or more individual HMOs. In some embodiments, the mixture of HMOs comprises five individual HMOs.

[0059] Suitable HMOs for use in the present invention and abundant in human milk may include 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), 3-fucosyllactose (3FL), lacto-N-fucopentaose-I (LNFP-I), lacto-N-fucopentaose-II (LNFP-II), lacto-N-fucopentaose-III (LNFP-III), lacto-N-difucosyhexaose-I (LNDFH-I), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) and disialyllacto-N-tetraose (DSLNT).

[0060] The HMO used in the present invention can be obtained by any suitable method. Suitable methods for synthesizing HMO are well known to those skilled in the art. For example, methods for preparing HMO by microbial fermentation, enzymatic methods, chemical synthesis, or a combination of these techniques have been developed (see, for example, Zeuner et al., 2019. Molecules, 24(11), p.2033).

[0061] In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide and / or at least one sialylated oligosaccharide. In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide and at least one sialylated oligosaccharide.

[0062] In some embodiments, the mixture of HMOs comprises or consists of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

[0063] In some embodiments, the mixture of HMOs consists of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

[0064] Fucosylated oligosaccharides

[0065] In some embodiments, the mixture of HMOs comprises at least one fucosylated oligosaccharide.

[0066] Suitably, the at least one fucosylated oligosaccharide comprises or consists of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), 3-fucosyllactose (3FL), lacto-N-fucopentaose-I (LNFP-I), lacto-N-fucopentaose-II (LNFP-II), lacto-N-fucopentaose-III (LNFP-III), lacto-N-fucopentaose-V (LNFP-V), lacto-neofucopentaose V (LNnFP-V), lacto-N-difucosylhexaose-I (LNDFH-1), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyl lacto-n-hexaose-III (MFNLH-III), difucosyl lacto-N-hexaose-a (DFLNHa) and any combination thereof.

[0067] In some embodiments, the at least one fucosylated oligosaccharide comprises or consists of 2'-fucosyllactose (2'FL) and / or 2',3-difucosyllactose (diFL). In some embodiments, the at least one fucosylated oligosaccharide consists of 2'-fucosyllactose (2'FL) and 2',3-difucosyllactose (diFL).

[0068] The at least one fucosylated oligosaccharide can be obtained by any suitable method. For example, 2'FL can be prepared by biotechnological means using specific fucosyltransferases and / or fucosidases, by using enzyme-based (recombinant or natural enzyme) fermentation techniques or microbial fermentation techniques. In the latter case, the microorganism can express its natural enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose. diFL can be synthesized by enzymes, biotechnology and / or chemical methods.

[0069] N-acetylated oligosaccharides

[0070] In some embodiments, the mixture of HMOs comprises at least one N-acetylated oligosaccharide.

[0071] Suitably, the at least one N-acetylated oligosaccharide comprises or consists of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), N-acetyl-glucosamine, N-acetyl-galactosamine or any combination thereof.

[0072] In some embodiments, the at least one N-acetylated oligosaccharide consists of lacto-N-tetraose (LNT).

[0073] N-acetylated oligosaccharides can be obtained by any suitable method. For example, LNnT can be chemically synthesized by enzymatic transfer of sugar units from donor moieties to acceptor moieties using glycosyltransferases. Alternatively, LNnT can be prepared by chemically converting free or oligosaccharide (e.g., lactulose)-bound ketone-hexose (e.g., fructose) into N-acetylhexosamine or oligosaccharides containing N-acetylhexosamine. LNT can be synthesized by enzymes, biotechnology and / or chemical methods.

[0074] Sialyl oligosaccharides

[0075] In some embodiments, the mixture of HMOs comprises at least one sialylated oligosaccharide.

[0076] Suitably, the at least one sialylated oligosaccharide comprises or consists of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllacto-N-tetraose b (LSTb), sialyllacto-N-tetraose c (LSTc), disialyllacto-N-tetraose (DSLNT) and any combination thereof.

[0077] In some embodiments, the at least one sialylated oligosaccharide comprises or consists of: 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL). In some embodiments, the at least one sialylated oligosaccharide consists of: 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL).

[0078] Sialyl oligosaccharides can be obtained by any suitable method. For example, 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL) can be isolated from natural sources (such as animal milk) using chromatography or filtration techniques. Alternatively, specific sialyltransferases or sialidases, neuraminidase, can also be used, by biotechnological means, by fermentation techniques based on enzymes (recombinant enzymes or natural enzymes), by chemical synthesis or by microbial fermentation techniques, to prepare sialyllactose. In the latter case, the microorganism can express its natural enzyme and substrate, or can be engineered to produce the corresponding substrate and enzyme. Single microbial culture or mixed culture can be used. Sialyl oligosaccharides can be formed initially with an acceptor substrate of any degree of polymerization (DP), starting from DP=1. Alternatively, sialyllactose can be produced by chemical synthesis by lactose and free sialic acid.

[0079] Form of administration

[0080] The mixture of HMOs can be administered in any suitable form. For example, the mixture of HMOs can be administered in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement.

[0081] In some embodiments, the mixture of HMOs is administered in the form of a nutritional composition. As used herein, "nutritional composition" may refer to a composition that provides nutrients to a subject. Nutritional compositions are typically administered orally or intravenously, and they typically include a lipid or fat source and a protein source.

[0082] The nutritional composition may be a synthetic nutritional composition. As used herein, "synthetic nutritional composition" may refer to a mixture obtained by chemical and / or biological methods, the chemical properties of which may be identical to the mixture naturally occurring in mammalian milk (ie, the synthetic composition is not breast milk).

[0083] The nutritional composition may be any suitable nutritional composition into which the mixture of HMOs may be incorporated, such as a nutritional composition in the form of a food or beverage product, a nutritional supplement, a nutraceutical composition or a pharmaceutical composition. The nutritional composition may be in solid (e.g., powder), liquid or semi-liquid form. Suitably, the nutritional composition is in a form suitable for feeding an infant, such as an infant formula, a milk fortifier or a supplement. The nutritional composition may also be in a form for young children, such as a yogurt or a medical food.

[0084] In a preferred embodiment, the mixture of HMOs is administered in the form of an infant formula. The infant formula may be a newborn infant formula, a premature infant formula, a milk fortifier, an older infant formula, a baby food formula, an infant cereal formula, or a growing-up milk. As used herein, the term "infant formula" may refer to a food intended for specific nutritional use for infants during the first few months of life and which itself meets the nutritional needs of such people (in accordance with Article 2(c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of December 22, 2006 for infant formula and older infant formula). It may also refer to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and infant specialties (including foods for special medical purposes).

[0085] In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula.Generally speaking, a "first infant formula" is intended for use as a breast milk substitute for newborn infants.

[0086] In some embodiments, the mixture of HMOs is administered in the form of a follow-on formula. A "follow-on formula" or "second infant formula" can be given starting from the sixth month. It can constitute the main liquid element in the gradually diversified diet of such people.

[0087] In some embodiments, the mixture of HMOs is administered in the form of a growing-up milk. As used herein, the term "growing-up milk" (or GUM) may refer to a milk formula product provided after one year. It is typically a dairy beverage adapted to the specific nutritional needs of young children.

[0088] In some embodiments, the mixture of HMOs is administered in the form of a premature infant formula.As used herein, the term "preterm infant formula" may refer to an infant formula intended for premature infants.

[0089] In some embodiments, the mixture of HMOs is administered in the form of a dairy fortifier.As used herein, the term "dairy fortifier" may refer to a liquid or solid nutritional composition suitable for mixing with infant formula.

[0090] In some embodiments, the mixture of HMOs is administered in the form of a baby food formula.As used herein, the term "baby food formula" may refer to a food material intended for specific nutritional use by infants or children, such as toddlers, during the first few years of life.

[0091] In some embodiments, the mixture of HMOs is administered in the form of an infant cereal composition.As used herein, the term "infant cereal composition" may refer to a food material intended for specific nutritional use by infants or children, such as toddlers, during the first few years of life.

[0092] In some embodiments, the mixture of HMOs is administered in at least the form of a newborn formula, a follow-on formula, and / or a growing-up milk. In some embodiments, the mixture of HMOs is administered in at least the form of a newborn formula. In some embodiments, the mixture of HMOs is administered in at least the form of a newborn formula and a follow-on formula. In some embodiments, the mixture of HMOs is administered in at least the form of a newborn formula, a follow-on formula, and a growing-up milk.

[0093] In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula and an follow-on formula. In some embodiments, the mixture of HMOs is administered in the form of a newborn infant formula, a follow-on formula, and a growing-up milk.

[0094] In other embodiments, the mixture of HMOs is administered in the form of a fortifier. The fortifier may be a formula fortifier, such as an infant formula fortifier. The fortifier may be a particularly advantageous embodiment when the infant or toddler is premature.

[0095] In other embodiments, the mixture of HMOs is administered in the form of a supplement. As used herein, a "supplement" or "dietary supplement" can be used to supplement a subject's nutrition (it is typically used as such, but it can also be added to any kind of composition intended to be ingested by a subject).

[0096] When the composition is a supplement, it can be provided in unit dosage form. Supplements are generally present in the form of liquids, gels, powders, tablets or capsules. Powder supplements generally encompass supplements to be dissolved in water or sprayed on food or beverages. Such supplements are intended to provide additional nutrition and / or health benefits to the subject consuming it. Supplements can be used to provide nutrients and / or health benefits to humans and animals, as defined above. Supplements include, for example, powder supplements added to breast milk, for example, for premature or low birth weight infants.

[0097] In other embodiments, the mixture of HMOs is administered in the form of a pharmaceutical product. Pharmaceutical products include, for example, drops, syrups, powders, tablets, or capsule products intended to treat or prevent an adverse medical condition in a subject in need thereof. In other embodiments, the mixture of HMOs is administered in the form of a nutraceutical product.

[0098] Other components

[0099] The nutritional composition of the present invention, in particular an infant formula, typically comprises, in addition to the mixture of HMOs, a protein source, a carbohydrate source and a lipid source.

[0100] Nutritional compositions according to the present invention, especially infant formula milk powder of the present invention, can contain protein source.Protein can be present with the amount of about 1.5g / 100kcal to about 3.0g / 100kcal, about 1.5g / 100kcal to about 2.5g / 100kcal, about 1.6g / 100kcal to about 2.5g / 100kcal or about 1.6g / 100kcal to about 2.25g / 100kcal.In some embodiments, protein amount is with about 2.0g / 100kcal or less, for example, about 1.8g / 100kcal to about 2.0g / 100kcal, or about 1.9g / 100kcal amount exists.

[0101] Protein sources based on, for example, whey, casein, and mixtures thereof may be used, as may plant-based (e.g., soy-based) protein sources. As far as whey protein is concerned, the protein source may be based on acid whey or sweet whey, or mixtures thereof, and may contain alpha-lactalbumin and beta-lactoglobulin in any desired proportions. In some embodiments, the protein source is predominantly whey (i.e., more than 50% of the protein is derived from whey protein, such as 60% or more or 70% or more).

[0102] The protein may be intact or hydrolyzed, or a mixture of intact and hydrolyzed proteins.

[0103] In the context of the present invention, the term "intact" may mean that the main part of the protein is intact, i.e. the molecular structure is not changed, for example at least 80% of the protein is not changed, such as at least 85% of the protein is not changed, preferably at least 90% of the protein is not changed, even more preferably at least 95% of the protein is not changed, such as at least 98% of the protein is not changed. In a specific embodiment, 100% of the protein is not changed.

[0104] In the context of the present invention, the term "hydrolyzed" may mean a protein that has been hydrolyzed or broken down into its component amino acids. The protein may be fully hydrolyzed or partially hydrolyzed. If hydrolyzed protein is desired, a hydrolysis process may be performed as desired and as known in the art. For example, a whey protein hydrolyzate may be prepared by enzymatic hydrolysis of a whey fraction in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source. In a specific embodiment, the protein of the composition is hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be 2 to 20, 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, 20, 40, 60, 80 or 90. At least 70%, 80%, 85%, 90%, 95% or 97% of the protein may be hydrolyzed. In a particular embodiment, 100% of the protein is hydrolyzed.

[0105] Nutritional compositions according to the present invention, especially infant formula milk powder of the present invention, can contain carbohydrate source.In this case, any carbohydrate source that is usually present in infant formula can be used, such as lactose, sucrose, saccharose, maltodextrin, starch and their mixture, but one of preferred carbohydrate sources of infant formula is lactose.The amount of carbohydrate can be about 8g to about 15g / 100kcal or about 9g to about 14g / 100kcal.In some embodiments, carbohydrate exists with the amount of about 10g / 100kcal to about 13g / 100kcal or about 11.1g / 100kcal.

[0106] According to the nutritional composition of the present invention, especially the infant formula milk powder of the present invention, lipids and essential fatty acids can be contained. Non-limiting examples of lipids include palm olein, high oleic sunflower oil, high oleic safflower oil, canola oil, fish oil, coconut oil, milk fat and combinations thereof. Non-limiting examples of essential fatty acids include: linoleic acid (LA), alpha-linolenic acid (ALA). The composition of the present invention may also contain gangliosides, monosialoganglioside-3 (GM3) and disialoganglioside 3 (GD3) and combinations thereof. The amount of lipids may be from about 3.0 g / 100 kcal to about 8.0 g / 100 kcal, from about 4.0 g / 100 kcal to about 6.0 g / 100 kcal, or from about 4.5 g / 100 kcal to about 5.5 g / 100 kcal. In some embodiments, the lipid is present in an amount of about 5.0 g / 100 kcal to about 5.5 g / 100 kcal, or about 5.3 g / 100 kcal.

[0107] The nutritional composition of the present invention, especially the infant formula milk powder of the present invention, can also contain all vitamins and minerals that are considered to be necessary for daily diet and in a significant amount of nutrition. The minimum requirements of certain vitamins and minerals have been determined. The examples of minerals, vitamins and other nutrients optionally present in the composition of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin E, vitamin K1, vitamin K2, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in the form of salts. The presence and amount of specific minerals and other vitamins will vary according to the target group. If necessary, the nutritional composition of the present invention can contain emulsifiers and stabilizers, such as soybeans, lecithin, citric acid monoglyceride and citric acid diglyceride, etc.

[0108] Suitably, the nutritional composition of the present invention, in particular the infant formula of the present invention, may have an energy density of about 60 kcal / 100 mL to about 72 kcal / 100 mL or about 67 kcal / 100 mL.

[0109] Preparation of composition

[0110] The compositions according to the invention may be prepared by any known or other suitable means.

[0111] For example, a nutritional composition (e.g., an infant formula) can be completed by blending a protein source with a carbohydrate source and a lipid source in appropriate proportions. If an emulsifier is used, it can be included at this stage. Vitamins and minerals can be added at this stage, but can also be added later to avoid thermal degradation. Water (preferably reverse osmosis water or deionized water) can then be added and mixed to form a liquid mixture. The mixing temperature is preferably room temperature, but can also be higher. The liquid mixture can then be heat treated to reduce the bacterial load. The mixture can then be homogenized.

[0112] If it is desired to produce a powdered composition, the homogenized mixture can be dried in a suitable drying apparatus, such as a spray dryer or a freeze dryer, and converted into a powder.

[0113] Methods for preparing infant and toddler formulas are based on the concept that products must be nutritionally adequate and safe to eat in terms of microbiology. Therefore, steps to eliminate or limit microbial growth are essential for production methods. Generally speaking, for powder products, processing techniques involve preserving oil-in-water (o / w) emulsions by dehydration, or for ready-to-eat or concentrated liquid products, processing techniques involve sterilization. Powdered infant formulas can be produced by various methods, such as dry mixing of dehydrated ingredients to form uniform formulas, or hydrating and wet mixing of a mixture of constant ingredients (such as fat, protein and carbohydrate ingredients), and then evaporating the resulting mixture and spray drying. A combination of the above two methods can be used, wherein a basic powder is first prepared by wet mixing and spray drying of all or some constant ingredients, and then dry mixing of the remaining ingredients (including carbohydrates, minerals and vitamins and other trace nutrients) to obtain a final formula. Liquid formulas are provided in ready-to-eat form or as concentrated liquids, the latter needing to be diluted with water, usually 1:1. The manufacturing methods for these products are similar to those for making reconstituted milk.

[0114] If it is desired to produce a liquid infant formula, the homogenised mixture can be filled into suitable containers, preferably under aseptic conditions. However, the liquid composition can also be cooked in a container, and suitable equipment for carrying out such filling and cooking is commercially available.

[0115] Dosage of HMO

[0116] The mixture of HMOs can be administered at any dose effective to induce convergence of the infant's gut microbiome trajectory with a reference gut microbiome trajectory and maintain or continue convergence with a reference gut microbiome trajectory. The effective dose can vary, for example, according to the weight and / or age of the infant.

[0117] If the mixture of HMOs is administered in the form of an infant formula, the infant formula can be administered normally (e.g., based on the weight of the infant or child), and the appropriate amount of the individual HMOs (e.g., 2'FL, diFL, LNT, 3'-SL, 6'-SL) can be based on the amount found in human breast milk produced for an infant or child of the same age (particularly by a well-nourished mother). The amount in the infant formula may vary, depending on, for example, the bioavailability of the HMO from the infant formula compared to human breast milk. The exemplary concentrations of the HMOs described herein may refer to the concentration of the composition after reconstitution, for example, with water.

[0118] The amounts of 2'FL, diFL, LNT, 3'-SL, and 6'-SL in human breast milk may fall within the following ranges: the amount of 2'FL is about 0.5 g / L to about 3 g / L (e.g., about 1.8 g / L); the amount of diFL is about 0.1 g / L to about 0.5 g / L (e.g., about 0.26 g / L); the amount of LNT is about 0.05 g / L to about 0.3 g / L (e.g., about 0.77 g / L); the amount of 3'SL is about 0.1 g / L to about 0.4 g / L (e.g., about 0.22 g / L); and the amount of 6'SL is about 0.05 g / L to about 0.75 g / L (e.g., about 0.47 g / L).

[0119] Suitably, the mixture of HMOs (e.g. 2'FL, diFL, LNT, 3'-SL and / or 6'-SL) is administered in a total amount of about 0.1 g / day to about 10 g / day. Suitably, the mixture of HMOs is administered in a total amount of about 0.5 g / day or more, about 1.0 g / day or more, or about 1.5 g / day or more. Suitably, the mixture of HMOs is administered in a total amount of about 5.0 g / day or less, 4.5 g / day or less, 4.0 g / day or less, 3.5 g / day or less, 3.0 g / day or less, or 2.5 g / day or less. Suitably, the mixture of HMOs is administered in a total amount of about 0.5 g / day to about 5.0 g / day, about 1.0 g / day to about 3.0 g / day, or about 1.4 g / day to about 2.5 g / day. In some embodiments, the mixture of HMOs is administered in a total amount of about 1.2 g / day to about 1.8 g / day (e.g., about 1.46 g / day) or about 2.0 g / day to about 3.0 g / day (e.g., about 2.44 g / day). In some embodiments, the mixture of HMOs is administered in a total amount of about 1.2 g / day to about 1.8 g / day. In some embodiments, the mixture of HMOs is administered in a total amount of about 1.46 g / day.

[0120] Suitably, when the mixture of HMOs is administered in the form of a newborn infant formula, the mixture of HMOs is administered in a total amount of about 0.5 g / day to about 5.0 g / day, about 1.0 g / day to about 3.0 g / day, or about 1.5 g / day to about 2.5 g / day (e.g., about 1.5 g / day or about 2.5 g / day). Suitably, when the mixture of HMOs is administered in the form of a follow-on formula, the mixture of HMOs is administered in a total amount of about 0.1 g / day to about 2.0 g / day, about 0.2 g / day to about 1.0 g / day, about 0.3 g / day to about 0.7 g / day, or about 0.5 g / day. Suitably, when the mixture of HMOs is administered in the form of a growing-up milk, the mixture of HMOs is administered in a total amount of about 0.05 g / day to about 0.5 g / day, about 0.1 g / day to about 0.3 g / day, or about 0.2 g / day.

[0121] Suitably, the mixture of HMOs is applied in the following proportions, based on the total weight of the HMOs: (i) 2'FL in an amount of about 55 wt % to about 60 wt % (e.g. about 58 wt %); (ii) diFL in an amount of about 5 wt % to about 7 wt % (e.g. about 6 wt %); (iii) LNT in an amount of about 18 wt % to about 20 wt % (e.g. about 19 wt %); (iv) 3'-SL in an amount of about 6 wt % to about 8 wt % (e.g. about 7 wt %); and (v) 6'-SL in an amount of about 9 wt % to about 11 wt % (e.g. about 10 wt %).

[0122] HMO concentration

[0123] Nutritional compositions (such as infant formula) comprising a mixture of HMOs may comprise the mixture of HMOs in any suitable concentration to provide an effective dosage.

[0124] As a guide, for example for infant formula, a mixture of HMOs (e.g. 2'FL, diFL, LNT, 3'-SL and / or 6'-SL) may be present in a total amount of about 0.1 g / L to about 10 g / L. Suitably, the composition comprises a mixture of HMOs in a total amount of about 0.5 g / L or more, about 1.0 g / L or more, or about 1.5 g / L or more. Suitably, the composition comprises a mixture of HMOs in a total amount of about 5.0 g / L or less, 4.5 g / L or less, 4.0 g / L or less, 3.5 g / L or less, 3.0 g / L or less, or 2.5 g / L or less. Suitably, the composition comprises a mixture of HMOs in a total amount of about 0.5 g / L to about 5.0 g / L, about 1.0 g / L to about 3.0 g / L, about 1.2 g / L to 3.0 g / L, about 1.5 g / L to about 2.5 g / L. In some embodiments, the composition comprises a mixture of HMOs in a total amount of about 1.2 g / L to about 1.8 g / L (e.g., about 1.5 g / L) or about 2.0 g / L to about 3.0 g / L (e.g., about 2.5 g / L). In some embodiments, the composition comprises a mixture of HMOs in a total amount of about 1.2 g / L to about 1.8 g / L. In some embodiments, the composition comprises a mixture of HMOs in a total amount of about 1.5 g / L.

[0125] Suitably, the newborn formula milk powder comprises the total amount of HMO of about 0.5g / L to about 5.0g / L, about 1.0g / L to about 3.0g / L, about 1.2g / L to 3.0g / L or about 1.5g / L to about 2.5g / L. Suitably, the older infant formula milk powder comprises the total amount of HMO of about 0.1g / L to about 2.0g / L, about 0.2g / L to about 1.0g / L, about 0.3g / L to about 0.8g / L, about 0.35g / L to about 0.65g / L or about 0.5g / L. Suitably, the growing-up milk comprises the total amount of HMO of about 0.1g / L to about 1.0g / L, about 0.2g / L to about 0.8g / L, about 0.28g / L to about 0.52g / L, about 0.3g / L to about 0.5g / L or about 0.4g / L.

[0126] Suitably, the mixture of HMOs (e.g. in the form of a nutritional composition such as an infant formula) comprises or consists of: (i) 2'FL in an amount of about 50 wt% to about 65 wt%; (ii) diFL in an amount of about 2 wt% to about 10 wt%; (iii) LNT in an amount of about 15 wt% to about 25 wt%; (iv) 3'-SL in an amount of about 4 wt% to about 10 wt%; and (v) 6'-SL in an amount of about 5 wt% to about 15 wt%, based on the total weight of the HMOs.

[0127] In some embodiments, a mixture of HMOs (e.g., in the form of a nutritional composition such as an infant formula) comprises or consists of: (i) 2'FL in an amount of about 55 wt % to about 60 wt % (e.g., about 58 wt %); (ii) diFL in an amount of about 5 wt % to about 7 wt % (e.g., about 6 wt %); (iii) LNT in an amount of about 18 wt % to about 20 wt % (e.g., about 19 wt %); (iv) 3'-SL in an amount of about 6 wt % to about 8 wt % (e.g., about 7 wt %); and (v) 6'-SL in an amount of about 9 wt % to about 11 wt % (e.g., about 10 wt %), based on the total weight of the HMOs.

[0128] As a guide, for example, for infant formula, the one or more fucosylated oligosaccharides (e.g., 2'FL and / or diFL) may be present in a total amount of about 0.1 g / L to about 4 g / L. Suitably, the one or more fucosylated oligosaccharides are present in an amount of about 0.1 g / L to about 3.5 g / L, about 0.15 g / L to about 3 g / L, about 0.2 g / L to about 2.5 g / L, about 0.3 g / L to about 2 g / L, about 0.4 g / L to about 2 g / L, or about 0.5 g / L to about 2 g / L. In some embodiments, 2'FL is present in an amount of about 0.5 g / L to about 3.0 g / L (e.g., about 0.87 g / L or about 1.45 g / L). In some embodiments, 2'FL is present in an amount of about 0.87 g / L. In some embodiments, diFL is present in an amount of about 0.05 g / L to about 0.3 g / L (eg, about 0.10 g / L or about 0.14 g / L). In some embodiments, diFL is present in an amount of about 0.10 g / L.

[0129] As a guide, for example, for infant formula, the one or more N-acetylated oligosaccharides (e.g., LNT) may be present in a total amount of about 0.05 g / L to about 1.0 g / L. Suitably, the one or more N-acetylated oligosaccharides are present in an amount of about 0.1 g / L to about 0.5 g / L or about 0.2 g / L to about 0.5 g / L. In some embodiments, LNT is present in an amount of about 0.1 g / L to about 1.0 g / L (e.g., about 0.29 g / L or about 0.48 g / L). In some embodiments, LNT is present in an amount of about 0.29 g / L.

[0130] As a guide, for example, for infant formula, the one or more sialylated oligosaccharides (e.g., 3'SL and / or 6'SL) may be present in a total amount of about 0.05g / L to about 1g / L. Suitably, the one or more sialylated oligosaccharides are present in an amount of about 0.05g / L to about 0.5g / L or about 0.1g / L to about 0.5g / L. In some embodiments, 3'SL is present in an amount of about 0.05g / L to about 0.3g / L (e.g., about 0.11g / L or about 0.18g / L). In some embodiments, 3'SL is present in an amount of about 0.11g / L. In some embodiments, 6'SL is present in an amount of about 0.05g / L to about 0.5g / L (e.g., about 0.14g / L or about 0.24g / L). In some embodiments, 6'SL is present in an amount of about 0.14g / L.

[0131] Suitably, the mixture of HMOs (e.g. in the form of a nutritional composition such as an infant formula) may comprise or consist of: (i) 2'FL in an amount of about 0.5 g / L to about 3.0 g / L (e.g. about 0.87 g / L or about 1.45 g / L); (ii) diFL in an amount of about 0.05 g / L to about 0.3 g / L (e.g. about 0.10 g / L or about 0.14 g / L); (iii) ) LNT in an amount of about 0.1 g / L to about 1.0 g / L (e.g., about 0.29 g / L or about 0.48 g / L); (iv) 3'-SL in an amount of about 0.05 g / L to about 0.3 g / L (e.g., about 0.11 g / L or about 0.18 g / L); and (v) 6'-SL in an amount of about 0.05 g / L to about 0.5 g / L (e.g., about 0.14 g / L or about 0.24 g / L).

[0132] In some embodiments, the mixture of HMOs (e.g., in the form of a nutritional composition such as a newborn infant formula) comprises or consists of: (i) 2'FL in an amount of about 0.70 g / L to about 1.05 g / L, preferably about 0.87 g / L; (ii) diFL in an amount of about 0.05 g / L to about 0.11 g / L, preferably about 0.10 g / L; (iii) LNT in an amount of about 0.23 g / L to about 0.36 g / L, preferably about 0.29 g / L; (iv) 3'-SL in an amount of about 0.09 g / L to about 0.13 g / L, preferably about 0.11 g / L; and (v) 6'-SL in an amount of about 0.12 g / L to about 0.17 g / L, preferably about 0.14 g / L.

[0133] In other embodiments, the mixture of HMOs (e.g., in the form of a nutritional composition such as a newborn infant formula) comprises or consists of: (i) 2'FL in an amount of about 1.16 g / L to about 1.74 g / L, preferably about 1.45 g / L; (ii) diFL in an amount of about 0.12 g / L to about 0.18 g / L, preferably about 0.14 g / L; (iii) LNT in an amount of about 0.39 g / L to about 0.58 g / L, preferably about 0.48 g / L; (iv) 3'-SL in an amount of about 0.14 g / L to about 0.21 g / L, preferably about 0.18 g / L; and (v) 6'-SL in an amount of about 0.19 g / L to about 0.28 g / L, preferably about 0.24 g / L.

[0134] In other embodiments, the mixture of HMOs (e.g., in the form of a nutritional composition such as an infant follow-on formula) comprises or consists of: (i) 2'FL in an amount of about 0.19 g / L to about 0.34 g / L, preferably about 0.26 g / L; (ii) diFL in an amount of about 0.03 g / L to about 0.05 g / L, preferably about 0.04 g / L; (iii) LNT in an amount of about 0.06 g / L to about 0.11 g / L, preferably about 0.09 g / L; (iv) 3'-SL in an amount of about 0.04 g / L to about 0.09 g / L, preferably about 0.06 g / L; and (v) 6'-SL in an amount of about 0.03 g / L to about 0.06 g / L, preferably about 0.05 g / L.

[0135] In other embodiments, the mixture of HMOs (e.g., in the form of a nutritional composition such as a growing-up milk) comprises or consists of: (i) 2'FL in an amount of about 0.15 g / L to about 0.28 g / L, preferably about 0.21 g / L; (ii) diFL in an amount of about 0.01 g / L to about 0.04 g / L, preferably about 0.03 g / L; (iii) LNT in an amount of about 0.05 g / L to about 0.09 g / L, preferably about 0.07 g / L; (iv) 3'-SL in an amount of about 0.03 g / L to about 0.08 g / L, preferably about 0.06 g / L; and (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L, preferably about 0.04 g / L.

[0136] Period of application

[0137] The mixture of HMOs can be administered over any suitable period. For example, if administered in the form of at least a newborn infant formula, the HMO can be administered until at least about 6 months after birth. For example, if administered in the form of at least a newborn infant formula and an follow-on formula, the HMO can be administered until at least about 12 months after birth. For example, if administered in the form of at least a newborn infant formula, a follow-on formula, and a growing-up milk, the HMO can be administered until at least about 15 months after birth.

[0138] The HMO mixture may be administered from at least about 0 months to about 6 months after birth. Suitably, administration of the HMO mixture begins from about 7 to about 21 days after birth. Suitably, the mixture of HMOs is administered until about 6 to about 18 months after birth, until about 6 to about 15 months after birth, or until about 6 to about 12 months after birth. In some embodiments, the mixture of HMOs is administered from about 7 to about 21 days after birth until about 15 months after birth, from about 7 to about 21 days after birth until about 12 months after birth, from about 7 to about 21 days after birth until about 9 months after birth, or from about 7 to about 21 days after birth until about 6 months after birth.

[0139] Interested baby

[0140] The mixture of HMOs can be administered to any infant who has, or is at risk of having, a gut microbiome trajectory that differs from a reference gut microbiome (e.g., a reference gut microbiome from a term, vaginally delivered and exclusively human milk fed infant).

[0141] The infant may be formula-fed. As used herein, a "formula-fed" infant may refer to an infant who receives all or some of his or her nutrition from an infant formula (e.g., a newborn infant formula) during early infancy. In some embodiments, the infant receives all or substantially all of his or her nutrition from an infant formula during early infancy. The feeding pattern of an infant has a recognized influence on the composition and function of the intestinal microbiome.

[0142] The infant may be a full-term infant or a premature infant. As used herein, a "premature infant" may refer to an infant born before about 37 weeks of gestation. As used herein, a "full-term infant" may refer to an infant born at about 37 weeks of gestation or later. In some embodiments, the infant is full-term.

[0143] The infant may be delivered vaginally, or by caesarean section. The inventors have shown that the benefits obtained with the present invention may be more pronounced in formula-fed infants delivered by caesarean section. In some embodiments, the infant is delivered by caesarean section.

[0144] Suitably, the infant is from about 0 to about 2 years old, from about 0 to about 1 year old, or from about 0 to about 0.5 years old. Suitably, the infant is from about 0 months old to about 24 months old, from about 0 months old to about 18 months old, from about 0 months old to about 15 months old, from about 0 months old to about 12 months old, from about 0 months old to about 9 months old, or from about 0 months old to about 6 months old.

[0145] Gut microbiome trajectories

[0146] The present invention provides methods for inducing a gut microbiome trajectory of an infant to converge with a reference gut microbiome trajectory and maintaining or sustaining convergence with a reference gut microbiome trajectory. These methods may include administering to the infant any of the HMO mixtures described herein.

[0147] "Gut microbiota" is the composition of microorganisms (including bacteria, archaea and fungi) living in the digestive tract. The term "gut microbiome" may include "gut microbiota" and their "activity sites", which may include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolites (signal molecules, toxins, organic and inorganic molecules) and molecules produced by coexisting hosts and structured by surrounding environmental conditions (see, for example, Berg, G. et al., 2020. Microbiome, 8 (1), pp. 1 to 22). In the present invention, the term "gut microbiome" can be used interchangeably with the term "gut microbiota".

[0148] The "gut microbiome age" of a subject may refer to the predicted age of the subject based on its gut microbiome data. For example, an artificial intelligence method based on machine learning can be used to predict the actual / chronological age of a subject from gut microbiome data obtained from a fecal sample. The term "gut microbiome age" covers both the "gut microbiome composition age" and the "gut microbiome functional age" of a subject. "Gut microbiome composition age" may refer to the gut microbiome age determined using gut microbiome composition data (such as at the genus level or species level composition data). "Gut microbiome functional age" may refer to the gut microbiome age determined using gut function data (such as pathway modules / submodules or metabolite data, such as CAZyme abundance). The "gut microbiome maturity index" or "gut microbiome maturity age" of a subject may be obtained from gut composition or function data as described above, or from gut composition and function data (and other similar data).

[0149] The "gut microbiome diversity" of a subject may refer to the diversity of the subject's gut microbiome. The "gut microbiome diversity" of a subject may refer to the number of different taxa (e.g., "richness") present in the subject's gut microbiome. It may also refer to the "evenness" of the gut microbiome, i.e., taking into account the abundance or relative abundance of each taxa. Suitably, the gut microbiome diversity of a subject may refer to the alpha diversity of the subject's gut microbiome. Alpha diversity may be the diversity of a single sample (such as a stool sample), and may take into account the number of different taxa and their relative abundance.

[0150] "Gut microbiome trajectory" may refer to a fitted curve describing the relationship between "gut microbiome age", "gut microbiome maturity index", "gut microbiome maturity age", or "gut microbiome diversity" and age, and may cover gut microbiome age trajectory and gut microbiome diversity trajectory. The curve may be fitted using another cohort or data subset (for external validation purposes) by methods such as LOESS or smoothing splines. Any suitable method may be used to provide a gut microbiome trajectory (see, e.g., Dogra, SK, Banjac, J., and Sprenger, N., 2022.bioRxiv2022.02.14.479826).

[0151] A "reference gut microbiome trajectory" (e.g., age predictor) can be obtained by training a regression model based on gut microbiome data from a reference population or determining a relationship therefrom. The reference population can be any suitable reference population. In some embodiments, the reference population is an infant that is exclusively fed human milk. In some embodiments, the reference population is an infant that is vaginally delivered and exclusively fed human milk. In some embodiments, the reference population is an infant that is vaginally delivered and exclusively fed human milk at term. In some embodiments, the reference population is an infant that is primarily fed human milk. In some embodiments, the reference population is an infant that is vaginally delivered and primarily fed human milk. In some embodiments, the reference population is an infant that is vaginally delivered and primarily fed human milk. In some embodiments, the reference population is an infant that is vaginally delivered and primarily fed human milk at term.

[0152] Gut microbiome age trajectories

[0153] In some embodiments, the invention provides methods of inducing convergence of an infant's gut microbiome age trajectory with a reference gut microbiome age trajectory.

[0154] A "gut microbiome age trajectory" may refer to a fitted curve describing the relationship between "gut microbiome age", "gut microbiome maturity index" or "gut microbiome maturity age" and actual age. Reference to a "gut microbiome age trajectory" may also be referred to as an "age predictor".

[0155] Any suitable method may be used to provide a reference gut microbiome age trajectory. For example, a method for providing a reference gut microbiome age trajectory may include:

[0156] (a) providing gut microbiome data from a reference population; and

[0157] (b) training a regression model based on the gut microbiome data, wherein the age of the reference population at the time of data collection is regressed based on one or more features provided by the gut microbiome data.

[0158] Any suitable feature obtained from the intestinal microbiome data can be used to train the regression model. In some embodiments, the one or more features include or consist of one or more microbial abundances, one or more microbial ratios, or one or more carbohydrate active enzymes (CAZyme) abundances. Features can be transformed (e.g., logarithmic transformation) in any manner suitable for training a regression model.

[0159] In some embodiments, the one or more features comprise or consist of one or more microbial abundances.As used herein, "microbial abundance" may refer to the relative abundance of a microbial taxon or the absolute abundance of a microbial taxon.

[0160] In some embodiments, the one or more features comprise or consist of one or more microbial ratios.As used herein, a "microbial ratio" refers to the ratio of the abundance of one microbial taxon to the abundance of another microbial taxon.

[0161] Microbial taxa can be classified according to any suitable classification, see, for example, Pitt, TL and Barer, MR, 2012. Medical Microbiology, page 24. Microbial taxa can be classified by the same classification system or by one or more different classification systems. Microbial taxa can be classified by taxonomy and / or by function.

[0162] In some embodiments, microbial groups are classified by taxonomy. Microbial taxonomy refers to the classification based on the level of microorganisms. In the scientific classification established by Carl Linnaaeus, each species must be assigned to a genus, and the genus is the lower level in the hierarchical level (family, suborder, order, subclass, class, division / phyla, kingdom and domain). The prokaryotic groups that have been correctly described are reviewed in, for example, Bergey's Manual of Systematic Bacteriology. Suitably, microbial taxa are classified by door, class, order, family, genus and / or species. Suitably, microbial taxa are classified by door, genus and / or species. Suitably, microbial taxa are classified by genus and / or species. In some embodiments, microbial taxa are classified by genus. In some embodiments, microbial taxa are classified by species.

[0163] In some embodiments, microbial groups are classified by function. For example, microbial groups can be classified by one or more phenotypic classification systems (e.g., Gram staining, morphology, growth requirements, biochemical reactions, serological systems, environmental libraries, etc.). In some embodiments, microbial groups are classified according to biological or metabolic pathways, protein domains or families, functional modules, complex carbohydrate metabolism, antibiotic resistance, virulence factors, bacterial drug targets and endotoxins, mobile genetic elements and / or any other functional characteristics, such as Kultima, JR et al., 2016. Bioinformatics, 32 (16), pp. 2520-2523 and Overbeek, R. et al., 2014. Nucleic acids research, 42 (D1), those described in pp. D206-D214.

[0164] Suitable microbial taxa can be determined by any suitable method. For example, the suitability of the microbial taxa can be based on simulated performance statistics, the availability or ease of testing, or based on the infant of interest. Suitably, the microbial taxa are bacterial taxa. Any suitable bacterial taxa can be used, see, for example, Rinninella, E. et al., 2019. Microorganisms, 7(1), p. 14. For example, (e.g., if the microbial taxa are taxonomically classified by genus) the microbial taxa can include one or more bacterial taxa selected from the group consisting of Escherichia, Roseburia, Faecalibacterium, Sutterella, SMB53, Collinsella, Ruminococcus, Akkermansia, Veillonellam, Parabacteroides, Clostridium, Oscillospira, Megasphaera, Fusobacterium, Bacteroides, Citrobacter, Neisseria, Bifidobacterium, m), Lachnospira, Dialister, Ruminococcus, Blautia, Streptococcus, Eggerthella, Paraprevotella, Corynebacterium, Atopobium, Lactobacillus, Enterococcus, Staphylococcus, Sphingobacterium, Tannerella, Alistipes, Prevotella, Shigella, Desulfovibrio, Bilophila, and Helicobacter.For example, (e.g., if the microbial taxa are taxonomically classified by species) the microbial taxa may include one or more bacterial taxa selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Clostridium spp., Roseburia intestinalis, Ruminococcus faecis, Dialister invisus, Lactobacillus reuteri, Enterococcus faecium, Staphylococcus leei, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides dissimilaris, distasonis, Alistipes finegoldii, Prevotella spp., Escherichia coli, Shigella flexneri, Desulfovibrio intestinalis, Helicobacter pylori, Fusobacterium nucleatum, and Akkermansia muciniphilia.

[0165] In some embodiments, the one or more features comprise or consist of one or more CAZyme abundances. As used herein, "CAZyme abundance" may refer to the abundance of CAZyme genes in intestinal microbiome data. The abundance may be relative abundance and / or absolute abundance. Suitably, the abundance is relative abundance, for example, the abundance may be calculated relative to total bacterial genes (see, e.g., Kaur, K. et al., 2020. PloS one, 15(4), p.e0231197) or relative to total CAZyme abundance. Suitably, the abundance is calculated relative to total bacterial genes.

[0166] Carbohydrate active enzyme (CAZyme) may refer to an enzyme involved in the synthesis, metabolism and transport of carbohydrates. CAZyme may include glycoside hydrolases (GH), glycosyltransferases (GT), polysaccharide lyases (PL), carbohydrate esterases (CE) and carbohydrate binding modules (CBM). Suitably, CAZyme is a microbial CAZyme. CAZyme can be classified according to any suitable classification system, see, for example, Lombard, V. et al., 2014. Nucleic acids research, 42 (D1), pages D490-D495. CAZyme can be classified by the same classification system or by one or more different classification systems. Suitably, CAZyme is classified by clan, family and / or subfamily. Suitably, CAZyme is classified by family.

[0167] Suitably, the CAZyme comprises one or more (e.g., 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more) of the following: GH1, GH2, GH3, GH4, GH5, GH6, GH7, GH8, GH9, GH10, GH11, GH12, GH13, GH14, GH15, GH16, GH17, GH18, GH19, GH20, GH21, GH22, GH23, GH24, GH25, GH26, GH27, GH28, GH29, GH30, GH31, GH32, GH33, GH34, GH35, GH36, GH37, GH38, GH39, GH40, GH41, GH42, GH43, GH44, GH45, GH46, GH47, GH48, GH49, GH50, GH51, GH52, GH53, GH54, GH55, GH56, GH57, GH58, GH59, GH60 , GH41, GH42, GH43, GH44, GH45, GH46, GH47, GH48, GH49, GH50, GH51, GH52, GH53, GH54, GH55, GH56, GH57, GH58, GH59, GH60, GH61, GH62, GH63, GH64, GH65 , GH66, GH67, GH68, GH69, GH70, GH71, GH72, GH73, GH74, GH75, GH76, GH77, GH78, GH79, GH80, GH81, GH82, GH83, GH84, GH85, GH86, GH87, GH88, GH89, GH90 ,GH91,GH92,GH93,GH94,GH95,GH96,GH97,GH98,GH99,GH100,GH101,GH102,GH103,GH104,GH105,GH106,GH107,GH108,GH109,GH110,GH111,GH112,G H113, GH114, GH115, GH116, GH117, GH118, GH119, GH120, GH121, GH122, GH123, GH124, GH125, GH126, GH127, GH128, GH129, GH130, GH131, GH132, GH133, GH134, GH135, GH136, GH137, GH138, GH139, GH140, GH141, GH142, GH143, GH144, GH145, GH146, GH147, GH148, GH149, GH150, GH151, GH152, GH153, GH154 , GH155, GH156, GH157, GH158, GH159, GH160, GH161, GH162, GH163, GH164, GH165, GH166, GH167, GH168, GH169, GH170, GH171, GH172, GT1, GT2, GT3, GT4,GT5、GT6、GT7、GT8、GT9、GT10、GT11、GT12、GT13、GT14、GT15、GT16、GT17、GT18、GT19、GT20、GT21、GT22、GT23、GT24、GT25、GT26、GT27、GT28、GT29、GT30、GT31、GT32、GT33、GT34、GT35、GT36、GT37、GT38、GT39、GT40、GT41、GT42、GT43、GT44、GT45、GT46、GT47、GT48、GT49、GT50、GT51、GT52、GT53、GT54、GT55、GT56、GT57、GT58、GT59、GT60、GT61、GT62、GT63、GT64、GT65、GT66、GT67、GT68、GT69、GT70、GT71、GT72、GT73、GT74、GT75、GT76、GT77、GT78、GT79、GT80、GT81、GT82、GT83、GT84、GT85、GT86、GT87、GT88、GT89、GT90、GT91、GT92、GT93、GT94、GT95、GT96、GT97、GT98、GT99、GT100、GT101、GT102、GT103、GT104、GT105、GT106、GT107、GT108、GT109、GT110、GT111、GT112、GT113、GT114、PL1、PL2、PL3、PL4、PL5、PL6、PL7、PL8、PL9、PL10、PL11、PL12、PL13、PL14、PL15、PL16、PL17、PL18、PL19、PL20、PL21、PL22、PL23、PL24、PL25、PL26、PL27、PL28、PL29、PL30、PL31、PL32、PL33、PL34、PL35、PL36、PL37、PL38、PL39、PL40、PL41、PL42、CE1、CE2、CE3、CE4、CE5、CE6、CE7、CE8、CE9、CE10、CE11、CE12、CE13、CE14、CE15、CE16、CE17、CE18、CE19、CBM1、CBM2、CBM3、CBM4、CBM5、CBM6、CBM7、CBM8、CBM9、CBM10、CBM11、CBM12、CBM13、CBM14、CBM15、CBM16、CBM17、CBM18、CBM19、CBM20、CBM21、CBM22、CBM23、CBM24、CBM25、CBM26、CBM27、CBM28, CBM29, CBM30, CBM31, CBM32, CBM33, CBM34, CBM35, CBM36, CBM37, CBM38, CBM39, CBM40, CBM41, CBM42, CBM43 , CBM44, CBM45, CBM46, CBM47, CBM48, CBM49, CBM50, CBM51, CBM52, CBM53, CBM54, CBM55, CBM56, CBM57, CBM58, CBM5 9. CBM60, CBM61, CBM62, CBM63, CBM64, CBM65, CBM66, CBM67, CBM68, CBM69, CBM70, CBM71, CBM72, CBM73, CBM74, CBM75, CBM76, CBM77, CBM78, CBM79, CBM80, CBM81, CBM82, CBM83, CBM84, CBM85, CBM86, CBM87 and CBM88, consist essentially of, or consist of.

[0168] Any suitable number of features can be used to train the regression model. Suitably, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more features can be used to train the regression model. For example, 10 microbial abundances (e.g., if the microbial taxa are taxonomically classified by genus), 20 or 25 microbial abundances (e.g., if the microbial taxa are taxonomically classified by species), or 30 CAZyme abundances can be used to train the regression model.

[0169] Gut microbiome diversity trajectories

[0170] In some embodiments, the present invention provides a method for inducing a gut microbiome diversity trajectory of an infant to converge with a reference gut microbiome diversity trajectory. "Gut microbiome diversity trajectory" may refer to a fitted curve, which is obtained to describe the relationship between gut diversity and age.

[0171] Any suitable method may be used to provide a reference gut microbiome diversity trajectory. For example, a method for providing a reference gut microbiome age diversity trajectory may include:

[0172] (a) providing gut microbiome data from a reference population; and

[0173] (b) Determine the relationship between the mean gut microbiome diversity of the reference population and the age of the reference population at the time of data collection.

[0174] Alpha diversity can be determined using a richness index, a phylogenetic diversity index, or a Shannon index. These indices can be determined using conventional methods in the art, such as using the R package Phyloseq (McMurdie and Holmes, 2013, PLoS One, 8, Article e61217). The alpha diversity index can be calculated based on 16rRNA sequencing data and / or whole-genome shotgun metagenomic sequencing. β diversity can be calculated using the Whittaker index (e.g., Jaccard or Sorensen), the Min-Max index (e.g., Simpson, β-2, or β-3), the Cody index, or an abundance index (e.g., Bray-Curtis or BD TOTAL ). Beta diversity indices can be calculated based on 16rRNA sequencing data and / or whole genome shotgun metagenomic sequencing. These indices can be determined using conventional methods in the art, such as, for example, using the R package Phyloseq (see, for example, McMurdie and Holmes, 2013, PLoS One, 8, Article e61217).

[0175] The microbial taxa used to determine the diversity of the intestinal microbiome can be classified according to any suitable classification described herein. Suitably, the microbial taxa are taxonomically classified by genus and / or species. In some embodiments, the microbial taxa are taxonomically classified by genus. In some embodiments, the microbial taxa are taxonomically classified by species.

[0176] Gut microbiome data

[0177] Gut microbiome data from a reference population can be used to determine a reference gut microbiome trajectory (e.g., a reference gut microbiome age trajectory or a reference gut microbiome diversity trajectory).

[0178] Gut microbiome data can be any data suitable for determining a reference gut microbiome trajectory, for example, microbiome data can include gut microbial abundance, gut metagenomic data, gut metabolite data, etc. Suitably, gut microbiome data is gut metagenomic data. The subject's "gut metagenomic data" ("gut metagenomic data" or "gut metagenome data") can refer to all genetic contents of the subject's intestine, including all genomes and genes from the gut microbiota (see, for example, Berg, G. et al., 2020. Microbiome, 8 (1), pp. 1-22; Pasolli, E. et al., 2019. Cell, 176 (3), pp. 649-662; and Qin, J. et al. 2010. Nature, 464 (7285), pp. 59-65).

[0179] The intestinal microbiome data can be obtained by or can be obtained by any suitable sampling method. For example, the intestinal microbiome data can be obtained by or can be obtained by any method described in the following document: Tang, Q. et al., 2020. Frontiers in cellular and infection microbiology, 10, p. 151. The intestinal microbiome data can be obtained from or can be obtained from stool samples, endoscopic samples (e.g., biopsy samples, lumen brush samples, laser capture microdissection samples), aspirated intestinal fluid samples, surgical samples, or by in vivo models or smart capsules.

[0180] Suitably, gut microbiome data may or can be obtained from stool samples. Stool samples are naturally collected, non-invasive and can be sampled repeatedly. Immediate freezing of stool material at -80°C to maintain microbial integrity without preservatives is widely considered the gold standard for gut metagenomics, but other storage methods with or without preservatives may also be used to obtain metagenomic data similar to fresh samples.

[0181] The intestinal microbiome data can be obtained or can be obtained from the sample by any suitable method. For example, the intestinal microbiome data can be obtained or can be obtained from the sample by a sequencing method (e.g., a next generation sequencing (NGS) method). NGS enables the genomic DNA of all microorganisms present in the sample to be analyzed. The NGS method may include a shotgun sequencing method, such as described in Poussin, C. et al., 2018. Drug discovery today, 23 (9), pp. 1644-1657.

[0182] The reference population may include any number of infants suitable for training a regression model or determining a relationship. The reference population may include at least 10 infants, at least 20 infants, at least 30 infants, at least 40 infants, at least 50 infants, at least 60 infants, at least 80 infants, or at least 100 infants. Suitably, the reference population may include 500 infants or less, 100 infants or less, or 50 infants or less. Suitably, the reference population may include 10 to 500 infants.

[0183] The intestinal microbiome data from the reference population may comprise any number of samples suitable for training a regression model. Suitably, the intestinal microbiome data comprises at least 50 samples, at least 100 samples, at least 200 samples, at least 300 samples, at least 400 samples, at least 500 samples or at least 1000 samples.

[0184] The gut microbiome data from the reference population may comprise any number of samples from any number of infants suitable for training a regression model. Suitably, the gut microbiome data from the reference population may comprise at least 50 samples from at least 10 infants.

[0185] The infant's gut microbiome trajectory may be or can be obtained by any suitable sampling method described herein. The infant's gut microbiome data may be or can be obtained by the same method as the gut microbiome data from the reference population or by or can be obtained by a different method.

[0186] Regression analysis

[0187] Reference gut microbiome trajectories can be determined using regression analysis to relate the age of the reference population at the time of data collection to one or more features provided by their metagenomic data.

[0188] Regression analysis is a set of statistical procedures used to estimate the relationship between a dependent variable (e.g., the age of a reference population at the time of data collection) and one or more independent variables (e.g., one or more features from gut microbiome data). Regression analysis can be used to provide a trained (or fitted) regression model (i.e., a reference gut microbiome trajectory).

[0189] Any suitable regression model can be used to perform the regression analysis. Suitable regression models are well known to those skilled in the art. Exemplary regression models include decision tree regression, linear regression, polynomial regression, quantile regression, ridge regression, lasso regression, elastic net regression, and support vector regression.

[0190] Suitably, regression analysis is performed using machine learning methods. Exemplary machine learning methods include tree-based regression models (e.g., random forest regression models), recursive partitioning, regularization and shrinkage methods, boosting and gradient descent, and Bayesian methods. Suitably, the regression model is a tree-based regression model (e.g., random forest regression model). In some embodiments, the regression model is a random forest regression model. In some embodiments, the regression model is an xgboost regression model.

[0191] Regression analysis can be performed by training a regression model based on the intestinal microbiome data. For example, regression analysis can be performed by training a regression model using the age of the reference population at the time of data collection and one or more features provided by the intestinal microbiome data.

[0192] As used herein, "training" or "fitting" a regression model may refer to determining the function that most closely fits the data according to appropriate statistical criteria. For example, ordinary least squares may be used to calculate a function that minimizes the sum of the squared differences between the true data and the function.

[0193] Convergence of gut microbiome trajectories

[0194] The inventors have shown in the present invention that a mixture of human milk oligosaccharides (HMOs) can induce an infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory and maintain or continue to converge with a reference gut microbiome trajectory.

[0195] As used herein, "converging" gut microbiome trajectories tend to move closer together and meet at some point. Suitably, gut microbiome trajectories can be considered to be converging when they no longer have significant statistical differences. For example, if the infant is not an outlier in the reference gut microbiome trajectory and / or if the infant conforms to the reference gut microbiome trajectory, the infant's gut microbiome trajectory can be considered to be converging with the reference gut microbiome trajectory. In some embodiments, the mixture of HMOs induces the infant's gut microbiome trajectory to remain or continue to converge with the reference gut microbiome trajectory.

[0196] As used herein, a mixture of HMOs "inducing" an infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory may mean that the infant's gut microbiome trajectory converges with a reference gut microbiome trajectory earlier than an infant to whom the mixture of HMOs is not administered (i.e., in the absence of the mixture of HMOs). The term "induce" may be used interchangeably with the term "promote." The phrase "induce convergence of an infant's gut microbiome trajectory with a reference gut microbiome trajectory" may be used interchangeably with the phrase "accelerate convergence of an infant's gut microbiome trajectory with a reference gut microbiome trajectory."

[0197] The infant's gut microbiome trajectory may converge with a reference gut microbiome trajectory at least about 1 month, at least about 2 months, or at least about 3 months earlier than an infant not administered a mixture of HMOs (i.e., in the absence of a mixture of HMOs). The infant's gut microbiome trajectory may converge with a reference gut microbiome trajectory at least about 2 months earlier than an infant not administered a mixture of HMOs (i.e., in the absence of a mixture of HMOs).

[0198] Suitably, the infant's gut microbiome trajectory converges with a reference gut microbiome trajectory at about 12 months of age or earlier, about 11 months of age or earlier, about 10 months of age or earlier, about 9 months of age or earlier, about 8 months of age or earlier, or about 7 months of age or earlier.

[0199] Suitably, the infant's gut microbiome trajectory converges with a reference gut microbiome trajectory at about 6 months of age or later, about 7 months of age or later, about 8 months of age or later, about 9 months of age or later, about 10 months of age or later, about 11 months of age or later, or about 12 months of age or later.

[0200] Suitably, the infant's gut microbiome trajectory converges with the reference gut microbiome trajectory at about 6 months to about 12 months, about 6 months to about 11 months, about 6 months to about 10 months, or about 6 months to about 9 months. Suitably, the infant's gut microbiome trajectory converges with the reference gut microbiome trajectory at about 7 months to about 12 months, about 7 months to about 11 months, about 7 months to about 10 months, or about 7 months to about 9 months. Suitably, the infant's gut microbiome trajectory converges with the reference gut microbiome trajectory at about 8 months to about 12 months, about 8 months to about 11 months, about 8 months to about 10 months, or about 8 months to about 9 months. Suitably, the infant's gut microbiome trajectory converges with the reference gut microbiome trajectory at about 8 months to about 10 months or about 9 months.

[0201] Outliers in reference gut microbiome trajectories

[0202] In some embodiments, the mixture of human milk oligosaccharides (HMOs) induces the infant to not be an outlier in a reference gut microbiome trajectory.

[0203] Any suitable statistical method can be used to determine whether the infant is an outlier in the reference gut microbiome trajectory (see, for example, Hodge, V. and Austin, J., 2004. Artificial intelligence review, 22 (2), pp. 85-126). For example, based on the standard error, confidence interval, prediction interval and / or standard deviation in the reference gut microbiome trajectory, the infant can be determined to be an outlier. Suitably, based on the standard error, confidence interval, prediction interval and / or standard deviation of the reference gut microbiome trajectory, if the infant's gut microbiome data is significantly different from the reference gut microbiome trajectory (e.g., age predictor), the infant can be determined to be an outlier.

[0204] Suitable cutoff values ​​are well known to those skilled in the art. For example, three standard deviations from the mean are common cutoff values ​​used in practice to identify outliers in Gaussian or quasi-Gaussian distributions.

[0205] In some embodiments, the infant is determined to be an outlier based on the standard error of the reference gut microbiome trajectory. The standard error (SE) represents the average distance between the observed value and the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data differs from the reference gut microbiome trajectory by -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, -3SE or less or 3SE or more, -3.5SE or less or 3.5SE or more, or -4SE or less or 4SE or more, the infant is an outlier. Suitably, if the infant's gut microbiome data differs from the reference gut microbiome trajectory by -3SE or less or 3SE or more, the infant is an outlier.

[0206] In some embodiments, based on the confidence interval of the reference intestinal microbiome trajectory, it is determined that the infant is an outlier. The confidence interval can be determined by any suitable method, such as using a resampling method (e.g., bootstrap resampling). Suitably, if the infant's intestinal microbiome data falls outside the 90% confidence interval, 95% confidence interval, 98% confidence interval, or 99% confidence interval in the reference intestinal microbiome trajectory, the infant is an outlier. Suitably, if the infant's intestinal microbiome data falls outside the 95% confidence interval in the reference intestinal microbiome trajectory, the infant is an outlier.

[0207] In some embodiments, an infant is determined to be an outlier based on a prediction interval of a reference gut microbiome trajectory. Suitably, an infant is an outlier if the infant's gut microbiome data falls outside the 90% prediction interval, 95% prediction interval, 98% prediction interval, or 99% prediction interval in the reference gut microbiome trajectory. Suitably, an infant is an outlier if the infant's gut microbiome data falls outside the 95% prediction interval in the reference gut microbiome trajectory.

[0208] In some embodiments, an infant is determined to be an outlier based on the standard deviation of a reference gut microbiome trajectory. For example, a Z score can be used to determine whether an infant is an outlier. A Z score is the number of standard deviations above and below the mean. Suitably, an infant is an outlier if it has a Z score of -2 or less or 2 or more, a Z score of -2.5 or less or 2.5 or more, a Z score of -3 or less or 3 or more, a Z score of -3.5 or less or 3.5 or more, or a Z score of -4 or less or 4 or more in a reference gut microbiome trajectory. Suitably, an infant is an outlier if it has a Z score of -3 or less or 3 or more in a reference gut microbiome trajectory.

[0209] In some embodiments, an infant is determined to be an outlier if the infant's gut microbiome data differs from a reference gut microbiome trajectory by -3 SE or less or 3 SE or more, if the infant's gut microbiome data falls outside a 95% confidence interval in a reference gut microbiome trajectory, if the infant's gut microbiome data falls outside a 95% prediction interval in a reference gut microbiome trajectory, and / or if the infant has a Z score of -3 or less or 3 or greater in a reference gut microbiome trajectory.

[0210] In some embodiments, an infant is determined to be an outlier if the infant's gut microbiome data track has a Z-score of -3 or less or 3 or greater in a reference gut microbiome track.

[0211] Conformity to reference gut microbiome trajectory

[0212] In some embodiments, a mixture of human milk oligosaccharides (HMOs) induces an infant to conform to a reference gut microbiome trajectory.

[0213] Suitably, if the infant's gut microbiome data is not significantly different from the reference gut microbiome trajectory, then the infant meets the reference gut microbiome trajectory, and / or if the infant's gut microbiome data is significantly different from the reference gut microbiome trajectory, then the infant does not meet the reference gut microbiome trajectory.

[0214] Any suitable method can be used to determine whether the infant meets the reference gut microbiome trajectory. For example, based on the standard error, confidence interval, prediction interval and / or standard deviation of the reference gut microbiome trajectory, it can be determined that the infant meets the reference gut microbiome trajectory.

[0215] In some embodiments, based on the standard error (SE) of the reference gut microbiome trajectory, it is determined that the infant does not conform to the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data differs from the reference gut microbiome trajectory by -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, -3SE or less or 3SE or more, -3.5SE or less or 3.5SE or more, or -4SE or less or 4SE or more, the infant does not conform to the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data differs from the reference gut microbiome trajectory by -3SE or less or 3SE or more, the infant does not conform to the reference gut microbiome trajectory.

[0216] In some embodiments, based on the confidence interval of the reference gut microbiome trajectory, it is determined that the infant does not meet the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data falls outside the 90% confidence interval, 95% confidence interval, 98% confidence interval, or 99% confidence interval of the reference gut microbiome trajectory, the infant does not meet the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data falls outside the 95% confidence interval of the reference gut microbiome trajectory, the infant does not meet the reference gut microbiome trajectory.

[0217] In some embodiments, based on the prediction interval of the reference gut microbiome trajectory, it is determined that the infant does not meet the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data falls outside the 90% prediction interval, 95% prediction interval, 98% prediction interval, or 99% prediction interval of the reference gut microbiome trajectory, the infant does not meet the reference gut microbiome trajectory. Suitably, if the infant's gut microbiome data falls outside the 95% prediction interval of the reference gut microbiome trajectory, the infant does not meet the reference gut microbiome trajectory.

[0218] In some embodiments, an infant is determined to be noncompliant with a reference gut microbiome trajectory based on the standard deviation of the reference gut microbiome trajectory. For example, a Z score can be used to determine whether an infant does not conform to a reference gut microbiome trajectory. Suitably, an infant is an outlier if it has a Z score of -2 or less or 2 or greater, a Z score of -2.5 or less or 2.5 or greater, a Z score of -3 or less or 3 or greater, a Z score of -3.5 or less or 3.5 or greater, or a Z score of -4 or less or 4 or greater. Suitably, an infant does not conform to a reference gut microbiome trajectory if it has a Z score of -3 or less or 3 or greater.

[0219] In some embodiments, an infant is determined to not meet a reference gut microbiome trajectory if the infant's gut microbiome data differs from the reference gut microbiome trajectory by -3 SE or less or 3 SE or more, if the infant's gut microbiome data falls outside the 95% confidence interval of the reference gut microbiome trajectory, if the infant's gut microbiome data falls outside the 95% prediction interval of the reference gut microbiome trajectory, and / or if the infant has a Z-score of -3 or less or 3 or greater.

[0220] In some embodiments, an infant is determined to not meet the reference gut microbiome trajectory if the infant has a Z-score of -3 or less or 3 or greater.

[0221] Intestinal maturation

[0222] The method of the present invention can promote intestinal maturation by inducing the infant's intestinal microbiome trajectory to converge with a reference intestinal microbiome trajectory.

[0223] Intestinal maturation during normal development involves a series of structural and functional changes that culminate during the weaning period when complex foods are introduced. As used herein, "promoting" intestinal maturation may refer to the maturation of an infant's intestine in a manner more appropriate for its age. Intestinal maturation may refer to the maturation of the intestinal microbiome, the maturation of intestinal metabolism, the maturation of intestinal barrier function, and / or the maturation of intestinal immune function.

[0224] A mixture of HMOs can promote the maturation of the intestinal microbiome. The characteristics of early intestinal microbiome maturation can be the acquisition, colonization and selection of microorganisms with different functional characteristics at specific times over time. This coordinated microbial sequence occurs during the first few years after birth, before the composition and function of the microbiome reach adult levels between the ages of 3 and 5. More and more people believe that these different steps in the development of the microbiome are key windows of opportunity for long-term health, mainly related to proper immune and metabolic development (see, for example, Dogra, SK et al., 2021. Microorganisms, 9 (10), p. 2110). For example, the maturation of the intestinal microbiome during the first year of life can contribute to a protective farm effect against childhood asthma (see, for example, Depner, M. et al., 2020. Nature medicine, 26 (11), p. 1766-1775).

[0225] Mixtures of HMOs can promote the maturation of intestinal metabolism. Changes in microbiome composition also reflect microbial functional capabilities to a certain extent, as illustrated by significant changes in the abundance of microbial carbohydrate-active enzymes (CAZymes) and other metabolic pathways (see, e.g., Stewart, CJ et al., 2018. Nature, 562 (7728), pp. 583-588).

[0226] The mixture of HMOs can promote the maturation of intestinal barrier function. The intestinal barrier, composed of mucus and underlying epithelial cells, is primarily considered a physical barrier that, together with many immune defense components, helps regulate the microbiome and host relationship (see, e.g., Dogra, SK et al., 2021. Microorganisms, 9(10), p. 2110).

[0227] Mixtures of HMOs promote maturation of intestinal immune function. Intestinal immune components, such as secretory immunoglobulin (Ig) A and defensins, as well as epithelial and mucus glycosylation patterns change during intestinal development and may play an important role in creating conditions for the development of host-microbiome mutualism (see, e.g., Dogra, SK et al., 2021. Microorganisms, 9(10), p. 2110).

[0228] A mixture of HMOs can modulate the abundance of one or more microorganisms and / or microbial metabolic pathways that are associated with age-appropriate intestinal maturation and related health benefits. For example, HMO-stimulated Bifidobacterium species may help prevent future respiratory infections (see, e.g., Dogra, SK et al., 2021. Microorganisms, 9(9), p. 1939) and members of the Lachnospiraceae family and the genera Faecalibacterium and Listeria are associated with reduced risk of atopy (see, e.g., Galazzo, G et al., 2020. Gastroenterology, 158(6), pp. 1584-1596).

[0229] In one aspect, the invention provides a mixture of HMOs to promote intestinal maturation in an infant by inducing convergence of the infant's intestinal microbiome trajectory with a reference intestinal microbiome trajectory.

[0230] In one aspect, the invention provides a method of promoting intestinal maturation in an infant in need thereof, wherein the method comprises administering a therapeutically effective amount of a mixture of HMOs, thereby inducing the infant's intestinal microbiome trajectory to converge with a reference intestinal microbiome trajectory.

[0231] Methods for determining intestinal maturity in infants

[0232] The present invention provides methods for determining the intestinal maturation state of an infant. Suitably, the methods include: (a) providing a reference intestinal microbiome trajectory; and (b) providing intestinal microbiome data from an infant and determining whether the infant is an outlier in the reference intestinal microbiome trajectory, or whether the infant conforms to the reference intestinal microbiome trajectory.

[0233] The infant can be any suitable infant, for example any infant described in the section entitled "Infants of Interest" above. Suitably, the infant is formula fed. In some embodiments, the infant is full term. In some embodiments, the infant is delivered by caesarean section. A mixture of HMOs can be administered to the infant, for example as described in the section entitled "Mixture of Human Milk Oligosaccharides (HMOs)" above.

[0234] The reference gut microbiome trajectory can be any suitable reference gut microbiome trajectory, such as any of the reference gut microbiome trajectories described above in the section entitled “Gut Microbiome Trajectory.” In some embodiments, the reference gut microbiome trajectory is obtained from a full-term vaginally delivered infant that is exclusively fed human milk.

[0235] Any suitable method can be used to determine whether an infant is an outlier in a reference gut microbiome trajectory or whether a formula-fed infant conforms to a reference gut microbiome trajectory, such as described above in the sections entitled "Outliers in Reference Gut Microbiome Trajectory" and "Conformance to Reference Gut Microbiome Trajectory."

[0236] Suitably, if the infant is not an outlier in the reference gut microbiome trajectory, then the infant's gut maturation state is normal, and / or if the infant is an outlier in the reference gut microbiome trajectory, then the infant's gut maturation state is abnormal. In this context, a "normal" gut maturation state may mean that the infant's gut metagenome is not significantly different from that of the reference population.

[0237] Computer program and computer readable medium

[0238] The method described herein for determining the intestinal maturity state of an infant may be a computer-implemented method.

[0239] In one aspect, the present invention provides a data processing system comprising means for performing the method described herein for determining the intestinal maturity state of an infant.

[0240] In one aspect, the present invention provides a data processing device comprising a processor configured to perform the method described herein for determining the intestinal maturity state of an infant.

[0241] In one aspect, the present invention provides a computer program comprising instructions which, when executed by a computer, cause the computer to perform the method described herein for determining the intestinal maturity state of an infant.

[0242] In one aspect, the present invention provides a computer readable medium comprising instructions that, when executed by a computer, cause the computer to perform the method described herein for determining the intestinal maturity state of an infant.

[0243] In one aspect, the invention provides a computer-readable data carrier on which the computer program of the invention has been stored.

[0244] In one aspect, the invention provides a data carrier signal carrying the computer program of the invention.

[0245] In one aspect, the present invention provides a computer-implemented method for determining the intestinal maturation state of an infant, wherein the method comprises: (a) providing a reference intestinal microbiome trajectory and a microbiome-age predictor model associated therewith; (b) providing intestinal microbiome data from an infant; and (c) determining whether the infant is an outlier in the reference intestinal microbiome trajectory; wherein if the infant is not an outlier in the reference intestinal microbiome trajectory, the intestinal maturation state of the infant is normal, and / or wherein if the infant is an outlier in the reference intestinal microbiome trajectory, the intestinal maturation state of the infant is abnormal.

[0246] In one aspect, the present invention provides a computer-implemented method for determining the intestinal maturation state of an infant, wherein the method comprises: (a) providing a reference gut microbiome trajectory and a microbiome-age predictor model associated therewith; (b) providing gut microbiome data from an infant; and (c) determining whether the infant meets the reference gut microbiome trajectory; wherein if the infant's gut microbiome data is not significantly different from the reference gut microbiome trajectory, the infant meets the reference gut microbiome trajectory, and / or wherein if the infant's gut microbiome data is significantly different from the reference gut microbiome trajectory, the infant does not meet the reference gut microbiome trajectory.

[0247] In one aspect, the present invention provides a data processing system comprising means for determining the intestinal maturation state of an infant given a reference gut microbiome trajectory, a microbiome-age predictor model associated therewith, and the infant's gut microbiome data, as described herein.

[0248] In one aspect, the present invention provides a data processing apparatus comprising a processor configured to determine the intestinal maturation state of an infant given a reference intestinal microbiome trajectory, a microbiome-age predictor model associated therewith, and the infant's intestinal microbiome data, as described herein.

[0249] In one aspect, the invention provides a computer program comprising instructions that, when executed by a computer, cause the computer to determine the intestinal maturation state of an infant given a reference intestinal microbiome trajectory, a microbiome-age predictor model associated therewith, and intestinal microbiome data of the infant, as described herein.

[0250] In one aspect, the invention provides a computer-readable medium comprising instructions that, when executed by a computer, causes the computer to determine the intestinal maturation state of an infant given a reference intestinal microbiome trajectory, a microbiome-age predictor model associated therewith, and intestinal microbiome data of the infant, as described herein.

[0251] The systems described herein may display to the user a dashboard or other appropriate user interface that is customized based on the infant of interest, for example, based on the infant's gut metagenomic sample, the infant's determined gut maturation state, and personalized suggestions and recommendations for the infant, such as HMO supplementation to maintain or improve the infant's gut maturation state.

[0252] Use of gut microbiome trajectories

[0253] In another aspect, the invention provides for the use of one or more reference gut microbiome trajectories for determining the intestinal maturation state of an infant following administration of a mixture of HMOs.

[0254] The infant can be any suitable infant, for example any infant described in the section entitled "Infants of Interest" above. Suitably, the infant is formula fed. In some embodiments, the infant is full term. In some embodiments, the infant is delivered by caesarean section. Any suitable mixture of HMOs can be administered to the infant, for example as described in the section entitled "Human Milk Oligosaccharide (HMO) Mixture" above.

[0255] The one or more reference gut microbiome trajectories may include or consist of any suitable reference gut microbiome trajectories, such as any of the reference gut microbiome trajectories described above in the section entitled "Gut Microbiome Trajectories". In some embodiments, the one or more reference gut microbiome trajectories are obtained from a full-term, vaginally delivered infant that is exclusively fed human milk. In some embodiments, the one or more reference gut microbiome trajectories are obtained from a full-term, vaginally delivered infant that is primarily fed human milk.

[0256] The use may include any suitable method steps to determine the intestinal maturity state of an infant. For example, any method steps described above in the section entitled "Method for determining the intestinal maturity state of an infant".

[0257] Example

[0258] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to practice the present invention but are not intended to limit the scope of the present invention in any way.

[0259] Example 1 - Consumption of a product containing a mixture of HMOs helps infants converge with a reference gut microbiome trajectory and Keep Converging

[0260] Materials and methods

[0261] A randomized controlled trial (ClinicalTrials.gov identifier: NCT03722550) was conducted to evaluate the effects of human milk oligosaccharides (HMOs) in formula-fed infants. An overview of the study is provided in Figure 1 middle.

[0262] Healthy term infants (7-21 days of age) were randomly assigned to a standard bovine milk-based newborn infant formula (control group, CG, n=154); the same formula with 1.5 g / L HMO (test group 1, TG1, n=155); or the same formula with 2.5 g / L HMO (test group 2, TG2, n=153); or human milk feeding (reference, HMG, n=61).

[0263] Standard newborn is a whey-based term infant formula based on bovine milk, with 67 kcal / 100 mL reconstituted formula, consisting of 1.9 g complete protein (70% whey / 30% casein) / 100 kcal, 11.1 g carbohydrate / 100 kcal and 5.3 g lipid / 100 kcal. The concentrations of individual HMOs in TG1 and TG2 newborn formulas are shown in Table 1 below.

[0264] Table 1 – Concentrations of individual HMOs in TG1 and TG2 infant formulas

[0265] HMO Proportion(%) 2'-Fucosyllactose (2'FL) 58％ 2',3-Difucosyllactose (diFL) 6％ Lacto-N-tetraose (LNT) 19％ 3'-Sialyl lactose (3'-SL) 7％ 6'-Sialyl Lactose (6'-SL) 10％

[0266] Standard follow-on formula is a whey-based full-term infant formula based on bovine milk, with 67 kcal / 100 mL reconstituted formula, composed of 2 g complete protein (50% whey / 50% casein) / 100 kcal, 12.4 g carbohydrate / 100 kcal and 4.7 g lipid / 100 kcal. The concentration of total HMO in TG1 and TG2 follow-on formula is 0.5 g / L, which is the same as the blend of newborn formula.

[0267] Standard growing-up milk is a bovine milk-based growing-up milk with 67 kcal / 100 mL reconstituted formula consisting of 2.25 g complete protein (40% whey / 60% casein) / 100 kcal, 12.6 g carbohydrate / 100 kcal and 4.5 g lipid / 100 kcal. The concentration of total HMO in TG1 and TG2 growing-up milks is 0.4 g / L, the same as the blend of newborn formula.

[0268] Fecal samples collected at enrollment, 3 months, 6 months, 12 months, and 15 months of age were used for microbiome profiling. Microbial DNA was extracted from frozen feces, purified, and sequenced using 2×150 bp sequencing. Taxonomic relative abundance was calculated using the metagenomic species (MGS) method, which enables quantification of known characterized and uncharacterized microbial species.

[0269] Microbiome-age predictors were trained on data from vaginally delivered HMG infants (reference set: HMG-VD, n = 31) using genus-level data, metagenomic species-level (MGS) data, or CAZyme composition data and optimized using RSME ( Figure 2 These models were applied to CG, TG1, and TG2 to predict microbiome-age and identify outliers (microbiome-age Z-score: |MAZ|>3). The microbiome-age trajectories of CG, TG1, and TG2 were compared with the HMG-VD reference trajectory.

[0270] result

[0271] The 10 features selected in the genus-based model were Romboutsia, Blautia, Staphylococcus, Intestinibacter, Cutibacterium, Megasphaera, Enterococcus, Bifidobacterium, Flavonifractor, and Roseburia.

[0272] The 20 features selected in the species-based model are Romboutsia timonensis, Intestinibacter bartlettii, Staphylococcus hominis subsp. hominis, Staphylococcus epidermidis, Veillonella parvula, [Clostridium] spiroforme, Enterococcus faecalis, [Ruminococcus] gnavus, Flavonifractor plautii, Parabacteroides distasonis, Megasphaera micronuciformis, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, and breve, Collinsella aerofaciens, Ruminococcaceae bacterium, Haemophilus parainfluenzae, Veillonellasp., Clostridiaceae bacterium, Fusicatenibacter saccharivorans, and Clostridium perfringens.

[0273] The 25 features selected in another species-based model were Rombus timonii, Enterobacter butlerii, Staphylococcus hominis subsp. hominis, Staphylococcus epidermidis, Veillonella parvum, Clostridium spiralis, Flavonoids perfringens, Enterococcus faecalis, Ruminococcus active, Parabacteroides dissimilar, Megasphaera walnutii, Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium breve, Bacteroides dorei, Collinsella aerogenes, Ruminococcaceae, Veillonella spp., Clostridium family, Clostridium perfringens, Erysipelatoclostridium ramosum, Fusobacterium sucroseus, Haemophilus parainfluenzae, Peptostreptococcaceae sp.

[0274] The 30 features selected in the CAZyme-based model are GH13_9, GH39, GH73, GT5, CE2, CBM34, GH43_26, GT51, CBM32, GH105, GH38, GH95, GH112, GH33, GH26, GH18, GH109, ​​CBM41, GH43_34, GH31, GH13_31, GH146, GH25, CBM48, GH43_24, GH170, GH5, CE11, GH51, GH76.

[0275] In each model, the TG trajectory converges to the reference trajectory earlier than the CG trajectory. For example, using the genus-based model (with 10 features, R 2 = 0.862), the trajectories differ significantly until about 11.4 months (for CG), about 9.4 months (for TG1), about 9.6 months (for TG2) (see Figure 3A ). Using the MGS species-based model (with 20 features, R 2 = 0.881), the trajectories differ significantly until about 10.3 months (for CG), about 8.1 months (for TG1), about 5.6 months (for TG2) (see Figure 3B ). Using different MGS species-based models (see Figure 3C ), CAZyme-based methods (see Figure 3D ) and α-diversity-based methods (see Figure 3E ), similar results were observed. This effect was more pronounced in formula-fed infants delivered by cesarean section (CS), as observed by different trajectories (see, e.g. Figure 4A and Figure 4B ).

[0276] After the start of the intervention, the number of outliers in the genus-based model was significantly reduced in the TG compared with the CG using the Cochran-Armitage trend test (p = 0.0002) and at the time of visit (3-6 months, p = 0.0002; 12-15 months, p = 0.0377) (see Figure 5 and Table 2 below). Models trained on other data types indicate similar trends.

[0277] Table 2 - Number of infants that were outliers in the genus-based model (with 10 selected features)

[0278]

[0279] These data suggest that supplementing infant formula with a mixture of HMOs can induce convergence of the gut microbiome trajectory of formula-fed infants with that of human milk-fed, vaginally delivered reference infants.

[0280] Implementation

[0281] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paragraphs).

[0282] 1. A mixture of human milk oligosaccharides (HMOs) used to induce the gut microbiome trajectory of formula-fed infants to converge with a reference gut microbiome trajectory obtained from infants fed human milk.

[0283] 2. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide and at least one sialylated oligosaccharide.

[0284] 3. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs comprises or consists of: 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), optionally wherein, based on the total weight of the HMOs, the mixture of HMOs comprises or consists of: (i) 2'FL in an amount of about 55 wt % to about 60 wt %; (ii) diFL in an amount of about 5 wt % to about 7 wt %; (iii) LNT in an amount of about 18 wt % to about 20 wt %; (iv) 3'-SL in an amount of about 6 wt % to about 8 wt %; and (v) 6'-SL in an amount of about 9 wt % to about 11 wt %.

[0285] 4. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of an infant formula, optionally wherein the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk.

[0286] 5. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of a newborn formula comprising a total amount of HMOs of about 0.5 g / L to about 5.0 g / L, about 1.0 g / L to about 3.0 g / L or about 1.5 g / L to about 2.5 g / L; a follow-on formula comprising a total amount of HMOs of about 0.1 g / L to about 2.0 g / L, about 0.2 g / L to about 1.0 g / L, about 0.3 g / L to about 0.8 g / L, about 0.35 g / L to about 0.65 g / L; and / or a growing-up milk comprising a total amount of HMOs of about 0.1 g / L to about 1.0 g / L, about 0.2 g / L to about 0.8 g / L, about 0.28 g / L to about 0.52 g / L or about 0.3 g / L to about 0.5 g / L.

[0287] 6. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs

[0288] The composition is administered in the following form:

[0289] (a) Infant formula milk powder, comprising:

[0290] (i) 2'FL in an amount of about 0.5 g / L to about 3.0 g / L, preferably about 0.70 g / L to about 1.05 g / L or about 1.16 g / L to about 1.74 g / L;

[0291] (ii) diFL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.05 g / L to about 0.11 g / L or about 0.12 g / L to about 0.18 g / L;

[0292] (iii) LNT in an amount from about 0.1 g / L to about 1.0 g / L, preferably from about 0.23 g / L to about 0.36 g / L or from about 0.39 g / L to about 0.58 g / L;

[0293] (iv) 3'-SL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.09 g / L to about 0.13 g / L or about 0.14 g / L to about 0.21 g / L; and

[0294] (v) 6'-SL in an amount of about 0.05 g / L to about 0.5 g / L, preferably about 0.12 g / L to about 0.17 g / L or about 0.19 g / L to about 0.28 g / L;

[0295] (b) Follow-on formula comprising:

[0296] (i) 2'FL in an amount of about 0.19 g / L to about 0.34 g / L;

[0297] (ii) diFL in an amount from about 0.03 g / L to about 0.05 g / L;

[0298] (iii) LNT in an amount from about 0.06 g / L to about 0.11 g / L;

[0299] (iv) 3'-SL in an amount from about 0.04 g / L to about 0.09 g / L; and

[0300] (v) 6'-SL in an amount from about 0.03 g / L to about 0.06 g / L; and / or

[0301] (c) a growing-up milk comprising:

[0302] (i) 2'FL in an amount of about 0.15 g / L to about 0.28 g / L;

[0303] (ii) diFL in an amount from about 0.01 g / L to about 0.04 g / L;

[0304] (iii) LNT in an amount from about 0.05 g / L to about 0.09 g / L;

[0305] (iv) 3'-SL in an amount from about 0.03 g / L to about 0.08 g / L; and

[0306] (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L.

[0307] 7. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of HMOs of about 1.5 g / L, optionally wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising:

[0308] (i) about 0.87 g / L of 2'FL;

[0309] (ii) diFL in an amount of about 0.10 g / L;

[0310] (iii) LNT in an amount of about 0.29 g / L;

[0311] (iv) 3'-SL in an amount of about 0.11 g / L; and

[0312] (v) 6'-SL in an amount of about 0.14 g / L.

[0313] 8. A mixture of HMOs for use according to any one of paragraphs 1 to 6, wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of HMOs of about 2.5 g / L, optionally wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising:

[0314] (i) 2'FL in an amount of about 1.45 g / L;

[0315] (ii) diFL in an amount of about 0.14 g / L;

[0316] (iii) LNT in an amount of about 0.48 g / L;

[0317] (iv) 3'-SL in an amount of about 0.18 g / L; and

[0318] (v) 6'-SL in an amount of about 0.24 g / L.

[0319] 9. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of a follow-on formula comprising a total amount of HMOs of about 0.5 g / L, optionally wherein the mixture of HMOs is administered in the form of a follow-on formula comprising:

[0320] (i) about 0.26 g / L of 2'FL;

[0321] (ii) diFL in an amount of about 0.04 g / L;

[0322] (iii) LNT in an amount of about 0.09 g / L;

[0323] (iv) 3'-SL in an amount of about 0.06 g / L; and

[0324] (v) 6'-SL in an amount of about 0.05 g / L.

[0325] 10. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of a growing-up milk comprising a total amount of HMOs of about 0.4 g / L, optionally wherein the mixture of HMOs is administered in the form of a growing-up milk comprising:

[0326] (i) about 0.21 g / L of 2'FL;

[0327] (ii) diFL in an amount of about 0.03 g / L;

[0328] (iii) LNT in an amount of about 0.07 g / L;

[0329] (iv) 3'-SL in an amount of about 0.06 g / L; and

[0330] (v) 6'-SL in an amount of about 0.04 g / L.

[0331] 11. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk each comprising protein in an amount of about 60 kcal / 100 mL to about 80 kcal / 100 mL, protein in an amount of about 1.5 g / 100 kcal to about 2.5 g / 100 kcal, carbohydrate in an amount of about 8 g / 100 kcal to about 15 g / 100 kcal and lipid in an amount of about 3 g / 100 kcal to about 8 g / 100 kcal.

[0332] 12. A mixture of HMOs for use according to any preceding paragraph, wherein the mixture of HMOs is administered to the formula-fed infant until at least about 6 months of age, until at least about 9 months of age, until at least about 12 months of age, or until at least about 15 months of age.

[0333] 13. A mixture of HMOs for use according to any preceding paragraph, wherein at about 6 months of age or later, about 7 months of age or later, about 8 months of age or later, or about 9 months of age or later, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from a human milk-fed infant.

[0334] 14. A mixture of HMOs for use according to any preceding paragraph, wherein at about 12 months of age or earlier, about 11 months of age or earlier, about 10 months of age or earlier, or about 9 months of age or earlier, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from a human milk-fed infant.

[0335] 15. A mixture of HMOs for use according to any preceding paragraph, wherein at about 6 months to about 12 months of age, about 7 months to about 11 months of age, about 8 months to 10 months of age, or about 9 months of age, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from a human milk-fed infant.

[0336] 16. A mixture of HMOs for use according to any preceding paragraph, wherein the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory.

[0337] 17. A mixture of HMOs for use according to any preceding paragraph, wherein the gut microbiome trajectory is a gut microbiome age trajectory, optionally wherein the gut microbiome age trajectory is obtained using genus level data, species level data and / or functional data from gut microbiome data.

[0338] 18. A mixture of HMOs for use according to any preceding paragraph wherein the formula fed infant is full term.

[0339] 19. A mixture of HMOs for use according to any preceding paragraph wherein the formula fed infant is delivered by caesarean section.

[0340] 20. A mixture of HMOs for use according to any preceding paragraph wherein the human milk fed infant is a full term vaginally delivered human milk fed infant.

[0341] 21. A mixture of HMOs for use according to any preceding paragraph, wherein inducing convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant promotes intestinal maturation.

[0342] 22. A mixture of HMOs for use according to any preceding paragraph, wherein inducing convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant promotes maturation of the gut microbiome, gut metabolism, gut barrier function and / or gut immune function.

[0343] 23. A method for inducing convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant, the method comprising administering to the formula-fed infant an effective amount of a mixture of human milk oligosaccharides (HMOs).

[0344] 24. Use of a mixture of human milk oligosaccharides (HMOs) for inducing convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from an infant fed human milk.

[0345] 25. The method according to paragraph 23 or the use according to paragraph 24, wherein the mixture of HMOs comprises at least one fucosylated oligosaccharide, at least one N-acetylated oligosaccharide and at least one sialylated oligosaccharide.

[0346] 26. The method according to paragraph 23 or 25 or the use according to paragraph 24 or 25, wherein the mixture of HMOs comprises or consists of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), optionally wherein, based on the total weight of the HMOs, the mixture of HMOs comprises or consists of: (i) 2'FL in an amount of about 55 wt % to about 60 wt %; (ii) diFL in an amount of about 5 wt % to about 7 wt %; (iii) LNT in an amount of about 18 wt % to about 20 wt %; (iv) 3'-SL in an amount of about 6 wt % to about 8 wt %; and (v) 6'-SL in an amount of about 9 wt % to about 11 wt %.

[0347] 27. The method according to any one of paragraphs 23, 25 or 26 or the use according to any one of paragraphs 24 to 26, wherein the mixture of HMOs is administered in the form of an infant formula, optionally wherein the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk.

[0348] 28. The method according to any one of paragraphs 23 or 25 to 27 or the use according to any one of paragraphs 24 to 27, wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of HMOs of about 0.5 g / L to about 5.0 g / L, about 1.0 g / L to about 3.0 g / L, or about 1.5 g / L to about 2.5 g / L; a follow-on infant formula comprising a total amount of HMOs of about 0.1 g / L to about 1.0 g / L; A total amount of HMO of about 2.0 g / L, about 0.2 g / L to about 1.0 g / L, about 0.3 g / L to about 0.8 g / L, about 0.35 g / L to about 0.65 g / L; and / or a growing-up milk comprising a total amount of HMO of about 0.1 g / L to about 1.0 g / L, about 0.2 g / L to about 0.8 g / L, about 0.28 g / L to about 0.52 g / L or about 0.3 g / L to about 0.5 g / L.

[0349] 29. The method according to any one of paragraphs 23 or 25 to 28 or the use according to any one of paragraphs 24 to 28, wherein the mixture of HMOs is administered in the form of:

[0350] (a) Infant formula milk powder, comprising:

[0351] (i) 2'FL in an amount of about 0.5 g / L to about 3.0 g / L, preferably about 0.70 g / L to about 1.05 g / L or about 1.16 g / L to about 1.74 g / L;

[0352] (ii) diFL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.05 g / L to about 0.11 g / L or about 0.12 g / L to about 0.18 g / L;

[0353] (iii) LNT in an amount from about 0.1 g / L to about 1.0 g / L, preferably from about 0.23 g / L to about 0.36 g / L or from about 0.39 g / L to about 0.58 g / L;

[0354] (iv) 3'-SL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.09 g / L to about 0.13 g / L or about 0.14 g / L to about 0.21 g / L; and

[0355] (v) 6'-SL in an amount of about 0.05 g / L to about 0.5 g / L, preferably about 0.12 g / L to about 0.17 g / L or about 0.19 g / L to about 0.28 g / L;

[0356] (b) Follow-on formula comprising:

[0357] (i) 2'FL in an amount of about 0.19 g / L to about 0.34 g / L;

[0358] (ii) diFL in an amount from about 0.03 g / L to about 0.05 g / L;

[0359] (iii) LNT in an amount from about 0.06 g / L to about 0.11 g / L;

[0360] (iv) 3'-SL in an amount from about 0.04 g / L to about 0.09 g / L; and

[0361] (v) 6'-SL in an amount from about 0.03 g / L to about 0.06 g / L; and / or

[0362] (c) a growing-up milk comprising:

[0363] (i) 2'FL in an amount of about 0.15 g / L to about 0.28 g / L;

[0364] (ii) diFL in an amount from about 0.01 g / L to about 0.04 g / L;

[0365] (iii) LNT in an amount from about 0.05 g / L to about 0.09 g / L;

[0366] (iv) 3'-SL in an amount from about 0.03 g / L to about 0.08 g / L; and

[0367] (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L.

[0368] 30. The method according to any one of paragraphs 23 or 25 to 29 or the use according to any one of paragraphs 24 to 29, wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of about 1.5 g / L of HMOs, optionally wherein the mixture of HMOs

[0369] The composition is administered in the form of a newborn infant formula comprising:

[0370] (i) about 0.87 g / L of 2'FL;

[0371] (ii) diFL in an amount of about 0.10 g / L;

[0372] (iii) LNT in an amount of about 0.29 g / L;

[0373] (iv) 3'-SL in an amount of about 0.11 g / L; and

[0374] (v) 6'-SL in an amount of about 0.14 g / L.

[0375] 31. The method according to any one of paragraphs 23 or 25 to 30 or the use according to any one of paragraphs 24 to 30, wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising a total amount of about 2.5 g / L of HMOs, optionally wherein the mixture of HMOs is administered in the form of a newborn infant formula comprising:

[0376] (i) 2'FL in an amount of about 1.45 g / L;

[0377] (ii) diFL in an amount of about 0.14 g / L;

[0378] (iii) LNT in an amount of about 0.48 g / L;

[0379] (iv) 3'-SL in an amount of about 0.18 g / L; and

[0380] (v) 6'-SL in an amount of about 0.24 g / L.

[0381] 32. The method according to any one of paragraphs 23 or 25 to 31 or the use according to any one of paragraphs 24 to 31, wherein the mixture of HMOs is administered in the form of a follow-on formula comprising a total amount of about 0.5 g / L of HMOs, optionally wherein the mixture of HMOs

[0382] The composition is administered in the form of a follow-on formula comprising:

[0383] (i) about 0.26 g / L of 2'FL;

[0384] (ii) diFL in an amount of about 0.04 g / L;

[0385] (iii) LNT in an amount of about 0.09 g / L;

[0386] (iv) 3'-SL in an amount of about 0.06 g / L; and

[0387] (v) 6'-SL in an amount of about 0.05 g / L.

[0388] 33. The method according to any one of paragraphs 23 or 25 to 32 or the use according to any one of paragraphs 24 to 32, wherein the mixture of HMOs is administered in the form of a growing-up milk comprising a total amount of about 0.4 g / L of HMOs, optionally wherein the mixture of HMOs is administered in the form of a growing-up milk comprising:

[0389] (i) about 0.21 g / L of 2'FL;

[0390] (ii) diFL in an amount of about 0.03 g / L;

[0391] (iii) LNT in an amount of about 0.07 g / L;

[0392] (iv) 3'-SL in an amount of about 0.06 g / L; and

[0393] (v) 6'-SL in an amount of about 0.04 g / L.

[0394] 34. The method according to any one of paragraphs 23 or 25 to 33 or the use according to any one of paragraphs 24 to 33, wherein the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk each comprising protein in an amount of about 60 kcal / 100 mL to about 80 kcal / 100 mL, protein in an amount of about 1.5 g / 100 kcal to about 2.5 g / 100 kcal, carbohydrates in an amount of about 8 g / 100 kcal to about 15 g / 100 kcal and lipids in an amount of about 3 g / 100 kcal to about 8 g / 100 kcal.

[0395] 35. The method of any one of paragraphs 23 or 25 to 34 or the use of any one of paragraphs 24 to 34, wherein the mixture of HMOs is administered to the formula-fed infant until at least about 6 months of age, until at least about 9 months of age, until at least about 12 months of age, or until at least about 15 months of age.

[0396] 36. The method of any one of paragraphs 23 or 25 to 35 or the use of any one of paragraphs 24 to 35, wherein at about 6 months of age or later, about 7 months of age or later, about 8 months of age or later, about 9 months of age or later, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from an infant fed human milk.

[0397] 37. The method of any one of paragraphs 23 or 25 to 36 or the use of any one of paragraphs 24 to 36, wherein at about 12 months of age or earlier, about 11 months of age or earlier, about 10 months of age or earlier, or about 9 months of age or earlier, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from an infant fed human milk.

[0398] 38. The method of any one of paragraphs 23 or 25 to 37 or the use of any one of paragraphs 24 to 37, wherein at about 6 months to about 12 months of age, about 7 months to about 11 months of age, about 8 months to 10 months of age, or about 9 months of age, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from an infant fed human milk.

[0399] 39. The method according to any one of paragraphs 23 or 25 to 38 or the use according to any one of paragraphs 24 to 38, wherein the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory.

[0400] 40. The method of any one of paragraphs 23 or 25 to 39 or the use of any one of paragraphs 24 to 39, wherein the gut microbiome trajectory is a gut microbiome age trajectory, optionally wherein the gut microbiome age trajectory is obtained using genus level data, species level data and / or functional data from the gut microbiome data.

[0401] 41. The method of any one of paragraphs 23 or 25 to 40 or the use of any one of paragraphs 24 to 40, wherein the formula-fed infant is full-term.

[0402] 42. The method of any one of paragraphs 23 or 25 to 41 or the use of any one of paragraphs 24 to 41, wherein the formula-fed infant is delivered by caesarean section.

[0403] 43. The method of any one of paragraphs 23 or 25 to 42 or the use of any one of paragraphs 24 to 42, wherein the human milk fed infant is a full term vaginally delivered human milk fed infant.

[0404] 44. The method of any one of paragraphs 23 or 25 to 43, or the use of any one of paragraphs 24 to 43, wherein the method promotes intestinal maturation.

[0405] 45. The method according to any one of paragraphs 23 or 25 to 44 or the use according to any one of paragraphs 24 to 44, wherein the method promotes the maturation of the intestinal microbiome, intestinal metabolism, intestinal barrier function and / or intestinal immune function.

[0406] 46. ​​A method for determining the state of intestinal maturation in a formula-fed infant, wherein the method comprises:

[0407] (a) Provide gut microbiome data from a population of human milk-fed infants;

[0408] (b) training a regression model based on gut microbiome data; and

[0409] (c) providing gut microbiome data from formula-fed infants and determining whether the formula-fed infants are outliers in a trained regression model,

[0410] wherein if the formula-fed infant is not an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant is normal, and / or wherein if the formula-fed infant is an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant is abnormal.

[0411] 47. A method according to paragraph 46, wherein the formula-fed infant is an outlier based on the standard error (SE), confidence interval, prediction interval and / or standard deviation in the trained regression model, preferably wherein the intestinal microbiome data of the formula-fed infant is an outlier if it differs from the trained regression line by -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, or -3SE or less or 3SE or more, if the intestinal microbiome data of the formula-fed infant falls outside the 90%, 95% or 99% confidence interval in the trained regression model, if the intestinal microbiome data of the formula-fed infant falls within the 90%, 95% or 99% confidence interval in the trained regression model,

[0412] outside the 95% or 99% prediction interval, and / or if the formula-fed infant has a -2 or less or 2 or greater, -2.5 or less or 2.5 in the trained regression model.

[0413] or greater, or -3 or less, or a Z score of 3 or greater, then the formula-fed infant is an outlier.

[0414] 48. A method for determining the state of intestinal maturation in a formula-fed infant, wherein the method comprises:

[0415] (a) Provide gut microbiome data from a population of human milk-fed infants;

[0416] (b) training a regression model based on the gut microbiome data to provide a gut microbiome trajectory; and

[0417] (c) provide gut microbiome data from formula-fed infants and determine whether formula-fed infants conform to gut microbiome trajectories,

[0418] wherein if the formula-fed infant meets the gut microbiome trajectory, then the intestinal maturation state of the formula-fed infant is normal, and / or wherein if the formula-fed infant does not meet the gut microbiome trajectory, then the intestinal maturation state of the formula-fed infant is abnormal.

[0419] 49. A method according to paragraph 48, wherein the formula-fed infant is determined to not conform to the gut microbiome trajectory based on the standard error (SE), confidence interval, prediction interval and / or standard deviation of the gut microbiome trajectory, preferably, if the gut microbiome data of the formula-fed infant differs from the gut microbiome trajectory by -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, or -3SE or less or 3SE or more, if the gut microbiome data of the formula-fed infant falls outside the 90%, 95% or 99% confidence interval of the gut microbiome trajectory, if the gut microbiome data of the formula-fed infant falls outside the 90%, 95% or 99% prediction interval of the gut microbiome trajectory, and / or if the formula-fed infant has a Z score of -2 or less or 2 or greater, -2.5 or less or 2.5 or greater, or -3 or less or 3 or greater, then the formula-fed infant is determined to not conform to the gut microbiome trajectory.

[0420] 50. A data processing system comprising means for performing a method according to any of paragraphs 46 to 49.

[0421] 51. A data processing system comprising a processor configured to perform a method according to any of paragraphs 46 to 49.

[0422] 52. A computer-readable medium comprising instructions which, when executed by a computer, cause the computer to perform the method of any of paragraphs 46 to 49.

[0423] 53. A computer program comprising instructions which, when executed by a computer, cause the computer to perform a method according to any one of paragraphs 46 to 49.

[0424] 54. A computer-readable data carrier having stored thereon a computer program according to paragraph 53.

[0425] 55. A data carrier signal carrying a computer program according to paragraph 53.

[0426] 56. Use of one or more reference gut microbiome trajectories obtained from human milk fed infants for determining the state of intestinal maturation in formula fed infants following administration of a mixture of HMOs.

[0427] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the methods, compositions and uses disclosed in the present invention will be apparent to the skilled person without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the present invention protected by the claims should not be unduly limited to such specific embodiments. In fact, various modifications to the modes disclosed for practicing the present invention that are apparent to the skilled person are intended to fall within the scope of the following claims.

Claims

1. A mixture of human milk oligosaccharides (HMOs) for inducing a convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant, wherein the mixture of HMOs comprises or consists of composition: 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

2. A mixture of HMOs for use according to claim 1, wherein the mixture of HMOs comprises or consists of the following, based on the total weight of the HMOs: composition: (i) 2'FL in an amount of about 55 wt% to about 60 wt%; (ii) diFL in an amount of about 5 wt% to about 7 wt%; (iii) LNT in an amount of about 18 wt% to about 20 wt%; (iv) 3'-SL in an amount of about 6 wt% to about 8 wt%; and (v) 6'-SL in an amount of about 9 wt% to about 11 wt%.

3. A mixture of HMOs for use according to claim 1 or 2, wherein the mixture of HMOs is administered in the form of an infant formula, optionally wherein the mixture of HMOs is administered in the form of a newborn formula, a follow-on formula and / or a growing-up milk.

4. A mixture of HMOs for use according to any preceding claim, wherein the mixture of HMOs is administered in the form of: (a) a newborn infant formula comprising a total amount of HMOs in an amount of about 0.5 g / L to about 5.0 g / L, about 1.0 g / L to about 3.0 g / L, or about 1.5 g / L to about 2.5 g / L, optionally wherein the newborn infant formula comprises: (i) 2'FL in an amount of about 0.5 g / L to about 3.0 g / L, preferably about 0.70 g / L to about 1.05 g / L or about 1.16 g / L to about 1.74 g / L; (ii) diFL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.05 g / L to about 0.11 g / L or about 0.12 g / L to about 0.18 g / L; (iii) LNT in an amount from about 0.1 g / L to about 1.0 g / L, preferably from about 0.23 g / L to about 0.36 g / L or from about 0.39 g / L to about 0.58 g / L; (iv) 3'-SL in an amount of about 0.05 g / L to about 0.3 g / L, preferably about 0.09 g / L to about 0.13 g / L or about 0.14 g / L to about 0.21 g / L; and (v) 6'-SL in an amount of about 0.05 g / L to about 0.5 g / L, preferably about 0.12 g / L to about 0.17 g / L or about 0.19 g / L to about 0.28 g / L; (b) a follow-on formula comprising a total amount of HMO in an amount of from about 0.1 g / L to about 2.0 g / L, from about 0.2 g / L to about 1.0 g / L, from about 0.3 g / L to about 0.8 g / L, from about 0.35 g / L to about 0.65 g / L, optionally wherein the follow-on formula comprises: (i) 2'FL in an amount of about 0.19 g / L to about 0.34 g / L; (ii) diFL in an amount from about 0.03 g / L to about 0.05 g / L; (iii) LNT in an amount from about 0.06 g / L to about 0.11 g / L; (iv) 3'-SL in an amount from about 0.04 g / L to about 0.09 g / L; and (v) 6'-SL in an amount from about 0.03 g / L to about 0.06 g / L; and / or (c) a growing-up milk comprising a total amount of HMOs of about 0.1 g / L to about 1.0 g / L, about 0.2 g / L to about 0.8 g / L, about 0.28 g / L to about 0.52 g / L, or about 0.3 g / L to about 0.5 g / L, optionally wherein the growing-up milk comprises: (i) 2'FL in an amount of about 0.15 g / L to about 0.28 g / L; (ii) diFL in an amount from about 0.01 g / L to about 0.04 g / L; (iii) LNT in an amount from about 0.05 g / L to about 0.09 g / L; (iv) 3'-SL in an amount from about 0.03 g / L to about 0.08 g / L; and (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L.

5. A mixture of HMOs for use according to any preceding claim, wherein the mixture of HMOs is administered in the form of: (a) a newborn infant formula comprising a total amount of HMO of about 1.5 g / L or 2.5 g / L, Optionally wherein the newborn infant formula comprises: (i) 2'FL in an amount of about 0.87 g / L or about 1.45 g / L; (ii) diFL in an amount of about 0.10 g / L or about 0.14 g / L; (iii) LNT in an amount of about 0.29 g / L or about 0.48 g / L; (iv) 3'-SL in an amount of about 0.11 g / L or about 0.18 g / L; and (v) 6'-SL in an amount of about 0.14 g / L or about 0.24 g / L; (b) a follow-on formula comprising a total amount of HMO of about 0.5 g / L, optionally wherein the follow-on formula comprises: (i) about 0.26 g / L of 2'FL; (ii) diFL in an amount of about 0.04 g / L; (iii) LNT in an amount of about 0.09 g / L; (iv) 3'-SL in an amount of about 0.06 g / L; and (v) 6'-SL in an amount of about 0.05 g / L; and / or (c) a growing-up milk comprising a total amount of HMO of about 0.4 g / L, optionally wherein the growing-up milk comprises: (i) about 0.21 g / L of 2'FL; (ii) diFL in an amount of about 0.03 g / L; (iii) LNT in an amount of about 0.07 g / L; (iv) 3'-SL in an amount of about 0.06 g / L; and (v) 6'-SL in an amount of about 0.04 g / L.

6. The mixture of HMOs for use according to any preceding claim, wherein the mixture of HMOs is administered to the formula-fed infant until at least about 6 months of age, until at least about 9 months of age, until at least about 12 months of age, or until at least about 15 months of age.

7. A mixture of HMOs for use according to any preceding claim, wherein at about 6 months to about 12 months of age, about 7 months to about 11 months of age, about 8 months to 10 months of age or about 9 months of age, the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from a human milk-fed infant.

8. A mixture of HMOs for use according to any preceding claim, wherein inducing convergence of the gut microbiome trajectory of the formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant promotes intestinal maturation.

9. A method for inducing convergence of a gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant, the method comprising administering to the formula-fed infant an effective amount of a mixture of human milk oligosaccharides (HMOs), wherein the mixture of HMOs comprises or consists of composition: 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

10. Use of a mixture of human milk oligosaccharides (HMOs) for inducing convergence of the gut microbiome trajectory of a formula-fed infant with a reference gut microbiome trajectory obtained from a human milk-fed infant, wherein the mixture of HMOs comprises or consists of composition: 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

11. A method for determining the state of intestinal maturation in a formula-fed infant, wherein the method include: (a) Provide gut microbiome data from a population of human milk-fed infants; (b) training a regression model based on the intestinal microbiome data; as well as (c) providing gut microbiome data from the formula-fed infant and determining whether the formula-fed infant is an outlier in the trained regression model, wherein if the formula-fed infant is not an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant is normal, and / or wherein if the formula-fed infant is an outlier in the trained regression model, then the intestinal maturation state of the formula-fed infant is abnormal.

12. A method for determining the state of intestinal maturation in a formula-fed infant, wherein the method include: (a) Provide gut microbiome data from a population of human milk-fed infants; (b) training a regression model based on the gut microbiome data to provide a gut microbiome trajectory; as well as (c) providing gut microbiome data from the formula-fed infant and determining whether the formula-fed infant meets the gut microbiome trajectory, Wherein if the formula-fed infant complies with the gut microbiome trajectory, the intestinal maturation state of the formula-fed infant is normal, and / or if the formula-fed infant does not comply with the gut microbiome trajectory, the intestinal maturation state of the formula-fed infant is abnormal.

13. Computer program comprising instructions which, when said program is executed by a computer, cause said computer to carry out the method according to claim 11 or 12.

14. A data carrier signal carrying a computer program according to claim 13.

15. Use of one or more reference gut microbiome trajectories obtained from human milk fed infants for determining the state of intestinal maturation in formula fed infants following administration of a mixture of HMOs.

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