Human milk oligosaccharides for improving the gut microbiome and methods for determining gut maturation status

Abstract

The present invention provides a method for inducing a gut microbiome trajectory of a formula-fed infant to converge with a reference gut microbiome trajectory obtained from a human breast-fed infant, the method comprising administering to the formula-fed infant an effective amount of a human milk oligosaccharide (HMO) mixture.The present invention also provides a method for determining the gut maturation status of a formula-fed infant, the method comprising providing a reference gut microbiome trajectory obtained from a human breast-fed infant.

Description

[Technical Field]

[0001] The present invention relates to methods for inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory. The present invention also relates to methods for determining the gut maturation status of an infant and / or young child. [Background technology]

[0002] Breast milk generally provides the reference nutrition for all infants, and exclusive breastfeeding for the first six months is typically recommended. During early infancy, full or partial breastfeeding has well-recognized effects on the composition and function of the gut microbiome compared with non-breastfeeding. For example, longer periods of exclusive breastfeeding are associated with reduced diarrhea-related gut microbiota dysbiosis. Furthermore, differences in the gut microbiota between breastfed and non-breastfed infants can persist even after six months of age (see, e.g., Ho, NT, et al., 2018, Nature Communications, 9(1), pp. 1-13).

[0003] Health benefits observed in breastfed infants may include protection against infections and potentially reduced risk of overweight and diabetes later in life. This benefit indicates that nutritional alterations to microbiome maturation early in life can have health consequences later in life. Numerous observational cohort studies and randomized controlled trials have shown that the composition of infant formula ingested has a significant impact on the microbiome (see, for example, Dogra, SK, et al., 2021. Microorganisms, 9(10), p. 2110).

[0004] For example, adding two specific human milk oligosaccharides, 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), to starter infant formula has been shown to more closely resemble the gut microbiome composition of breastfed infants. However, at 12 months, the gut microbiome composition of the breastfed group remained significantly different from that of the formula group (see, e.g., Berger, B., et al., 2020. Mbio, 11(2), pp. e03196-19).

[0005] [Summary of the Invention] We have shown that adding a human milk oligosaccharide (HMO) mixture to starter infant formula, follow-up formula, and growing-up milk can induce the gut microbiome trajectories of formula-fed infants to converge with, and remain converged with, a reference gut microbiome trajectory obtained from a human breast-fed group. The gut microbiome trajectories converged more quickly, and the number of outlier formula-fed infants and toddlers was significantly reduced when the HMO mixture was added to the starter infant formula, follow-up formula, and growing-up milk.

[0006] In one aspect, the invention provides a human milk oligosaccharide (HMO) mixture for use in inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory.

[0007] In another aspect, the invention provides a method for inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory, comprising administering to the infant an effective amount of a human milk oligosaccharide (HMO) mixture.

[0008] In another aspect, the invention provides for the use of a human milk oligosaccharide (HMO) mixture to induce an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory.

[0009] The HMO mixture may comprise any suitable HMO, which may be administered in any suitable form, in any suitable amount, and for any suitable period of time. Preferably, the HMO mixture comprises at least one fucosyl oligosaccharide, at least one N-acetyl oligosaccharide, and at least one sialyloligosaccharide. Preferably, the HMO mixture 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). Preferably, the HMO mixture is administered in the form of infant formula and / or growing-up milk. Preferably, the HMO mixture comprises or consists of, based on the total weight of the HMO, (i) 2'FL in an amount of about 55% to about 60% by weight, (ii) diFL in an amount of about 5% to about 7% by weight, (iii) LNT in an amount of about 18% to about 20% by weight, (iv) 3'-SL in an amount of about 6% to about 8% by weight, and (v) 6'-SL in an amount of about 9% to about 11% by weight. Preferably, the HMO mixture is administered to the infant until at least about 6 months of age, at least about 9 months of age, at least about 12 months of age, or at least about 15 months of age.

[0010] In a preferred embodiment, the HMO mixture is administered in the form of a starter infant formula, a follow-up formula, and / or a growing-up milk. Preferably, the HMO mixture is administered in the form of a starter infant formula containing 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, or about 1.5 g / L to about 2.5 g / L; a follow-up formula containing HMOs in a total amount 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, or about 0.35 g / L to about 0.65 g / L; and / or a growing-up milk containing HMOs in a total amount 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] Preferably, the HMO mixture is administered in the form of a starter 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 HMO mixture is administered in the form of a starter 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. Preferably, the HMO mixture is administered in the form of a follow-up 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. Preferably, the HMO mixture is administered in the form of a growing-up milk containing: (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 HMO mixture is administered in the form of a starter infant formula comprising a total amount of HMOs of about 1.5 g / L. In some embodiments, the HMO mixture is administered in the form of a starter 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 HMO mixture is administered in the form of a starter infant formula comprising a total amount of HMOs of about 2.5 g / L. In some embodiments, the HMO mixture is administered in the form of a starter 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 HMO mixture is administered in the form of a follow-up formula comprising a total amount of HMOs of about 0.5 g / L. In some embodiments, the HMO mixture is administered in the form of a follow-up formula containing (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 HMO mixture is administered in the form of a growing-up milk containing a total amount of HMOs of about 0.4 g / L. In some embodiments, the HMO mixture is administered in the form of a growing-up milk containing (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] The starter infant formula, follow-up formula, and growing-up milk may each contain any other suitable ingredients. Preferably, the starter infant formula, follow-up formula, and growing-up milk each contain about 60 kcal / 100 mL to about 80 kcal / 100 mL, and contain 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.

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

[0015] The infant gut microbiome trajectory and the reference gut microbiome trajectory may be obtained by any suitable method. The reference gut microbiome trajectory may be obtained from a human breast-fed group (infants and / or young children). Preferably, the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory. Preferably, the gut microbiome trajectory is a gut microbiome age trajectory. Preferably, 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 with a gut microbiome composition that differs from the reference gut microbiome trajectory. The infant may be a formula-fed infant. Preferably, the infant is a full-term infant. Preferably, the infant is a Caesarean section infant.

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

[0018] In one aspect, the present invention provides a method for determining the intestinal maturity status of a formula-fed infant or toddler, the method comprising: (a) providing gut microbiome data from a cohort of human breast-fed infants and / or young children; (b) training a regression model on gut microbiome data; and (c) providing gut microbiome data from the formula-fed infant or toddler and determining whether the formula-fed infant or toddler is an outlier in the trained regression model; Including, A method is provided in which the intestinal maturation status of a formula-fed infant or toddler is normal if the formula-fed infant or toddler is not an outlier in the trained regression model, and / or the intestinal maturation status of a formula-fed infant or toddler is not normal if the formula-fed infant or toddler is an outlier in the trained regression model.

[0019] In another aspect, the present invention provides a method for determining the intestinal maturity status of a formula-fed infant or toddler, the method comprising: (a) providing gut microbiome data from a cohort of human breast-fed infants and / or young children; (b) training a regression model on the gut microbiome data to provide a gut microbiome trajectory; (c) providing gut microbiome data from the formula-fed infant or toddler to determine whether the formula-fed infant or toddler is on a gut microbiome trajectory; and Including, Methods are provided in which the formula-fed infant or toddler has a normal gut maturation status if the formula-fed infant or toddler is on a gut microbiome trajectory, and / or the formula-fed infant or toddler has a non-normal gut maturation status if the formula-fed infant or toddler is off a gut microbiome trajectory.

[0020] 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 on a gut microbiome trajectory. Preferably, the formula-fed infant or toddler is an outlier and / or off-trajectory based on the standard error (SE), confidence interval, prediction interval, and / or standard deviation in the trained regression model or gut microbiome trajectory. Preferably, a formula-fed infant or toddler is an outlier and / or off the trajectory if the formula-fed infant's or toddler's gut microbiome data is -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, or -3SE or less or 3SE or more from the trained regression line or gut microbiome trajectory; if the formula-fed infant's or toddler's gut microbiome data is outside the 90%, 95% or 99% confidence interval in the trained regression model or gut microbiome trajectory; if the formula-fed infant's or toddler's gut microbiome data is 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 more, -2.5 or less or 2.5 or more, or -3 or less or 3 or more on the trained regression model or gut microbiome trajectory.

[0021] In another aspect, the present invention provides a data processing system comprising means for carrying out the method for determining the intestinal maturity status of a formula-fed infant or young child according to the present invention.

[0022] In another aspect, the present invention provides a processor configured to carry out a method for determining the intestinal maturity status of a formula-fed infant or toddler according to the present invention.

[0023] In another aspect, the present invention provides a computer readable medium comprising instructions that, when executed by a computer, cause the computer to perform a method for determining the intestinal maturity status of a formula-fed infant or young child in accordance with the present invention.

[0024] In another aspect, the present invention provides a computer program comprising instructions which, when executed by a computer, cause the computer to carry out a method for determining the intestinal maturity status of a formula-fed infant or young child according to the present invention.

[0025] In another aspect, the present invention provides a computer readable data carrier having stored thereon a computer program according to the present invention.

[0026] In another aspect, the present invention provides a data carrier signal carrying a computer program according to the present invention.

[0027] In another aspect, the present invention provides the use of one or more reference gut microbiome trajectories to determine the gut maturation status of a formula-fed infant or toddler following administration of an HMO mixture. [Brief explanation of the drawings]

[0028] [Figure 1] Schematic of the HMO study. Healthy, full-term infants were randomly assigned to receive a standard cow's milk-based starter infant formula (control group, CG); the same formula containing 1.5 g / L of HMO (test group 1, TG1); or the same formula containing 2.5 g / L of HMO (test group 2, TG2); or a human breast-fed group (reference, HMG). Fecal samples were collected at enrollment and at 3, 6, 12, and 15 months of age. [Figure 2] Modeling gut microbiome age. The overall process for modeling microbiome age is presented. [Figure 3A]Exemplary Gut Microbiome Trajectories from the Model. LOESS fits microbiome age trajectories for each nutritional group using age predictors trained on data from the vaginally delivered HMG group (infants and / or young children) using: (A) genus-level data (10 selected features, R2 = 0.862); (B) MGS species-level data (20 selected features, R2 = 0.881); (C) MGS species-level data (25 selected features, R2 = 0.844); (D) CAZyme data (30 selected features, R2 = 0.658); (E) Shannon index by gene. Shaded regions indicate 95% confidence intervals. [Figure 3B] Exemplary Gut Microbiome Trajectories from the Model. LOESS fits microbiome age trajectories for each nutritional group using age predictors trained on data from the vaginally delivered HMG group (infants and / or young children) using: (A) genus-level data (10 selected features, R2 = 0.862); (B) MGS species-level data (20 selected features, R2 = 0.881); (C) MGS species-level data (25 selected features, R2 = 0.844); (D) CAZyme data (30 selected features, R2 = 0.658); (E) Shannon index by gene. Shaded regions indicate 95% confidence intervals. [Figure 3C] Exemplary Gut Microbiome Trajectories from the Model. LOESS fits microbiome age trajectories for each nutritional group using age predictors trained on data from the vaginally delivered HMG group (infants and / or young children) using: (A) genus-level data (10 selected features, R2 = 0.862); (B) MGS species-level data (20 selected features, R2 = 0.881); (C) MGS species-level data (25 selected features, R2 = 0.844); (D) CAZyme data (30 selected features, R2 = 0.658); (E) Shannon index by gene. Shaded regions indicate 95% confidence intervals. [Figure 3D]Exemplary Gut Microbiome Trajectories from the Model. LOESS fits microbiome age trajectories for each nutritional group using age predictors trained on data from the vaginally delivered HMG group (infants and / or young children) using: (A) genus-level data (10 selected features, R2 = 0.862); (B) MGS species-level data (20 selected features, R2 = 0.881); (C) MGS species-level data (25 selected features, R2 = 0.844); (D) CAZyme data (30 selected features, R2 = 0.658); (E) Shannon index by gene. Shaded regions indicate 95% confidence intervals. [Figure 3E] Exemplary Gut Microbiome Trajectories from the Model. LOESS fits microbiome age trajectories for each nutritional group using age predictors trained on data from the vaginally delivered HMG group (infants and / or young children) using: (A) genus-level data (10 selected features, R2 = 0.862); (B) MGS species-level data (20 selected features, R2 = 0.881); (C) MGS species-level data (25 selected features, R2 = 0.844); (D) CAZyme data (30 selected features, R2 = 0.658); (E) Shannon index by gene. Shaded regions indicate 95% confidence intervals. [Figure 4A] Exemplary Gut Microbiome Trajectories with Delivery Models. LOESS fits microbiome age trajectories for each feeding group using age predictors trained on data from the vaginally delivered HMG group (infants and / or infants) using CAZyme data (30 selected features) for (A) the vaginally delivered formula-fed group (infants and / or infants); (B) the Cesarean section delivered formula-fed group (infants and / or infants). Shaded regions indicate 95% confidence intervals. [Figure 4B]Exemplary Gut Microbiome Trajectories with Delivery Models. LOESS fits microbiome age trajectories for each feeding group using age predictors trained on data from the vaginally delivered HMG group (infants and / or infants) using CAZyme data (30 selected features) for (A) the vaginally delivered formula-fed group (infants and / or infants); (B) the Cesarean section delivered formula-fed group (infants and / or infants). Shaded regions indicate 95% confidence intervals. [Figure 5] Exemplary outliers in genus-level data. MAZ values ​​were calculated from an age predictor trained on data from the vaginally delivered HMG group (infants and / or young children) using genus-level data (10 selected features, optimized with RMSE, R2 = 0.862). DETAILED DESCRIPTION OF THE INVENTION

[0029] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example, it being understood that those skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0030] It should be noted that as used in this specification and 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," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," are inclusive, or open-ended, and do not exclude additional, unspecified members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0031] Numerical ranges are inclusive of the numbers defining the range. As used herein, the term "about" means approximately, in the region of, roughly, or in the vicinity of. When the term "about" is used in conjunction with a numerical value or range, the value or range modifies the value or range by extending the boundaries above and below the stated numerical value(s). In general, the terms "about" and "approximately" are used herein to adjust numerical value(s) above and below the stated value(s) by 10%.

[0032] The concentrations of ingredients in the compositions described herein may refer to the concentrations after the composition has been reconstituted, for example with water.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any reference cited herein should not be construed as an admission that such reference constitutes prior art to the claims appended hereto.

[0034] The methods and systems disclosed herein can be used by physicians, medical professionals, laboratory technicians, infant and / or child care providers, and the like.

[0035] Human milk oligosaccharide (HMO) mixture The present invention provides a human milk oligosaccharide (HMO) mixture for use in inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory.

[0036] Human milk oligosaccharides (HMO) Any suitable HMO mixture may be used in the present invention.

[0037] As used herein, "human milk oligosaccharides" or "HMOs" (also known as human milk glycans) are short-chain polymers of monosaccharides found in high concentrations in human breast milk. Many types of HMOs are found in human breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid, fucose, and / or N-acetylglucosamine, with a wide variety of linkages between them, resulting in a great diversity of oligosaccharides in human breast milk. Most HMOs have a lactose moiety at the reducing end, with sialic acid and / or fucose (if present) occupying the non-reducing terminal positions. HMOs can be acidic (e.g., charged sialic acid-containing oligosaccharides) or neutral (e.g., fucosyl oligosaccharides).

[0038] An HMO mixture may include two or more individual HMOs, three or more individual HMOs, four or more individual HMOs, or five or more individual HMOs. In some embodiments, an HMO mixture includes five or more individual HMOs. In some embodiments, an HMO mixture includes five individual HMOs.

[0039] HMOs abundant in human milk suitable for use in the present invention 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-difucosylhexaose-I (LNDFH-I), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL), and disialacto-N-tetraose (DSLNT).

[0040] 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, processes have been developed for producing HMO by microbial fermentation, enzymatic process, chemical synthesis, or a combination of these techniques (see Zeuner et al., 2019.Molecules, 24(11), p.2033).

[0041] In some embodiments, the HMO mixture comprises at least one fucosyl oligosaccharide, at least one N-acetyl oligosaccharide, and / or at least one sialyloligosaccharide, hi some embodiments, the HMO mixture comprises at least one fucosyl oligosaccharide, at least one N-acetyl oligosaccharide, and at least one sialyloligosaccharide.

[0042] In some embodiments, the HMO mixture 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).

[0043] In some embodiments, the HMO mixture 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).

[0044] Fucosyl oligosaccharides In some embodiments, the HMO mixture comprises at least one fucosyl oligosaccharide.

[0045] Preferably, the at least one fucosyl oligosaccharide is 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), 3-fucosyllactose (3 FL), 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-I), lacto-N-neodifucosylhexaose (LNnDFH), monofucosyllacto-N-hexaose-III (MFNLH-III), difucosyllacto-N-hexaose-a (DFLNHa), or any combination thereof.

[0046] In some embodiments, at least one fucosyl-oligosaccharide comprises or consists of 2'-fucosyllactose (2'FL) and / or 2',3-difucosyllactose (diFL). In some embodiments, at least one fucosyl-oligosaccharide consists of 2'-fucosyllactose (2'FL) and 2',3-difucosyllactose (diFL).

[0047] At least one fucosyl-oligosaccharide can be obtained by any suitable method. For example, 2'FL can be produced by biotechnological means using specific fucosyltransferases and / or fucosidases, either through enzyme-based fermentation techniques (recombinant or natural enzymes) or through the use of microbial fermentation techniques. In the latter case, microorganisms can express the natural enzymes and substrates or can be engineered to produce the respective substrates and enzymes. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose. diFL can be synthesized by enzymatic, biotechnological, and / or chemical processes.

[0048] N-acetyl oligosaccharides In some embodiments, the HMO mixture comprises at least one N-acetyl oligosaccharide.

[0049] Preferably, the at least one N-acetyl oligosaccharide comprises or consists of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), N-acetyl-glucosamine, N-acetyl-galactosamine, or any combination thereof.

[0050] In some embodiments, at least one N-acetyl oligosaccharide consists of lacto-N-tetraose (LNT).

[0051] N-acetyl oligosaccharides can be obtained by any suitable method. For example, LNnTs can be chemically synthesized by the enzymatic transfer of sugar units from a donor moiety to an acceptor moiety using glycosyltransferases. Alternatively, LNnTs can be prepared by chemically converting a ketohexose (e.g., fructose) that is free or bound to an oligosaccharide (e.g., lactulose) into an N-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharide. LNTs can be synthesized by enzymatic, biotechnological, and / or chemical processes.

[0052] Sialyl oligosaccharides In some embodiments, the HMO mixture comprises at least one sialyloligosaccharide.

[0053] Preferably, the at least one sialyloligosaccharide comprises or consists of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetraose b (LSTb), sialyllactose-N-tetraose c (LSTc), disialacto-N-tetraose (DSLNT), or any combination thereof.

[0054] In some embodiments, at least one sialyloligosaccharide comprises or consists of 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL). In some embodiments, at least one sialyloligosaccharide consists of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0055] 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, by chromatography or filtration techniques. Alternatively, 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL) can be produced by enzyme-based fermentation techniques (recombinant or natural enzymes), chemical synthesis, or biotechnological means using specific sialyltransferases, sialidases, or neuraminidases, either by microbial fermentation techniques. In the latter case, microorganisms can express their native enzymes and substrates or can be engineered to produce the respective substrates and enzymes. Single microbial cultures or mixed cultures can be used. Sialyl oligosaccharide formation can be initiated by acceptor substrates of any degree of polymerization (DP), starting from DP = 1. Alternatively, sialyllactose can be produced by chemical synthesis from lactose and free sialic acid.

[0056] Dosage form The HMO mixture can be administered in any suitable form, for example, the HMO mixture can be administered in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement.

[0057] In some embodiments, the HMO mixture is administered in the form of a nutritional composition. As used herein, "nutritional composition" can refer to a composition that provides nutrition to a subject. Nutritional compositions are usually administered orally or intravenously and usually contain a lipid or fat source and a protein source.

[0058] 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 means, and such a mixture may be chemically identical to a mixture naturally occurring in mammalian milk (i.e., the synthetic composition is not human milk).

[0059] The nutritional composition may be any suitable nutritional composition that can incorporate the HMO mixture, such as a nutritional composition in the form of a food or beverage product, a dietary supplement, a functional food composition, or a pharmaceutical composition.The nutritional composition may be in the form of a solid (e.g., powder), liquid, or semi-liquid.Preferably, the nutritional composition is in a form suitable for feeding to infants, such as infant formula, milk fortifier, or supplement.The nutritional composition may also be in the form of a toddler food, such as yogurt or medical food.

[0060] In a preferred embodiment, the HMO mixture is administered in the form of an infant formula. The infant formula may be a starter infant formula, a preterm infant formula, a milk fortifier, a follow-up 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 in infants during the first month of life, where the formula itself meets the nutritional requirements of people in this category (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006, on infant formulas and follow-up formulas). This formula may also refer to nutritional compositions intended for infants, as defined in Codex Alimentarius (Codex STAN72-1981), and special infant foods (including foods for specific medical purposes).

[0061] In some embodiments, the HMO mixture is administered in the form of a starter infant formula. Generally, a starter infant formula is intended to be a breast milk substitute for infants from birth.

[0062] In some embodiments, the HMO mixture is administered in the form of a follow-up formula. A "follow-up formula" or "follow-on formula" may be given from month 6 onwards. This formula may constitute the main liquid component of an increasingly varied diet for individuals in this category.

[0063] In some embodiments, the HMO mixture 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 given after the first year of life. This milk is generally a dairy-based beverage specifically tailored to meet the specific nutritional needs of children.

[0064] In some embodiments, the HMO mixture is administered in the form of a preterm infant formula. As used herein, the term "preterm infant formula" may refer to an infant formula intended for preterm infants.

[0065] In some embodiments, the HMO mixture is administered in the form of a milk fortifier. As used herein, the term "milk fortifier" may refer to a liquid or solid nutritional composition suitable for mixing with infant formula.

[0066] In some embodiments, the HMO mixture is administered in the form of a baby food formula. As used herein, the term "baby food formula" may refer to a food product intended for specific nutritional use by an infant or child, e.g., a toddler, during the first year of life.

[0067] In some embodiments, the HMO mixture is administered in the form of an infant cereal composition. As used herein, the term "infant cereal composition" may refer to a food product intended for specific nutritional use by an infant or child, e.g., a toddler, during the first year of life.

[0068] In some embodiments, the HMO mixture is administered in the form of at least a starter infant formula, a follow-up formula, and / or a growing-up milk. In some embodiments, the HMO mixture is administered in the form of at least a starter infant formula. In some embodiments, the HMO mixture is administered in the form of at least a starter infant formula and a follow-up formula. In some embodiments, the HMO mixture is administered in the form of at least a starter infant formula, a follow-up formula, and a growing-up milk.

[0069] In some embodiments, the HMO mixture is administered in the form of a starter infant formula. In some embodiments, the HMO mixture is administered in the form of a starter infant formula and a follow-up formula. In some embodiments, the HMO mixture is administered in the form of a starter infant formula, a follow-up formula, and a growing-up milk.

[0070] In other embodiments, the HMO mixture is administered in the form of a fortifier. The fortifier may be a formula fortifier, for example, an infant formula fortifier. Fortifiers may be particularly advantageous embodiments when the infant or toddler is preterm.

[0071] In other embodiments, the HMO mixture 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 (and although typically used as such, a dietary supplement can also be added to any type of composition intended for ingestion by a subject).

[0072] When the composition is a supplement, it can be provided in the form of a unit dose.Supplements are typically in the form of liquid, gel, powder, tablet or capsule.Powdered supplements typically include supplements that are dissolved in water or sprinkled on food or drink.Such supplements are intended to provide additional nutrition and / or health benefits to the subject who takes the supplement.Supplements can be used to provide nutrition and / or health benefits to humans and animals as defined above.Supplements include, for example, powdered supplements that are added to breast milk for premature or low birth weight babies.

[0073] In another embodiment, the HMO mixture is administered in the form of a pharmaceutical product, such as a drop, syrup, powder, tablet, or capsule product intended to treat or prevent an adverse medical condition in a subject in need thereof, hi another embodiment, the HMO mixture is administered in the form of a functional food.

[0074] Other ingredients In addition to the HMO mixture, the nutritional compositions of the present invention, particularly infant formulas, generally contain a protein source, a carbohydrate source, and a lipid source.

[0075] Nutritional compositions according to the present invention, particularly infant formulas according to the present invention, can contain a protein source. The protein can be present in an amount of about 1.5 to about 3.0 g / 100 kcal, about 1.5 to about 2.5 g / 100 kcal, about 1.6 to about 2.5 g / 100 kcal, or about 1.6 to about 2.25 g / 100 kcal. In some embodiments, the protein amount is about 2.0 g / 100 kcal or less, e.g., about 1.8 to about 2.0 g / 100 kcal, or about 1.9 g / 100 kcal.

[0076] For example, protein sources based on whey, casein, and mixtures thereof can be used, as can plant-based protein sources, such as soybeans. With respect to whey protein, the protein source can be based on acid whey or sweet whey, or a mixture thereof, and can contain α-lactalbumin and β-lactoglobulin in any desired proportion. In some embodiments, the protein source is predominantly whey (i.e., more than 50%, e.g., more than 60% or more than 70% of the protein is derived from whey protein).

[0077] The protein may be in its native state or hydrolyzed, or a mixture of native and hydrolyzed proteins.

[0078] The term "intact" in the context of the present invention may mean that the majority of the protein is in its native state, i.e., that its molecular structure is unchanged, e.g., that at least 80% of the protein is unchanged, e.g., that at least 85% of the protein is unchanged, preferably that at least 90% of the protein is unchanged, even more preferably that at least 95% of the protein is unchanged, e.g., that at least 98% of the protein is unchanged. In certain embodiments, the protein is completely unchanged.

[0079] The term "hydrolyzed" in the context of the present invention can mean that a protein has been hydrolyzed or broken down into its constituent amino acids. The protein can be either fully hydrolyzed or partially hydrolyzed. When hydrolyzed protein is required, the hydrolysis process can be carried out as desired as known in the art. For example, whey protein hydrolysates can be prepared by enzymatically hydrolyzing a whey fraction in one or more steps. It has been found that if the whey fraction used as the starting material is substantially lactose-free, the protein undergoes significantly less lysine blackage during the hydrolysis process. This can reduce the degree of lysine blackage from about 15% by weight of the total lysine to less than about 10% by weight of the total lysine, for example, about 7% by weight of lysine, which significantly improves the nutritional value 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, or 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, greater than 20, greater than 40, greater than 60, greater than 80, or greater than 90. At least 70%, 80%, 85%, 90%, 95%, or 97% of the protein may be hydrolyzed. In certain embodiments, 100% of the protein is hydrolyzed.

[0080] Nutritional compositions according to the present invention, particularly infant formulas according to the present invention, may contain a carbohydrate source. While any carbohydrate source typically found in infant formulas can be used, such as lactose, sucrose, saccharose, maltodextrin, starch, and mixtures thereof, lactose is a preferred carbohydrate source for infant formulas. The carbohydrate may be present in an amount of about 8 to about 15 g / 100 kcal or about 9 to about 14 g / 100 kcal. In some embodiments, the carbohydrate is present in an amount of about 10 to about 13 g / 100 kcal, or about 11.1 g / 100 kcal.

[0081] Nutritional compositions according to the present invention, particularly infant formulas according to the present invention, may contain lipids and essential fatty acids. 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) and alpha-linolenic acid (ALA). The compositions of the present invention may further contain gangliosides, monosialoganglioside-3 (GM3) and disialoganglioside-3 (GD3), and combinations thereof. The lipids may be in an amount of about 3.0 to about 8.0 g / 100 kcal, about 4.0 to about 6.0 g / 100 kcal, or about 4.5 to about 5.5 g / 100 kcal. In some embodiments, lipids are present in an amount of about 5.0 to about 5.5 g / 100 kcal, or about 5.3 g / 100 kcal.

[0082] The nutritional composition of the present invention, and in particular the infant formula of the present invention, may also contain all vitamins and minerals that are considered essential in daily diets and in significant amounts for nutrition.Minimum required amounts have been established for certain vitamins and minerals.Examples of minerals, vitamins, and other nutrients that may optionally be 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 salt form.The presence and amount of specific minerals and other vitamins vary depending on the target population. If necessary, the nutritional compositions of the present invention may contain emulsifiers and stabilizers, such as soy, lecithin, citrate esters of mono- and diglycerides, and the like.

[0083] Suitably, the nutritional compositions of the present invention, particularly the infant formulas of the present invention, may have an energy density of about 60 to about 72 kcal per 100 mL or about 67 kcal per 100 mL.

[0084] Preparation of the Composition The compositions according to the invention can be prepared in any known or otherwise suitable manner.

[0085] For example, a nutritional composition, such as an infant formula, can be prepared by blending a protein source with a carbohydrate source and a fat source in an appropriate ratio. If used, an emulsifier can be included at this point. Vitamins and minerals can be added at this point, but can also be added later, usually to avoid thermal decomposition. Water, preferably reverse osmosis water or deionized water, can then be added and mixed to form a liquid mixture. The temperature of the mixture is preferably room temperature, but can also be higher. The liquid mixture can then be heat-treated to reduce bacterial content. The mixture can then be homogenized.

[0086] If it is desired to produce a powdered composition, the homogenized mixture may be dried to a powder in suitable drying equipment, such as a spray dryer or freeze dryer.

[0087] The processes used in the manufacture of infant and child formulas are based on the concept that products must be nutritionally adequate and microbiologically safe for consumption. Therefore, steps that eliminate or limit microbial growth are central to the manufacturing process. Processing techniques generally include preserving an oil-in-water (o / w) emulsion by dehydration in the case of powdered products, or sterilization in the case of ready-to-feed or concentrated liquid products. Powdered infant formulas can be manufactured using a variety of processes, such as dry-blending dehydrated ingredients to form a homogeneous formula, or hydrating and wet-blending a mixture of macroingredients, such as fat, protein, and carbohydrate ingredients, followed by evaporating and spray-drying the resulting mixture. A combination of the two processes may also be used, where a base powder is first produced by wet-blending and spray-drying all or some of the macroingredients, followed by dry-blending the remaining ingredients, including carbohydrates, minerals, vitamins, and other micronutrients, to create the final formula. Liquid formulas are available in ready-to-use forms or as concentrates that must usually be diluted 1:1 with water. The manufacturing processes used for these products are similar to those used to make recombined milk.

[0088] When it is desired to produce a liquid infant formula, the homogenized mixture may be filled, preferably aseptically, into a suitable container. However, the liquid composition may also be retorted within the container, and suitable equipment for this type of filling and retorting is commercially available.

[0089] HMO Dosage The HMO mixture may be administered at any dosage effective to induce the infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory. Effective dosages may vary depending, for example, on the weight and / or age of the infant.

[0090] When the HMO mixture is administered in the form of an infant formula, the infant formula may be administered normally (e.g., based on the weight of the infant or child), and the appropriate amount of each HMO, e.g., 2'FL, diFL, LNT, 3'-SL, 6'-SL, may be based on the amount found in human breast milk for an infant or child of the same age, particularly when produced by a nutritionally replete mother. The amount in the infant formula may vary depending, for example, on the bioavailability of the HMO from the infant formula compared to human breast milk. The exemplary concentrations of HMOs described herein may refer to the concentration after the composition is reconstituted, for example, with water.

[0091] The amounts of 2'FL, diFL, LNT, 3'-SL, and 6'-SL in human breast milk can be within the following ranges: 2'FL in an amount of about 0.5 to about 3 g / L (e.g., about 1.8 g / L); diFL in an amount of about 0.1 to about 0.5 g / L (e.g., about 0.26 g / L); LNT in an amount of about 0.05 to about 0.3 g / L (e.g., about 0.77 g / L); 3'SL in an amount of about 0.1 to about 0.4 g / L (e.g., about 0.22 g / L); and 6'SL in an amount of about 0.05 to about 0.75 g / L (e.g., about 0.47 g / L).

[0092] Preferably, 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. Preferably, the HMO mixture 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. Preferably, the HMO mixture 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. Preferably, the HMO mixture 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 HMO mixture 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 HMO mixture is administered in a total amount of about 1.2 g / day to about 1.8 g / day. In some embodiments, the HMO mixture is administered in a total amount of about 1.46 g / day.

[0093] Preferably, when the HMO mixture is administered in the form of a starter infant formula, the HMO mixture 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). Preferably, when the HMO mixture is administered in the form of a follow-up formula, the HMO mixture 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. Preferably, when the HMO mixture is administered in the form of a growing-up milk, the HMO mixture 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.

[0094] Preferably, the HMO mixture is administered in the following proportions, based on the total weight of the HMO: (i) 2'FL in an amount of about 55% to about 60% by weight (e.g., about 58%), (ii) diFL in an amount of about 5% to about 7% by weight (e.g., about 6%), (iii) LNT in an amount of about 18% to about 20% by weight (e.g., about 19%), (iv) 3'-SL in an amount of about 6% to about 8% by weight (e.g., about 7%), and (v) 6'-SL in an amount of about 9% to about 11% by weight (e.g., about 10%).

[0095] HMO concentration Nutritional compositions (eg, infant formula) that include an HMO mixture may include the HMO mixture at any suitable concentration to provide an effective dose.

[0096] As a guideline, for example, in infant formula, the 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. Preferably, the composition contains the HMO mixture 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. Preferably, the composition contains the HMO mixture 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. Preferably, the composition contains the HMO mixture 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, or about 1.5 g / L to about 2.5 g / L. In some embodiments, the composition comprises an HMO mixture 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 an HMO mixture in a total amount of about 1.2 g / L to about 1.8 g / L. In some embodiments, the composition comprises an HMO mixture in a total amount of about 1.5 g / L.

[0097] Preferably, the starter infant formula contains 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, about 1.2 g / L to 3.0 g / L, or about 1.5 g / L to about 2.5 g / L. Preferably, the follow-up formula contains 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, or about 0.5 g / L. Preferably, the growing up milk contains 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, about 0.3 g / L to about 0.5 g / L, or about 0.4 g / L.

[0098] Preferably, the HMO mixture (e.g., a nutritional composition, e.g., in the form of an infant formula) comprises or consists of, based on the total weight of the HMO, (i) 2'FL in an amount of about 50% to about 65% by weight, (ii) diFL in an amount of about 2% to about 10% by weight, (iii) LNT in an amount of about 15% to about 25% by weight, (iv) 3'-SL in an amount of about 4% to about 10% by weight, and (v) 6'-SL in an amount of about 5% to about 15% by weight.

[0099] In some embodiments, the HMO mixture (e.g., a nutritional composition, e.g., in the form of an infant formula) comprises, or consists of, based on the total weight of the HMO, (i) 2'FL in an amount of about 55% to about 60% by weight (e.g., about 58% by weight), (ii) diFL in an amount of about 5% to about 7% by weight (e.g., about 6% by weight), (iii) LNT in an amount of about 18% to about 20% by weight (e.g., about 19% by weight), (iv) 3'-SL in an amount of about 6% to about 8% by weight (e.g., about 7% by weight), and (v) 6'-SL in an amount of about 9% to about 11% by weight (e.g., about 10% by weight).

[0100] As a guideline, for example, in infant formula, one or more fucosyl 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 fucosyl 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 (e.g., 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.

[0101] As a guideline, for example, in infant formula, one or more N-acetyl oligosaccharides (e.g., LNT) may be present in a total amount of about 0.05 g / L to about 1.0 g / L. Preferably, one or more N-acetyl 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.

[0102] As a guideline, for example, in infant formula, one or more sialyloligosaccharides (e.g., 3'SL and / or 6'SL) may be present in a total amount of about 0.05 g / L to about 1 g / L. Preferably, one or more sialyloligosaccharides are present in an amount of about 0.05 g / L to about 0.5 g / L, or about 0.1 g / L to about 0.5 g / L. In some embodiments, the 3'SL is present 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). In some embodiments, the 3'SL is present in an amount of about 0.11 g / L. In some embodiments, the 6'SL is present 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). In some embodiments, the 6'SL is present in an amount of about 0.14 g / L.

[0103] Preferably, the HMO mixture (e.g., in the form of a nutritional composition, e.g., infant formula) comprises (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), and (iii) 2'FL in an amount of about 0.1 g / L to about 1.0 g / L (e.g., about 0.10 g / L or about 0.14 g / L). It may comprise or consist of (iv) LNT in an amount of about 0.0 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).

[0104] In some embodiments, the HMO mixture (e.g., in the form of a nutritional composition, e.g., a starter 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 0.05 g / L to about 0.11 g / L, preferably about 0.10 g / L; (iii) LNT in an amount of 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.

[0105] In other embodiments, the HMO mixture (e.g., in the form of a nutritional composition, e.g., a starter 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 0.12 g / L to about 0.18 g / L, preferably about 0.14 g / L; (iii) LNT in an amount of 0.39 g to about 0.58 g, 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.

[0106] In other embodiments, the HMO mixture (e.g., in the form of a nutritional composition, e.g., a follow-up 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 0.03 g / L to about 0.05 g / L, preferably about 0.04 g / L; (iii) LNT in an amount of 0.06 g 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.

[0107] In other embodiments, the HMO mixture (e.g., a nutritional composition, e.g., in the form of 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 0.01 g / L to about 0.04 g / L, preferably about 0.03 g / L; (iii) LNT in an amount of 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.

[0108] Administration period The HMO mixture can be administered for any suitable period. For example, when administered in the form of at least a starter infant formula, the HMO can be administered until at least about 6 months of age. For example, when administered in the form of at least a starter infant formula and a follow-up formula, the HMO can be administered until at least about 12 months of age. For example, when administered in the form of at least a starter infant formula, a follow-up formula, and a growing-up milk, the HMO can be administered until at least about 15 months of age.

[0109] The HMO mixture may be administered from at least about 0 to about 6 months of age. Suitably, the HMO mixture is administered beginning at about 7 to 21 days of age. Preferably, the HMO mixture is administered from about 6 to about 18 months of age, from about 6 to about 15 months of age, or from about 6 to about 12 months of age. In some embodiments, the HMO mixture is administered beginning at about 7 to about 21 days of age until about 15 months of age, from about 7 to about 21 days of age until about 12 months of age, from about 7 to about 21 days of age until about 9 months of age, or from about 7 to about 21 days of age until about 6 months of age.

[0110] Target infants The HMO mixture 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 full-term, vaginally delivered, exclusively human milk-fed infants).

[0111] The infant may be a formula-fed infant. As used herein, a "formula-fed" infant may refer to an infant who receives all or part of their nutrition from infant formula (e.g., starter infant formula) during early infancy. In some embodiments, an infant receives all or substantially all of their nutrition from infant formula during early infancy. An infant's feeding mode has a well-recognized effect on the composition and function of the gut microbiome.

[0112] The infant may be full-term or preterm. As used herein, "preterm" may refer to an infant born before about 37 weeks of gestation. As used herein, "full-term" may refer to an infant delivered after about 37 weeks of gestation. In some embodiments, the infant is full-term.

[0113] The infant may be a vaginally delivered infant or a Cesarean section delivered infant. The inventors have shown that the benefits of the present invention may be more pronounced in Cesarean section delivered infants who are formula fed. In some embodiments, the infant is a Cesarean section delivered infant.

[0114] Preferably, the infant is about 0 to about 2 years old, about 0 to about 1 year old, or about 0 to about 0.5 years old. Preferably, the infant is about 0 to about 24 months old, about 0 to about 18 months old, about 0 to about 15 months old, about 0 to about 12 months old, about 0 to about 9 months old, or about 0 to about 6 months old.

[0115] The trajectory of the gut microbiome The present invention provides methods for inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory, which may comprise administering to the infant any mixture of HMOs described herein.

[0116] The "gut microbiota" is the composition of microorganisms (including bacteria, archaea, and fungi) living in the digestive tract. The term "gut microbiome" can encompass both the "gut microbiota" and their "theatre of activity," which can include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolic products (signaling molecules, toxins, organic molecules, and inorganic molecules), and molecules produced by the coexisting host and structured by the surrounding environmental conditions (e.g., Berg, G., et al., 2020. Microbiome, 8(1), pp. 1-22). In the present invention, the term "gut microbiome" can be used interchangeably with the term "gut microbiota."

[0117] A subject's "gut microbiome age" may refer to the subject's predicted age based on the subject's gut microbiome data. For example, a subject's actual / chronological age may be predicted from gut microbiome data obtained from a fecal sample using a machine learning-based artificial intelligence approach. The term "gut microbiome age" encompasses both a subject's "gut microbiome composition age" and "gut microbiome functional age." "Gut microbiome composition age" may refer to gut microbiome age determined using gut microbial composition data, e.g., genus-level or species-level composition data. "Gut microbiome functional age" may refer to gut microbiome age determined using gut function data, e.g., pathway module / submodule or metabolite data (e.g., CAZyme abundance). A subject's "gut microbiome maturity index" or "gut microbiome maturity age" can be obtained from gut composition or function data, as described above, or can be obtained from gut composition and function data (and other similar data).

[0118] The "gut microbiome diversity" of a subject may refer to the diversity of the subject's gut microbiome. The subject's gut microbiome diversity may refer to the number of different taxa (e.g., "richness") present in the subject's gut microbiome. Gut microbiome diversity may also refer to the "balance" of the gut microbiome, i.e., taking into account the abundance or relative abundance of each taxon. Suitably, the diversity of a subject's gut microbiome 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 fecal sample) and may take into account the number of different taxa and their relative abundance.

[0119] "Gut microbiome trajectory" may refer to a fitted curve describing the relationship between "gut microbiome age," "gut microbiome maturation index," "gut microbiome maturation age," or "gut microbiome diversity" and age, and may encompass gut microbiome age trajectories and gut microbiome diversity trajectories. Curves may be fitted by methods such as LOESS or smooth splines using a separate cohort or subset of data (for external validation purposes). Any suitable method can be used to prepare the gut microbiome trajectory (see, e.g., Dogra, S.K., Banjac, J. and Sprenger, N., 2022. bioRxiv 2022.02.14.479826).

[0120] A "reference gut microbiome trajectory" (e.g., age predictor) may be obtained by training a regression model on gut microbiome data from a reference population or by determining relationships from gut microbiome data from a reference population. The reference population may be any suitable reference population. In some embodiments, the reference population is exclusively human breastfed infants. In some embodiments, the reference population is vaginally delivered exclusively human breastfed infants. In some embodiments, the reference population is full-term vaginally delivered exclusively human breastfed infants. In some embodiments, the reference population is predominantly human milk-fed infants. In some embodiments, the reference population is vaginally delivered predominantly human breastfed infants. In some embodiments, the reference population is full-term vaginally delivered predominantly human breastfed infants.

[0121] Gut microbiome age trajectory In some embodiments, the present invention provides methods for guiding an infant's gut microbiome age trajectory to converge to a reference gut microbiome age trajectory.

[0122] A "gut microbiome age trajectory" may refer to a fitted curve that describes the relationship between "gut microbiome age," "gut microbiome maturation index," or "gut microbiome maturation age" and actual age. The reference "gut microbiome age trajectory" may also be referred to as an "age predictor."

[0123] Any suitable method can be used to prepare the reference gut microbiome age trajectory. For example, a method for preparing a reference gut microbiome age trajectory can include: (a) providing gut microbiome data from a reference population; and (b) training a regression model on the gut microbiome data, wherein the age of the reference population at the time of data collection is regressed against one or more features provided from the gut microbiome data; may include:

[0124] Any suitable feature obtained from gut microbiome data can be used to train the regression model. In some embodiments, the one or more feature comprises or consists of one or more microbial abundances, one or more microbial ratios, or one or more carbohydrate-associated enzyme (CAZyme) abundances. The feature may be transformed (e.g., log-transformed) in any manner suitable for training the regression model.

[0125] In some embodiments, the one or more features comprise or consist of one or more microbial abundances. As used herein, "microbial abundance" can refer to the relative abundance of a microbial taxon or the absolute abundance of a microbial taxon.

[0126] 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.

[0127] Microbial taxa can be classified according to any suitable classification (see, for example, Pitt, TL and Barer, MR, 2012. Medical Microbiology, p. 24). Microbial taxa can be classified by the same classification system(s) or by one or more different classification systems. Microbial taxa can be classified taxonomically and / or functionally.

[0128] In some embodiments, microbial taxa are classified taxonomically. Microbial taxonomy refers to the rank-based classification of microorganisms. In the scientific classification established by Carl Linnaeus, each species is assigned to a genus, and genera are sub-levels in a hierarchy of ranks (family, suborder, order, subclass, class, division / phyla, kingdom, and domain). Prokaryotic taxa, as accurately described to date, are outlined, for example, in Bergey's manual of Systematic Bacteriology. Preferably, microbial taxa are taxonomically classified by phylum, class, order, family, genus, and / or species. Preferably, microbial taxa are taxonomically classified by phylum, genus, and / or species. Preferably, microbial taxa are taxonomically classified by genus and / or species. In some embodiments, microbial taxa are taxonomically classified by genus. In some embodiments, microbial taxa are taxonomically classified by species.

[0129] In some embodiments, microbial taxa are functionally classified. For example, microbial taxa can be classified by one or more phenotypic classification systems (e.g., Gram staining, morphology, growth requirements, biochemical reactions, serological systems, environmental reservoirs, etc.). In some embodiments, microbial taxa can be classified according to biological or metabolic pathways, protein domains or families, functional modules, glycoconjugate metabolism, antibiotic resistance, virulence factors, bacterial drug targets and endotoxins, mobile genetic elements, and / or any other functional characteristics (see, e.g., Kultima, JR, et al., 2016. Bioinformatics, 32(16), pp. 2520-2523 and Overbeek, R., et al., 2014. Nucleic acids research, 42(D1), pp. D206-D214).

[0130] Suitable microbial taxa can be determined by any suitable method. For example, the suitability of the microbial taxa can be based on modeling performance statistics, availability or ease of testing, or the target infant. Preferably, the microbial taxa is a 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., when microbial taxa are taxonomically classified by genus) microbial taxa may include the genera Escherichia, Roseburia, Faecalibacterium, Sutterella, SMB53, Collinsella, Ruminococcus, Akkermansia, Veillonella, Pseudomonas, and the like. Parabacteroides, Clostridium, Oscillospira, Megasphaera, Fusobacterium, Bacteroides, Citrobacter, Neisseria, Bifidobacterium, Lachnospira pira, Dialister, Ruminococcus, Blautia, Streptococcus, Eggerthella, Paraprevotella, Corynebacterium, Atopobium, Lactobacillus, Enterococcus cus, Staphylococcus, Sphingobacterium, Tannella, Alistipes, Prevotella, Shigella, Desulfovibrio, Bilophila, and Helicobacter.For example, (e.g., when microbial taxa are taxonomically classified by species) the microbial taxa may be Bifidobacterium longum, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Clostridium spp., Roseburia intestinalis, Ruminococcus faecis, Dialister invisus, Lactobacillus reuteri, Enterococcus faecium, Staphylococcus leei, Bacteroides fragilis, Bacteroides spp. ... The bacterial strains may include one or more bacterial taxa selected from Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides distasonis, Alistipes finegoldii, Prevotella spp., Escherichia coli, Shigella flexneri, Desulfovibrio intestinalis, Helicobacter pylori, Fusobacterium nucleatum, and Akkermansia muciniphilia.

[0131] In some embodiments, the one or more features include or consist of one or more CAZyme abundances. As used herein, "CAZyme abundance" may refer to the abundance of CAZyme genes in gut microbiome data. Abundance may be relative abundance and / or absolute abundance. Suitably, abundance is relative abundance, for example, 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, abundance is calculated relative to all bacterial genes.

[0132] Carbohydrate-associated enzymes (CAZymes) may refer to enzymes involved in the synthesis, metabolism, and transport of carbohydrates. CAZymes may include glycoside hydrolases (GHs), glycosyltransferases (GtSs), polysaccharide lyases (pLSs), carbohydrate esterases (CEs), and carbohydrate-binding modules (CBMs). Suitably, CAZymes are microbial CAZymes. CAZymes may be classified according to any suitable classification system, see, for example, Lombard, V., et al., 2014. Nucleic acids research, 42(D1), pp. D490-D495. CAZymes may be classified by the same classification system(s) or by one or more different classification systems. Suitably, CAZymes are classified by clan, family, and / or subfamily. Suitably, CAZymes are classified by family.

[0133] Appropriately、CAZymeは、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、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、GH113、GH114、GH115、GH116、GH117、GH118、GH119、GH12 0、GH121、GH122、GH123、GH124、GH125、GH126、GH127、GH128、GH129、GH130、GH131、GH132、GH133、GH134、GH135、GH136、GH137、GH138、GH139、GH140、GH1 41、GH142、GH143、GH144、GH145、GH146、GH147、GH148、GH149、GH150、GH151、GH152、GH153、GH154、GH155、GH156、GH157、GH158、GH159、GH160、GH161 162、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, C BM51, CBM52, CBM53, CBM54, CBM55, CBM56, CBM57, CBM58, CBM59, CBM60, CBM61, CBM62, CBM63, CBM64, CBM65, CBM66, CBM 67, CBM68, CBM69, CBM70, CBM71, CBM72, CBM73, CBM74, CBM75, CBM76, CBM77, CBM78, CBM79, CBM80, CBM81, CBM82, CBM83, CBM84, CBM85, CBM86, CBM87, and CBM88 (e.g., 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more).

[0134] Any suitable number of features can be used to train the regression model. Preferably, 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, the regression model can be trained using 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.

[0135] The trajectory of gut microbiome diversity In some embodiments, the present invention provides methods for guiding an infant's gut microbiome diversity trajectory to converge to a reference gut microbiome diversity trajectory. "Gut microbiome diversity trajectory" may refer to a fitted curve obtained to describe the relationship between gut diversity and age.

[0136] Any suitable method can be used to provide the reference gut microbiome diversity trajectory. For example, a method for providing a reference gut microbiome age-diversity trajectory can include: (a) providing gut microbiome data from a reference population; and (b) determining the relationship between the average gut microbiome diversity of the reference population at the time of data collection and the age of the reference population.

[0137] Alpha diversity can be determined using a richness index, a phylogenetic diversity index, or a Shannon index. These indices can be determined using methods routine in the art, such as using the R package Phyloseq (McMurdie and Holmes, 2013, PLoS One, 8, Article e61217). Alpha diversity indices can be calculated based on 16 rRNA sequencing data and / or whole-genome shotgun metagenomics sequencing. Beta 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 the Abundance index (e.g., Bray-Curtis or BD TOTAL ) can be used to determine the beta diversity index. Beta diversity indices can be calculated based on 16rRNA sequencing data and / or whole genome shotgun metagenomics sequencing. These indices can be determined using methods routine in the art, such as using the R package Phyloseq (see, e.g., McMurdie and Holmes, 2013, PLoS One, 8, Article e61217).

[0138] The microbial taxa used to determine gut microbiome diversity may be classified according to any suitable classification described herein. Preferably, 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.

[0139] Gut microbiome data A reference gut microbiome trajectory (e.g., a reference gut microbiome age trajectory or a reference gut microbiome diversity trajectory) can be determined using gut microbiome data from a reference population.

[0140] The gut microbiome data may be any data suitable for determining the trajectory of a reference gut microbiome. For example, the microbiome data may include gut microbial abundance, gut metagenomic data, gut metabolite data, etc. Preferably, the gut microbiome data is gut metagenomic data. The "gut metagenomics data" or "gut metagenomic data" of a subject may refer to the entire genetic content of the subject's gut, 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).

[0141] The gut microbiome data may be obtained or may be obtainable by any suitable sampling method. For example, the gut microbiome data may be obtained or may be obtainable by any method described in Tang, Q., et al., 2020, Frontiers in Cellular and Infection Microbiology, 10, p. 151. The gut microbiome data may be obtained or may be obtainable from a fecal sample, an endoscopic sample (e.g., a biopsy sample, a luminal brush sample, a laser capture microdissection sample), an aspirated intestinal fluid sample, or a surgical sample, or by an in vivo model or an intelligent capsule.

[0142] Preferably, gut microbiome data may be obtained or obtainable from fecal samples. Fecal samples are naturally collected, non-invasive, and can be sampled repeatedly. While flash-frozen fecal material at -80°C maintains microbial consistency without preservatives and has been widely recognized as the gold standard for gut metagenomics, other preservation methods, with or without preservatives, can also be used to obtain metagenomic data similar to that of fresh samples.

[0143] Gut microbiome data can be obtained or can be obtained from a sample by any suitable method. For example, gut microbiome data can be obtained or can be obtained from a sample by sequencing (e.g., next-generation sequencing (NGS)). NGS allows profiling of the genomic DNA of all microorganisms present in a sample. NGS methods can include, for example, shotgun sequencing approaches such as those described in Poussin, C., et al., 2018. Drug discovery today, 23(9), pp.1644-1657.

[0144] 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. Preferably, the reference population may include no more than 500 infants, no more than 100 infants, or no more than 50 infants. Preferably, the reference population may include between 10 and 500 infants.

[0145] The gut microbiome data from the reference population can include any number of samples suitable for training a regression model. Preferably, the gut microbiome data includes 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.

[0146] The gut microbiome data from the reference population may include any number of samples from any number of infants suitable for training a regression model. Preferably, the gut microbiome data from the reference population may include at least 50 samples from at least 10 infants.

[0147] The infant's gut microbiome trajectory may be obtained or obtainable by any suitable sampling method described herein. The infant's gut microbiome data may be obtained or obtainable by the same method as the gut microbiome data from the reference population, or by a different method.

[0148] Regression analysis The trajectory of the reference gut microbiome 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.

[0149] Regression analysis is a set of statistical processes for estimating 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 prepare a trained (or fitted) regression model (i.e., the trajectory of the reference gut microbiome).

[0150] The regression analysis can be performed using any suitable regression model. 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.

[0151] Suitably, the regression analysis is performed using a machine learning technique. Examples of machine learning techniques include tree-based regression models (e.g., random forest regression models), recursive partitioning, regularization and shrinkage, 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.

[0152] The regression analysis can be performed by training a regression model on the gut microbiome data. For example, the 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 from the gut microbiome data.

[0153] As used herein, "training" with respect to "fitting" a regression model may mean determining the function that most closely fits the data according to appropriate statistical criteria. For example, ordinary least squares may be used to calculate the function that minimizes the sum of squares of the differences between the true data and the function.

[0154] Converging trajectories of the gut microbiome The inventors have shown in the present invention that a human milk oligosaccharide (HMO) mixture can induce an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory and to remain converged or converged to the reference gut microbiome trajectory.

[0155] As used herein, "convergent" gut microbiome trajectories tend to converge toward one another. Preferably, gut microbiome trajectories may be considered convergent when they are no longer statistically significantly different. For example, an infant's gut microbiome trajectory may be considered convergent to a reference gut microbiome trajectory if the infant is not an outlier in the reference gut microbiome trajectory and / or if the infant is on the reference gut microbiome trajectory. In some embodiments, the HMO mixture induces the infant's gut microbiome trajectory to remain converged or converged to the reference gut microbiome trajectory.

[0156] As used herein, an HMO mixture "inducing" an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory can mean that the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory more quickly than an infant not receiving the HMO mixture (i.e., in the absence of the HMO mixture). The term "inducing" can be used interchangeably with the term "promoting." The phrase "inducing an infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory" can be used interchangeably with the phrase "accelerating the convergence of an infant's gut microbiome trajectory to a reference gut microbiome trajectory."

[0157] The infant's gut microbiome trajectory may converge to the reference gut microbiome trajectory at least about 1 month, at least about 2 months, or at least about 3 months earlier than infants not receiving the HMO mixture (i.e., in the absence of the HMO mixture).The infant's gut microbiome trajectory may converge to the reference gut microbiome trajectory at least about 2 months earlier than infants not receiving the HMO mixture (i.e., in the absence of the HMO mixture).

[0158] Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory within about 12 months of age, within about 11 months of age, within about 10 months of age, within about 9 months of age, within about 8 months of age, or within about 7 months of age.

[0159] Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory at about 6 months of age or older, at about 7 months of age or older, at about 8 months of age or older, at about 9 months of age or older, at about 10 months of age or older, at about 11 months of age or older, or at about 12 months of age or older.

[0160] Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory at about 6 to about 12 months of age, about 6 to about 11 months of age, about 6 to about 10 months of age, or about 6 to about 9 months of age. Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory at about 7 to about 12 months of age, about 7 to about 11 months of age, about 7 to about 10 months of age, or about 7 to about 9 months of age. Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory at about 8 to about 12 months of age, about 8 to about 11 months of age, about 8 to about 10 months of age, or about 8 to about 9 months of age. Preferably, the infant's gut microbiome trajectory converges to the reference gut microbiome trajectory at about 8 to about 10 months of age, or at about 9 months of age.

[0161] Outliers in the baseline gut microbiome trajectory In some embodiments, the human milk oligosaccharide (HMO) mixture induces the infant to be an outlier in the trajectory of the reference gut microbiome.

[0162] Any suitable statistical method may be used to determine whether an 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, an infant may be determined to be an outlier based on the standard error, confidence interval, prediction interval, and / or standard deviation in the reference gut microbiome trajectory. Preferably, an infant may be determined to be an outlier if the infant's gut microbiome data significantly differs from the line of the reference gut microbiome trajectory (e.g., age predictor) based on the standard error, confidence interval, prediction interval, and / or standard deviation of the reference gut microbiome trajectory.

[0163] Suitable cutoffs are well known to those skilled in the art, for example, 3 standard deviations from the mean is a common cutoff in practice for identifying outliers in Gaussian or Gaussian-like distributions.

[0164] In some embodiments, an 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 that an observed value deviates from the reference gut microbiome trajectory. Preferably, an infant is an outlier if their gut microbiome data is less than or equal to -2SE or more than 2SE, less than or equal to -2.5SE or more than 2.5SE, less than or equal to -3SE or more than 3SE, less than or equal to -3.5SE or more than 3.5SE, or less than or equal to -4SE and more than 4SE from the reference gut microbiome trajectory. Preferably, an infant is an outlier if their gut microbiome data is less than or equal to -3SE or more than 3SE from the reference gut microbiome trajectory.

[0165] In some embodiments, an infant is determined to be an outlier based on the confidence interval of the reference gut microbiome trajectory. The confidence interval may be determined by any suitable method, for example, using a resampling technique (e.g., bootstrap resampling). Preferably, an infant is an outlier if the infant's gut microbiome data falls outside the 90%, 95%, 98%, or 99% confidence interval of the reference gut microbiome trajectory. Preferably, an infant is an outlier if the infant's gut microbiome data falls outside the 95% confidence interval of the reference gut microbiome trajectory.

[0166] In some embodiments, an infant is determined to be an outlier based on the prediction interval of the reference gut microbiome trajectory. Preferably, an infant is an outlier if the infant's gut microbiome data falls outside the 90%, 95%, 98%, or 99% prediction interval of the reference gut microbiome trajectory. Preferably, an infant is an outlier if the infant's gut microbiome data falls outside the 95% prediction interval of the reference gut microbiome trajectory.

[0167] In some embodiments, an infant is determined to be an outlier based on the standard deviation of the 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. Preferably, an infant is an outlier if they have a Z-score of -2 or less or 2 or more, -2.5 or less or 2.5 or more, -3 or less or 3 or more, -3.5 or less or 3.5 or more, or -4 or less or 4 or more on the reference gut microbiome trajectory. Preferably, an infant is an outlier if they have a Z-score of -3 or less or 3 or more on the reference gut microbiome trajectory.

[0168] In some embodiments, an infant is determined to be an outlier if the infant's gut microbiome data is less than or equal to -3SE or more than 3SE from the reference gut microbiome trajectory, if the infant's gut microbiome data is outside the 95% confidence interval for the reference gut microbiome trajectory, if the infant's gut microbiome data is outside the 95% prediction interval for the reference gut microbiome trajectory, and / or if the infant has a Z-score less than or equal to -3 or more than 3 for the reference gut microbiome trajectory.

[0169] In some embodiments, an infant is determined to be an outlier if their gut microbiome data has a Z-score of −3 or less or 3 or more on the reference gut microbiome trajectory.

[0170] Whether or not it is on the trajectory of the reference gut microbiome In some embodiments, the human milk oligosaccharide (HMO) mixture induces the infant to be on a reference gut microbiome trajectory.

[0171] Preferably, an infant is on a reference gut microbiome trajectory if the infant's gut microbiome data is not significantly different from the reference gut microbiome trajectory, and / or an infant is off a reference gut microbiome trajectory if the infant's gut microbiome data is significantly different from the reference gut microbiome trajectory.

[0172] Any suitable method may be used to determine whether an infant is on a reference gut microbiome change trajectory. For example, an infant may be determined to be on a reference gut microbiome trajectory based on the standard error, confidence interval, prediction interval, and / or standard deviation of the reference gut microbiome trajectory.

[0173] In some embodiments, an infant is determined to be off the reference gut microbiome trajectory based on the standard error (SE) of the reference gut microbiome trajectory. Preferably, an infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is less than or equal to -2SE or more than 2SE, less than or equal to -2.5SE or more than 2.5SE, less than or equal to -3SE or more than 3SE, less than or equal to 3.5SE or more than 3.5SE, or less than or equal to -4SE and more than 4SE from the reference gut microbiome trajectory. Preferably, an infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is less than or equal to -3SE or more than 3SE from the reference gut microbiome trajectory.

[0174] In some embodiments, the infant is determined to be off the reference gut microbiome trajectory based on the confidence interval of the reference gut microbiome trajectory. Preferably, the infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is outside the 90% confidence interval, 95% confidence interval, 98% confidence interval, or 99% confidence interval of the reference gut microbiome trajectory. Preferably, the infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is outside the 95% confidence interval of the reference gut microbiome trajectory.

[0175] In some embodiments, the infant is determined to be off the reference gut microbiome trajectory based on the prediction interval of the reference gut microbiome trajectory. Preferably, the infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is outside the 90% prediction interval, 95% prediction interval, 98% prediction interval, or 99% prediction interval of the reference gut microbiome trajectory. Preferably, the infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is outside the 95% prediction interval of the reference gut microbiome trajectory.

[0176] In some embodiments, an infant is determined to be off the 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 is off the reference gut microbiome trajectory. Preferably, an infant is an outlier if they have 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. Preferably, an infant is off the reference gut microbiome trajectory if they have a Z-score of -3 or less or 3 or more.

[0177] In some embodiments, an infant is determined to be off the reference gut microbiome trajectory if the infant's gut microbiome data is less than or equal to -3SE or more than 3SE from the reference gut microbiome trajectory, if the infant's gut microbiome data is outside the 95% confidence interval of the reference gut microbiome trajectory, if the infant's gut microbiome data is outside the 95% prediction interval of the reference gut microbiome trajectory, and / or if the infant has a Z-score less than or equal to -3 or greater than or equal to 3.

[0178] In some embodiments, an infant is determined to be off the reference gut microbiome trajectory if they have a Z-score below -3 or above 3.

[0179] intestinal maturation The methods of the present invention may promote gut maturation by inducing the trajectory of an infant's gut microbiome to converge to a reference gut microbiome trajectory.

[0180] Intestinal maturation during normal development involves a series of structural and functional changes, culminating during the weaning period when complex foods are introduced. As used herein, "promoting" intestinal maturation may mean that an infant's intestine matures in a more age-appropriate manner. 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.

[0181] HMO mixtures may promote the maturation of the gut microbiome. Early gut microbiome maturation can be characterized by the sequential acquisition, colonization, and selection of specific microorganisms, resulting in distinct functional characteristics over time. This orchestrated microbial sequence occurs during the first few years of life, after which the microbiome reaches an adult-like composition and function between the ages of 3 and 5. These various steps in microbiome development are increasingly recognized as critical time points for long-term health, primarily related to appropriate immune and metabolic development (see, e.g., Dogra, SK, et al., 2021, Microorganisms, 9(10), p. 2110). For example, gut microbiome maturation during the first year of life may be involved in the farm effect, which protects against childhood asthma (see, e.g., Depner, M., et al., 2020, Nature Medicine, 26(11), pp. 1766-1775).

[0182] HMO mixtures can promote the maturation of intestinal metabolism. The compositional changes of the microbiome also reflect the functional capabilities of microorganisms to some extent, as indicated by significant changes in the abundance of microbial carbohydrate-associated enzymes (CAZymes) and other metabolic pathways (see, for example, Stewart, CJ, et al., 2018. Nature, 562(7728), pp. 583-588).

[0183] HMO mixtures may promote the maturation of intestinal barrier function. The intestinal barrier, composed of mucus and underlying epithelial cells, is considered primarily a physical barrier, and together with numerous immune defense components, contributes to regulating the relationship between the microbiome and the host (see, for example, Dogra, SK, et al., 2021. Microorganisms, 9(10), p. 2110).

[0184] HMO mixtures may promote the maturation of intestinal immune function. Gut immune components, such as secretory immunoglobulin (Ig) A and defensins, along with epithelial and mucosal glycosylation patterns, change during intestinal development and are thought to play an important role in setting the stage for the development of host-microbiome mutualism (see, e.g., Dogra, SK, et al., 2021. Microorganisms, 9(10), p. 2110).

[0185] HMO mixtures may modulate the abundance of one or more microorganisms and / or microbial metabolic pathways associated with age-dependent intestinal maturation and associated health benefits. For example, HMO-stimulated Bifidobacterium species may contribute to the prevention of subsequent respiratory tract 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 Dialister are associated with a reduced risk of atopy (see, e.g., Galazzo, G., et al., 2020. Gastroenterology, 158(6), pp. 1584-1596).

[0186] In one aspect, the present invention provides HMO mixtures to promote gut maturation in infants by inducing the infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory.

[0187] In one aspect, the invention provides a method of promoting intestinal maturation in an infant in need thereof, comprising administering a therapeutically effective amount of an HMO mixture, thereby inducing the infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory.

[0188] Methods for determining intestinal maturity status in infants The present invention also provides a method for determining the gut maturation status of an infant, preferably comprising: (a) providing a reference gut microbiome trajectory; and (b) providing gut microbiome data from the infant and determining whether the infant is an outlier in the reference gut microbiome trajectory or whether the infant is on the reference gut microbiome trajectory.

[0189] The infant may be any suitable infant, for example, any infant described in the section above entitled "Target Infants." Preferably, the infant is a formula-fed infant. In some embodiments, the infant is a full-term infant. In some embodiments, the infant is a Cesarean section-born infant. The infant may be administered an HMO mixture, for example, as described in the section above entitled "Human Milk Oligosaccharide (HMO) Mixture."

[0190] The reference gut microbiome trajectory can be any suitable reference gut microbiome trajectory, for example, any of the reference gut microbiome trajectories described in the section above entitled "Gunt Microbiome Trajectories." In some embodiments, the reference gut microbiome trajectory is obtained from a full-term, vaginally delivered, exclusively human breastfed infant.

[0191] Any suitable method may be used to determine whether an infant is an outlier in a reference gut microbiome trajectory or whether a formula-fed infant is on a reference gut microbiome trajectory, for example, as described above in the sections entitled "Outliers in a Reference Gut Microbiome Trajectory" and "Whether on a Reference Gut Microbiome Trajectory."

[0192] Suitably, an infant's gut maturation status is normal if the infant is not an outlier in the reference gut microbiome trajectory, and / or the infant's gut maturation status is not normal if the infant is an outlier in the reference gut microbiome trajectory. In this context, a "normal" gut maturation status may mean that the infant has a gut metagenome that is not significantly different from the gut metagenome of a reference population.

[0193] Computer program and computer-readable medium The methods for determining the intestinal maturation status of an infant described herein may be computer-implemented methods.

[0194] In one aspect, the present invention provides a data processing system comprising means for carrying out the method for determining the intestinal maturation status of an infant described herein.

[0195] In one aspect, the present invention provides a data processing apparatus comprising a processor configured to carry out the method for determining the intestinal maturity status of an infant described herein.

[0196] In one aspect, the present invention provides a computer program comprising instructions that, when executed by a computer, cause the computer to carry out a method for determining the intestinal maturity status of an infant as described herein.

[0197] In one aspect, the invention provides a computer readable medium comprising instructions that, when executed by a computer, cause the computer to perform a method for determining the intestinal maturity status of an infant as described herein.

[0198] In one aspect, the invention provides a computer readable data carrier having stored thereon the computer program of the invention.

[0199] In one aspect, the present invention provides a data carrier signal carrying the computer program of the present invention.

[0200] In one aspect, the invention provides a computer-implemented method for determining an infant's gut maturation status, the method comprising: (a) providing a reference gut microbiome trajectory and an associated microbiome age predictor model; (b) providing gut microbiome data from the infant; and (c) determining whether the infant is an outlier in the reference gut microbiome trajectory, wherein if the infant is not an outlier in the reference gut microbiome trajectory, the infant's gut maturation status is normal, and / or if the infant is an outlier in the reference gut microbiome trajectory, the infant's gut maturation status is not normal.

[0201] In one aspect, the invention provides a computer-implemented method for determining an infant's gut maturation status, the method comprising: (a) providing a reference gut microbiome trajectory and an associated microbiome age predictor model; (b) providing gut microbiome data from the infant; and (c) determining whether the infant is on the reference gut microbiome trajectory, wherein the infant is on the reference gut microbiome trajectory if the infant's gut microbiome data is not significantly different from the reference gut microbiome trajectory, and / or the infant is off the reference gut microbiome trajectory if the infant's gut microbiome data is significantly different from the reference gut microbiome trajectory.

[0202] In one aspect, the invention provides a data processing system comprising a means for determining an infant's gut maturation status given a reference gut microbiome trajectory, an associated microbiome age predictor model, and the infant's gut microbiome data, as described herein.

[0203] In one aspect, the present invention provides a data processing apparatus comprising a processor configured to determine the gut maturation status of an infant given a reference gut microbiome trajectory, an associated microbiome age predictor model, and the infant's gut microbiome data, as described herein.

[0204] In one aspect, the invention provides a computer program comprising instructions that, when executed by a computer, cause the computer to determine the gut maturation status of an infant given a reference gut microbiome trajectory, its associated microbiome age predictor model, and the infant's gut microbiome data, as described herein.

[0205] In one aspect, the invention provides a computer-readable medium comprising instructions that, when executed by a computer, cause a computer to determine an infant's gut maturation status given a reference gut microbiome trajectory, its associated microbiome age predictor model, and the infant's gut microbiome data, as described herein.

[0206] The systems described herein may display to the user a dashboard or other suitable 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 status, and the infant's personalized advice and recommendations, such as HMO supplementation to maintain or improve the infant's gut maturation status.

[0207] Uses of gut microbiome trajectories In another aspect, the present invention provides the use of one or more reference gut microbiome trajectories to determine the intestinal maturation status of an infant following administration of an HMO mixture.

[0208] The infant may be any suitable infant, for example, any infant described in the section above entitled "Target Infants." Preferably, the infant is a formula-fed infant. In some embodiments, the infant is a full-term infant. In some embodiments, the infant is a Cesarean section-born infant. For example, any suitable mixture of HMOs may be administered to the infant, as described in the section above entitled "Human Milk Oligosaccharide (HMO) Mixtures."

[0209] The one or more reference gut microbiome trajectories can include or consist of any suitable reference gut microbiome trajectory, for example, any of the reference gut microbiome trajectories described in the section above entitled "Gunt Microbiome Trajectories." In some embodiments, the one or more reference gut microbiome trajectories are obtained from full-term, vaginally delivered, exclusively breastfed infants. In some embodiments, the one or more reference gut microbiome trajectories are obtained from full-term, vaginally delivered, mixed-feeding infants primarily on human breast milk.

[0210] The use may include any suitable method steps for determining the intestinal maturation status of an infant, for example, any of the method steps described in the section above entitled "Methods for determining the intestinal maturation status of an infant." [Example]

[0211] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention, and are not intended to limit the scope of the invention in any way.

[0212] Example 1 Consumption of a product containing an HMO mixture helps infants converge and remain converged to a reference gut microbiome trajectory.

[0213] Materials and Methods A randomized controlled trial (ClinicalTrials.gov Identifier: NCT03722550) was conducted to evaluate the effects of human milk oligosaccharides (HMOs) in formula feeding. An overview of the trial is shown in Figure 1.

[0214] Healthy term infants (7–21 days old) were randomly assigned to receive a standard cow's milk-based starter infant formula (control group, CG, n = 154); the same formula containing 1.5 g / L HMOs (test group 1, TG1, n = 155); or the same formula containing 2.5 g / L HMOs (test group 2, TG2, n = 153); or a human breast milk-fed group (reference, HMG, n = 61).

[0215] The standard starter infant was a bovine milk-based whey predominant term infant formula consisting of 1.9 g undenatured protein (70% whey / 30% casein) / 100 kcal, 11.1 g carbohydrate / 100 kcal, and 5.3 g lipid / 100 kcal, for a total of 67 kcal per 100 mL of reconstituted formula. The individual HMO concentrations in the TG1 and TG2 starter infant formulas are shown in Table 1 below.

[0216] [Table 1]

[0217] The standard follow-up formula was a milk-based, whey-based, term formula consisting of 2 g undenatured protein (50% whey / 50% casein) / 100 kcal, 12.4 g carbohydrate / 100 kcal, and 4.7 g lipid / 100 kcal, for a total of 67 kcal per 100 mL of reconstituted formula. The total HMO concentration in the TG1 and TG2 follow-up formulas was 0.5 g per L, the same blend as in the starter infant formulas.

[0218] The standard growing-up milk was a cow's milk-based growing-up milk consisting of 2.25 g undenatured protein (40% whey / 60% casein) / 100 kcal, 12.6 g carbohydrate / 100 kcal, and 4.5 g lipid / 100 kcal, for a total of 67 kcal per 100 mL of reconstituted formula. The concentration of total HMOs in the TG1 and TG2 growing-up milks was 0.4 g per L, the same blend as the starter infant formula.

[0219] Fecal samples collected at enrollment and at 3, 6, 12, and 15 months of age were used for microbiome profiling. Microbial DNA was extracted from frozen feces, purified, and shotgun sequenced using a 2x150bp sequencing method. Taxonomic relative abundance was calculated using a metagenomic species (MGS) approach. This calculation allows for quantification of both known and uncharacterized microbial species.

[0220] Microbiome age predictors were trained on data from vaginally delivered HMG infants (reference set: HMG-VD, n = 31) using genus-level data, metagenomics species-level (MGS) data, or CAZyme composition data and optimized using RSME (as shown in Figure 2). These models were applied to CG, TG1, and TG2 to predict microbiome age and identify outliers (microbiome-to-age Z-score: MAZ > 3). Microbiome age trajectories for CG, TG1, and TG2 were compared against the HMG-VD reference trajectory.

[0221] result The 10 features selected for the genus-based model were Romboutsia, Blautia, Staphylococcus, Intestinibacter, Cutibacterium, Megasphaera, Enterococcus, Bifidobacterium, Flavonifractor, and Roseburia.

[0222] The 20 features selected in the species-based model were Romboutsia timonensis, Intestinibacter bartlettii, Staphylococcus hominis subsp. hominis, Staphylococcus epidermidis, Veillonella parvula, [Clostridium] spiroforme, Enterococcus faecalis, [Ruminococcus] gnavus, Flavonifractor plautii, and Parabacteroides distasonis. distasonis, Megasphaera micronuciformis, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Collinsella aerofaciens, Ruminococcus bacterium, Haemophilus parainfluenzae, Veillonella sp., Clostridiaceae bacterium, Fusicatenibacter saccharivorans, and Clostridium perfringens.

[0223] The 25 features selected in the separate species-based model were Rombautzia zimonensis, Intestinibacter bartholettii, Staphylococcus hominis subsp. hominis, Staphylococcus epidermidis, Veillonella parvula, [Clostridium] spiroforme, Flavonifracter proutii, Enterococcus faecalis, [Ruminococcus] gnavus, Parabacteroides distasonis, Megasphaera micronuciformis, Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bacteroides dorei. dorei, Collinsella aerofaciens, Ruminococcus aeruginosa, Veillonella spp., Clostridiaceae aeruginosa, Clostridium perfringens, Erysipelatoclostridium ramosum, Fusicatenibacter saccharivorans, Haemophilus parainfluenzae, and Peptostreptococcaceae sp.

[0224] The 30 features selected in the CAZymes-based model were 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, and GH76.

[0225] In each model, the trajectory of TG converged to the reference trajectory faster than that of CG. For example, the genus-based model (with 10 features, R 2Using the MGS species-based model (with 20 features, R 2 Using a variance (σ = 0.881), trajectories differed significantly up to approximately 10.3 months for CG, 8.1 months for TG1, and 5.6 months for TG2 (see Figure 3B). Similar results were seen using various MGS species-based models (see Figure 3C), CAZymes-based approaches (see Figure 3D), and α-diversity-based approaches (see Figure 3E). This effect was more pronounced in formula-fed cesarean section (CS)-born infants, as seen by the various trajectories (see, e.g., Figures 4A and 4B).

[0226] After starting the intervention, outliers in the genus-based model were significantly reduced in the TG compared to the CG using the Cochran-Armitage trend test (p=0.0002) and visits (3-6 months p=0.0002; 12-15 months p=0.0377) (Figure 5 and Table 2 below). Models trained on other data types showed similar trends.

[0227] [Table 2]

[0228] These data indicate that the addition of an HMO mixture to infant formula can induce the gut microbiome trajectory of formula-fed infants to converge with that of vaginally delivered reference infants receiving human breast milk.

[0229] Embodiment Various preferred features and embodiments of the present invention are described with reference to the following numbered paragraphs.

[0230] 1. A human milk oligosaccharide (HMO) mixture for use in inducing the gut microbiome trajectory of formula-fed infants to converge with the reference gut microbiome trajectory obtained from human breast-fed infants.

[0231] 2. An HMO mixture for use according to paragraph 1, wherein said HMO mixture comprises at least one fucosyl oligosaccharide, at least one N-acetyl oligosaccharide, and at least one sialyloligosaccharide.

[0232] 3. The HMO mixture for use according to paragraph 1 or 2, wherein the HMO mixture 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), and optionally the HMO mixture comprises or consists of, based on the total weight of the HMO, (i) 2'FL in an amount of about 55% to about 60% by weight, (ii) diFL in an amount of about 5% to about 7% by weight, (iii) LNT in an amount of about 18% to about 20% by weight, (iv) 3'-SL in an amount of about 6% to about 8% by weight, and (v) 6'-SL in an amount of about 9% to about 11% by weight.

[0233] 4. An HMO mixture for use according to any one of paragraphs 1 to 3, wherein the HMO mixture is administered in the form of an infant formula, optionally wherein the HMO mixture is administered in the form of a starter infant formula, a follow-up formula, and / or a growing-up milk.

[0234] 5. The HMO mixture for use according to any one of paragraphs 1 to 4, wherein the HMO mixture is administered in the form of a starter infant formula comprising 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, or about 1.5 g / L to about 2.5 g / L; a follow-up formula comprising HMOs in a total amount 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, or about 0.35 g / L to about 0.65 g / L; and / or a growing-up milk comprising HMOs in a total amount 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.

[0235] 6. The HMO mixture is (a) (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 of about 0.1 g / L to about 1.0 g / L, preferably 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 0.05 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; (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; Starter infant formula, including (b) (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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.06 g / L; Follow-up formula containing, and / or (c) (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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L; 6. The HMO mixture for use according to any one of paragraphs 1 to 5, wherein the HMO mixture is administered in the form of a growing-up milk comprising:

[0236] 7. The HMO mixture is administered in the form of a starter infant formula containing about 1.5 g / L total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.14 g / L; 7. The HMO mixture for use according to any one of paragraphs 1 to 6, administered in the form of a starter infant formula comprising:

[0237] 8. The HMO mixture is administered in the form of a starter infant formula containing about 2.5 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.24 g / L; 7. The HMO mixture for use according to any one of paragraphs 1 to 6, administered in the form of a starter infant formula comprising:

[0238] 9. The HMO mixture is administered in the form of a follow-up formula containing about 0.5 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.05 g / L; 9. The HMO mixture for use according to any one of paragraphs 1 to 8, administered in the form of a follow-up formula comprising:

[0239] 10. The HMO mixture is administered in the form of a growing-up milk containing about 0.4 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.04 g / L; 10. The HMO mixture for use according to any one of paragraphs 1 to 9, administered in the form of a growing-up milk comprising:

[0240] 11. The HMO mixture for use according to any one of paragraphs 1 to 10, wherein the mixture of HMOs is administered in the form of a starter infant formula, a follow-up formula, and / or a growing-up milk, each of the formulas being from about 60 kcal / 100 mL to about 80 kcal / 100 mL and comprising protein in an amount of from about 1.5 g / 100 kcal to about 2.5 g / 100 kcal, carbohydrates in an amount of from about 8 g / 100 kcal to about 15 g / 100 kcal, and lipids in an amount of from about 3 g / 100 kcal to about 8 g / 100 kcal.

[0241] 12. The HMO mixture for use according to any one of paragraphs 1 to 11, wherein the HMO mixture 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.

[0242] 13. The HMO mixture for use according to any one of paragraphs 1 to 12, wherein the gut microbiome trajectory of the formula-fed infant converges to a reference gut microbiome trajectory obtained from a human breast-fed infant at about 6 months of age or older, at about 7 months of age or older, at about 8 months of age or older, or at about 9 months of age or older.

[0243] 14. The HMO mixture for use according to any one of paragraphs 1 to 13, wherein the gut microbiome trajectory of the formula-fed infant converges to a reference gut microbiome trajectory obtained from a human breast-fed infant within about 12 months of age, within about 11 months of age, within about 10 months of age, or within about 9 months of age.

[0244] 15. The HMO mixture for use according to any one of paragraphs 1 to 14, wherein the gut microbiome trajectory of the formula-fed infant converges to a reference gut microbiome trajectory obtained from a human breast-fed infant at about 6 to about 12 months of age, at about 7 to about 11 months of age, at about 8 to 10 months of age, or at about 9 months of age.

[0245] 16. The HMO mixture for use according to any one of paragraphs 1 to 15, wherein the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory.

[0246] 17. The HMO mixture for use according to any one of paragraphs 1 to 16, wherein the gut microbiome trajectory is a gut microbiome age trajectory, and optionally the gut microbiome age trajectory is obtained using genus-level data, species-level data, and / or functional data from gut microbiome data.

[0247] 18. An HMO mixture for use according to any one of paragraphs 1 to 17, wherein the formula-fed infant is a full-term infant.

[0248] 19. An HMO mixture for use according to any one of paragraphs 1 to 18, wherein the formula-fed infant is a Caesarean section-born infant.

[0249] 20. An HMO mixture for use according to any one of paragraphs 1 to 19, wherein the human breastfed infant is a full-term vaginally delivered human breastfed infant.

[0250] 21. The HMO mixture for use according to any one of paragraphs 1 to 20, wherein inducing the formula-fed infant's gut microbiome trajectory to converge to a reference gut microbiome trajectory obtained from a human breast-fed infant promotes intestinal maturation.

[0251] 22. The HMO mixture for use according to any one of paragraphs 1 to 21, wherein inducing the formula-fed infant's gut microbiome trajectory to converge with a reference gut microbiome trajectory obtained from a human breast-fed infant promotes maturation of the gut microbiome, maturation of gut metabolism, maturation of gut barrier function, and / or maturation of gut immune function.

[0252] 23. A method for inducing the trajectory of the gut microbiome of a formula-fed infant to converge to a reference gut microbiome trajectory obtained from a human breast-fed infant, the method comprising administering to the formula-fed infant an effective amount of a human milk oligosaccharide (HMO) mixture.

[0253] 24. Use of a human milk oligosaccharide (HMO) mixture to induce the gut microbiome trajectory of formula-fed infants to converge with the reference gut microbiome trajectory obtained from human breast-fed infants.

[0254] 25. The method according to paragraph 23 or the use according to paragraph 24, wherein the HMO mixture comprises at least one fucosyloligosaccharide, at least one N-acetyloligosaccharide, and at least one sialyloligosaccharide.

[0255] 26. The method of paragraph 23 or 25 or the use of paragraph 24 or 25, wherein the HMO mixture 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), and optionally the HMO mixture comprises or consists of, based on the total weight of HMO, (i) 2'FL in an amount of about 55% to about 60% by weight, (ii) diFL in an amount of about 5% to about 7% by weight, (iii) LNT in an amount of about 18% to about 20% by weight, (iv) 3'-SL in an amount of about 6% to about 8% by weight, and (v) 6'-SL in an amount of about 9% to about 11% by weight.

[0256] 27. The method of any one of paragraphs 23, 25 or 26 or the use of any one of paragraphs 24 to 26, wherein the HMO mixture is administered in the form of an infant formula, optionally wherein the HMO mixture is administered in the form of a starter infant formula, a follow-up formula, and / or a growing-up milk.

[0257] 28. A starter infant formula, wherein the HMO mixture contains 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 formula containing 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, or about 0.35 g / L to about 0.65 g / L. 28. The method of any one of paragraphs 23 or 25 to 27, or the use of any one of paragraphs 24 to 27, administered in the form of a growing-up formula; and / or a growing-up milk containing HMOs in a total amount of from about 0.1 g / L to about 1.0 g / L, from about 0.2 g / L to about 0.8 g / L, from about 0.28 g / L to about 0.52 g / L, or from about 0.3 g / L to about 0.5 g / L.

[0258] 29. The HMO mixture is (a) (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 of about 0.1 g / L to about 1.0 g / L, preferably 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 0.05 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; (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; Starter infant formula, including (b) (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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.06 g / L; Follow-up formula containing, and / or (c) (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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L; 29. The method of any one of paragraphs 23 or 25 to 28, or the use of any one of paragraphs 24 to 28, wherein the method is administered in the form of a grow up milk comprising

[0259] 30. The HMO mixture is administered in the form of a starter infant formula containing about 1.5 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.14 g / L; 30. The method of any one of paragraphs 23 or 25 to 29 or the use of any one of paragraphs 24 to 29, administered in the form of a starter infant formula comprising

[0260] 31. The HMO mixture is administered in the form of a starter infant formula containing about 2.5 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.24 g / L; 31. The method of any one of paragraphs 23 or 25 to 30 or the use of any one of paragraphs 24 to 30, administered in the form of a starter infant formula comprising

[0261] 32. The HMO mixture is administered in the form of a follow-up formula containing about 0.5 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.05 g / L; 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 method is administered in the form of a follow-up formula comprising

[0262] 33. The HMO mixture is administered in the form of a growing-up milk containing about 0.4 g / L of total HMOs, and optionally, the HMO mixture is (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; (v) 6'-SL in an amount of about 0.04 g / L; 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 method is administered in the form of a growing up milk comprising

[0263] 34. The method of any one of paragraphs 23 or 25 to 33, or the use of any one of paragraphs 24 to 33, wherein the HMO mixture is administered in the form of a starter infant formula, a follow-up formula, and / or a growing-up milk, each of the formulas being about 60 kcal / 100 mL to about 80 kcal / 100 mL and comprising 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.

[0264] 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 HMO mixture is administered to a 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.

[0265] 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 the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from human breast-fed infants at about 6 months of age or older, at about 7 months of age or older, at about 8 months of age or older, or at about 9 months of age or older.

[0266] 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 the gut microbiome trajectory of the formula-fed infant converges to a reference gut microbiome trajectory obtained from human breast-fed infants by about 12 months of age, by about 11 months of age, by about 10 months of age, or by about 9 months of age.

[0267] 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 the gut microbiome trajectory of the formula-fed infant converges to a reference gut microbiome trajectory obtained from a human breast-fed infant at about 6 to about 12 months of age, at about 7 to about 11 months of age, at about 8 to 10 months of age, or at about 9 months of age.

[0268] 39. The method of any one of paragraphs 23 or 25 to 38, or the use of any one of paragraphs 24 to 38, wherein the gut microbiome trajectory is a gut microbiome age trajectory or a gut microbiome diversity trajectory.

[0269] 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, and optionally the gut microbiome age trajectory is obtained using genus-level data, species-level data, and / or functional data from gut microbiome data.

[0270] 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 a full-term infant.

[0271] 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 a Caesarean section born infant.

[0272] 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 breastfed infant is vaginally delivered at term and is a human breastfed infant.

[0273] 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.

[0274] 45. The method of any one of paragraphs 23 or 25 to 44 or the use of any one of paragraphs 24 to 44, wherein the method promotes maturation of the gut microbiome, maturation of gut metabolism, maturation of gut barrier function, and / or maturation of gut immune function.

[0275] 46. ​​A method for determining the intestinal maturity status of a formula-fed infant, said method comprising: (a) To provide gut microbiome data from a cohort of human breast-fed infants; and (b) training a regression model on the gut microbiome data; (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; Including, A method wherein the formula-fed infant's intestinal maturation status is normal if the formula-fed infant is not an outlier in the trained regression model, and / or the formula-fed infant's intestinal maturation status is not normal if the formula-fed infant is an outlier in the trained regression model.

[0276] 47. The method of 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 the formula-fed infant is an outlier if the formula-fed infant's gut microbiome data is -2SE or less or 2SE or more, -2.5SE or less or 2.5SE or more, or -3SE or less or 3SE or more from the trained regression line, if the formula-fed infant's gut microbiome data is outside a 90%, 95%, or 99% confidence interval in the trained regression model, if the formula-fed infant's gut microbiome data is outside a 90%, 95%, or 99% prediction interval in the trained regression model, and / or if the formula-fed infant has a Z-score of -2 or less or 2 or more, -2.5 or less or 2.5 or more, or -3 or less or 3 or more in the trained regression model.

[0277] 48. A method for determining the intestinal maturity status of a formula-fed infant, said method comprising: (a) To provide gut microbiome data from a cohort of human breast-fed infants; and (b) training a regression model on the gut microbiome data to provide a gut microbiome trajectory; (c) providing gut microbiome data from the formula-fed infant and determining whether the formula-fed infant is on a gut microbiome trajectory; Including, The method wherein the formula-fed infant has a normal intestinal maturation status if the formula-fed infant is on the gut microbiome trajectory, and / or the formula-fed infant has a non-normal intestinal maturation status if the formula-fed infant is off the gut microbiome trajectory.

[0278] 49. The formula-fed infant is determined to be deviating from 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 formula-fed infant's gut microbiome data is less than or equal to -2SE or more than 2SE, less than or equal to -2.5SE or more than 2.5SE, or less than or equal to -3SE or more than 3SE from the gut microbiome trajectory. 49. The method of paragraph 48, wherein the formula-fed infant is determined to be off the gut microbiome trajectory if the formula-fed infant's gut microbiome data is outside a 90%, 95%, or 99% confidence interval of the microbiome trajectory, if the formula-fed infant's gut microbiome data is outside a 90%, 95%, or 99% prediction interval of the gut microbiome trajectory, and / or if the formula-fed infant has a Z-score below -2 or above 2, below -2.5 or above 2.5, or below -3 or above 3.

[0279] 50. A data processing system comprising means for carrying out the method of any one of paragraphs 46 to 49.

[0280] 51. A data processing system comprising a processor configured to carry out the method of any one of paragraphs 46 to 49.

[0281] 52. A computer-readable medium comprising instructions that, when executed by a computer, cause the computer to perform the method of any one of paragraphs 46 to 49.

[0282] 53. A computer program comprising instructions that, when the program is run by a computer, cause the computer to carry out a method according to any one of paragraphs 46 to 49.

[0283] 54. A computer-readable data carrier storing a computer program according to paragraph 53.

[0284] 55. A data carrier signal carrying a computer program according to paragraph 53.

[0285] 56. Use of one or more reference gut microbiome trajectories obtained from human breast-fed infants to establish the intestinal maturation status of formula-fed infants after administration of HMO mixtures.

[0286] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed methods, compositions, and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. A human milk oligosaccharide (HMO) mixture for use in inducing the gut microbiome trajectory of a formula-fed infant to converge to the trajectory of a reference gut microbiome obtained from a human breast-fed infant, the HMO mixture comprising or consisting of 2'-fucosyllactose (2'FL), 2',3-difucosyllactose (diFL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

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

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

4. The HMO mixture comprises: (a) a starter infant formula comprising a total amount of HMOs from about 0.5 g / L to about 5.0 g / L, from about 1.0 g / L to about 3.0 g / L, or from about 1.5 g / L to about 2.5 g / L, optionally comprising: (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 of about 0.1 g / L to about 1.0 g / L, preferably 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 0.05 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; (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; Starter infant formula, including (b) a follow-up formula comprising HMOs in a total amount 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, optionally 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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.06 g / L; and / or a follow-up formula containing (c) a growing-up milk comprising a total amount of HMOs from about 0.1 g / L to about 1.0 g / L, from about 0.2 g / L to about 0.8 g / L, from about 0.28 g / L to about 0.52 g / L, or from about 0.3 g / L to about 0.5 g / L, and optionally 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; (v) 6'-SL in an amount of about 0.03 g / L to about 0.05 g / L; 4. The HMO mixture for use according to any one of claims 1 to 3, administered in the form of a growing-up milk comprising:

5. The HMO mixture comprises: (a) a starter infant formula comprising about 1.5 g / L or 2.5 g / L total HMOs, optionally comprising: (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; (v) 6'-SL in an amount of about 0.14 g / L or about 0.24 g / L; Starter infant formula, including (b) a follow-up formula comprising HMOs in a total amount of about 0.5 g / L, optionally 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; (v) 6'-SL in an amount of about 0.05 g / L; and / or a follow-up formula containing (c) a growing-up milk containing about 0.4 g / L total HMOs, optionally 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; (v) 6'-SL in an amount of about 0.04 g / L; 5. The HMO mixture for use according to any one of claims 1 to 4, administered in the form of a growing-up milk comprising:

6. 6. The HMO mixture for use according to any one of claims 1 to 5, wherein the HMO mixture 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. 7. The HMO mixture for use according to any one of claims 1 to 6, wherein the gut microbiome trajectory of the formula-fed infant converges with a reference gut microbiome trajectory obtained from a human breast-fed infant at about 6 to about 12 months of age, at about 7 to about 11 months of age, at about 8 to 10 months of age, or at about 9 months of age.

8. 8. The HMO mixture for use according to any one of claims 1 to 7, wherein inducing the formula-fed infant's gut microbiome trajectory to converge towards a reference gut microbiome trajectory obtained from a human breast-fed infant promotes intestinal maturation.

9. 1. A method for inducing the trajectory of the gut microbiome of a formula-fed infant to converge to a reference gut microbiome trajectory obtained from a human breast-fed infant, the method comprising administering to the formula-fed infant an effective amount of a human milk oligosaccharide (HMO) mixture, the HMO mixture comprising or consisting of 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 human milk oligosaccharide (HMO) mixture to induce the trajectory of the gut microbiome of a formula-fed infant to converge to the trajectory of a reference gut microbiome obtained from a human breast-fed infant, wherein the HMO mixture 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).

11. 1. A method for determining the intestinal maturity status of a formula-fed infant, said method comprising: (a) providing gut microbiome data from a population of human breast-fed infants; (b) training a regression model on the gut microbiome data; (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; Including, A method wherein the intestinal maturation status of the formula-fed infant is normal if the formula-fed infant is not an outlier in the trained regression model, and / or the intestinal maturation status of the formula-fed infant is not normal if the formula-fed infant is an outlier in the trained regression model.

12. 1. A method for determining the intestinal maturity status of a formula-fed infant, said method comprising: (a) providing gut microbiome data from a population of human breast-fed infants; (b) training a regression model on the gut microbiome data to provide a gut microbiome trajectory; (c) providing gut microbiome data from the formula-fed infant and determining whether the formula-fed infant is on the gut microbiome trajectory; Including, The method wherein the intestinal maturation status of the formula-fed infant is normal if the formula-fed infant is on the gut microbiome trajectory, and / or the intestinal maturation status of the formula-fed infant is not normal if the formula-fed infant is off the gut microbiome trajectory.

13. A computer program comprising instructions that, when said program is run by a computer, cause said computer to carry out the method according to claim 11 or 12.

14. 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 breast-fed infants to determine the intestinal maturation status of formula-fed infants following administration of an HMO mixture.

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