Composition for use

Abstract

The present invention provides a method for identifying infants or toddlers at risk of growth retardation, and a composition for use in preventing and / or treating growth retardation in infants or toddlers.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying infants or young children at risk of growth retardation, and to compositions for use in the prevention and / or treatment of growth retardation in infants or young children. [Background technology]

[0002] Studies investigating the impact of malnutrition on growth have primarily focused on weight-related outcomes. However, approaches aimed at identifying and correcting weight gain may lead to negative long-term outcomes; for example, weight gain can be associated with an increased risk of metabolic disorders.

[0003] Several studies have investigated the problem of undergrowth by using microbiome-targeted solutions (e.g., Chen RY et al. NEJM 2021;384:1517-28; Subramanian S, et al. Nature 2014;510(7505):417-421). However, most of these studies report increases in weight-for-age or weight-for-height. This increase may be related to the use of calorie-rich foods in these intervention trials and may have associated effects, such as an increased risk of subsequent metabolic disorders.

[0004] Further approaches and methods are needed to identify and / or treat or prevent growth retardation related to height or body length, for example, as an outcome of malnourishment in infants or young children. [Overview of the project]

[0005] This invention is at least in part based on the inventors' surprising conclusion that a decrease in the levels of Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) and / or Streptococcus thermophilus (S. thermophilus) in the gut microbiota is associated with linear growth faltering in a cohort of infants and young children. Furthermore, the inventors concluded that Bifidobacterium pseudocatenulatum can be proliferated by human milk oligosaccharides (HMOs). To the best of the inventors' knowledge, the relationship between linear growth faltering and the microbiome has not been investigated, or, if they do not have positive results, has not been identified. For example, no association or improvement in age-for-age z (HAZ) scores or age-for-age z (LAZ) scores had been concluded (Subramanian S, et al. Nature. 2014; 510(7505): 417-421), or LAZ correlations with only a small number of bacteria in the duodenal microbiome had been reported (Chen RY, et al. N Engl J Med. 2020; see above).

[0006] Therefore, the present invention provides an approach to identify infants or toddlers at risk of growth retardation by measuring the abundance of Bifidobacterium pseudocatenulatum and / or S. thermophilus in one or more samples obtained from infants. The present invention also provides an approach to prevent and / or treat growth retardation in infants or toddlers by promoting the growth of Bifidobacterium pseudocatenulatum and / or S. thermophilus in the gut microbiota. Thus, the present invention may include increasing the abundance and / or activity of Bifidobacterium pseudocatenulatum and / or S. thermophilus in the gut microbiota.

[0007] Accordingly, in a first aspect, the present invention provides a method for identifying infants or toddlers at risk of growth inhibition, comprising measuring the abundance of Bifidobacterium pseudocatenulatum and / or S. thermophilus in one or more samples obtained from infants.

[0008] Appropriately, this method involves measuring the amount of Bifidobacterium pseudocatenulatum present in one or more samples obtained from an infant.

[0009] Appropriately, infants or young children with reduced levels of B. pseudocatenulatum are identified as being at risk of growth retardation.

[0010] Growth inhibition may be impaired growth in height or body length. Appropriately, growth inhibition in height or body length may be defined as a decrease in the age-specific body length z-score (LAZ) or a decrease in the LAZ over time. Appropriately, growth inhibition in height or body length may be defined as a decrease in the age-specific height z-score (HAZ) or a decrease in the HAZ over time.

[0011] In another aspect, the present invention provides a composition for use in preventing and / or treating growth retardation in infants or young children, the composition promoting the growth of B. pseudocatenulatum in the intestinal microbiota of infants or young children.

[0012] The composition may contain the microorganism B. pseudocatenulatum. Preferably, the composition containing B. pseudocatenulatum may be administered in combination with a prebiotic.

[0013] The composition may contain prebiotics. Preferably, the prebiotic-containing composition may be administered in combination with the microorganism B. pseudocatenulatum.

[0014] The present invention further provides combinations of B. pseudocatenulatum microorganisms and prebiotics for use in preventing and / or treating growth retardation in infants or young children.

[0015] Prebiotics may be in the form of dietary compositions or nutritional compositions. Prebiotics may contain human milk oligosaccharides (HMOs).

[0016] The present invention further provides HMOs or combinations of HMOs for use in preventing and / or treating growth retardation in infants or young children, wherein the HMOs or combinations of HMOs promote the growth of B. pseudocatenulatum in the intestinal microbiota of infants or young children.

[0017] The HMO may be selected from the group consisting of 2'-FL, 3-FL, di-FL, 3'-SL, 6'-SL, LNT, and LNnT, and any combination thereof. The HMO may be any HMO or combination of HMOs as defined herein.

[0018] HMO can be selected from the group consisting of 2'-FL, di-FL, 6'-SL, and LNnT, and any combination thereof. HMO can be a combination of 2'-FL and di-FL. HMO can be a combination of 6'-SL and LNnT.

[0019] HMOs can be provided in combination with galactooligosaccharides (GOS).

[0020] The present invention also provides the microorganism B. pseudocatenulatum for use in preventing and / or treating growth retardation in infants or young children.

[0021] Treatment and / or prevention of growth inhibition may be associated with increased bone development and / or bone strength in the subject.

[0022] The present invention further provides a method for preventing and / or treating growth retardation in infants or young children, the method comprising administering to an infant or young child a composition that promotes the growth of B. pseudocatenulatum in the intestinal microbiota.

[0023] In a further aspect, the present invention provides the use of B. pseudocatenulatum microorganisms and / or prebiotics as defined herein in the manufacture of a medicament for preventing and / or treating growth inhibition in infants or young children.

[0024] The present invention also provides the use of a composition for regulating the abundance of B. pseudocatenulatum in the intestine of an infant or young child. The composition can be any composition described herein.

[0025] The present invention further provides a probiotic composition comprising B. pseudocatenulatum.

[0026] The present invention also provides a synbiotic composition comprising B. pseudocatenulatum and a prebiotic. The prebiotic can be a prebiotic as defined herein.

[0027] Suitably, for any aspect of the present invention, S. thermophilus can be provided as an alternative to B. pseudocatenulatum.

[0028] Suitably, any aspect of the present invention can relate to a combination of B. pseudocatenulatum and S. thermophilus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] [Figure 1] Schematic diagram of the Microhealth study. [Figure 2] Microbiome profiling of each group classified by differences in the dynamic changes over time of the LAZ score. Q1 is an infant with a negative slope of the age-specific length z (LAZ) score over time, defined as "stunted growth". Q4 is an infant with a positive slope of the LAZ score over time, defined as "reference". [Figure 3]Using both (A) dynamic changes and (B) static outcomes, Bifidobacterium pseudocatenulatum was identified as a bacterial signature of inadequate height profiles up to 24 months. [Figure 4] Using both (A) dynamic changes and (B) static outcomes, Streptococcus thermophilus was identified as a bacterial signature of inadequate height profiles up to 24 months of age. [Figure 5] An example of age-specific height (Z-score) data for girls aged 2-5 years, according to the WHO. [Modes for carrying out the invention]

[0030] Herein, various preferred features and embodiments of the present invention are described by non-limiting examples. Those skilled in the art will understand that all features of the present invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0031] When used in this specification and the appended claims, it should be noted that the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise indicated.

[0032] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” “contains,” and may be inclusive or open-ended, and do not exclude additional, unspecified members, elements, or process steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0033] A numerical range includes the number that defines that range.

[0034] The publications discussed herein are provided simply because they were disclosed prior to the filing date of this application. No document cited herein should be construed as constituting prior art with respect to the claims attached herein.

[0035] The methods and systems disclosed herein may be used by physicians, medical professionals, laboratory technicians, infant or child care providers, and others.

[0036] Growth inhibition The UNICEF and WHO definition of stunting or stunted growth refers to children who are too short for their age. These children may suffer serious and irreversible physical and cognitive impairments associated with stunting. The effects of stunting can last a lifetime and may even affect future generations (https: / / data.unicef.org / topic / nutrition / malnutrition). According to the 2021 UNICEF / WHO / World Bank Joint Child Malnutrition Estimates, approximately 22% of children under the age of five were affected by stunting in 2020. This is equivalent to 149.2 million children under the age of five being stunted. Growth retardation is observed in infants and young children, but can extend to preschool and school-aged children (Leroy JL, Ruel M, Habicht JP, Frongillo EA., J Nutr. 2014; Dutta A, et al. Food Nutr Bull. 2009).

[0037] Growth retardation in infants and young children can be caused by a number of factors, including poor intrauterine environment, exposure to aflatoxins, intergenerational effects, environmental intestinal dysfunction, lack of nutrition and dietary diversity, infections (e.g., Campylobacter infection), diarrhea, water, sanitation, and hygiene problems, as well as other causes, such as maternal factors. Several studies have investigated the problem of growth retardation by using microbiome-targeted solutions (e.g., Chen RY et al. NEJM 2021; Subramanian S, et al. Nature 2014). However, most of these studies report increases in age-specific or height-specific body weight. This increase may be related to the use of calorie-rich foods in these intervention trials and may lead to associated effects, such as the risk of subsequent metabolic disorders. To the best of our knowledge, the relationship between linear growth retardation and the microbiome has not been investigated, or cases where they do not yield positive results have not been identified. For example, either no association or improvement in age-specific height z-score (HAZ) or age-specific body length z-score (LAZ) has been evaluated (Subramanian S, et al. Nature. 2014; see above), or an LAZ correlation with only a small number of bacteria in the duodenal microbiota has been reported (Chen RY, et al. N Engl J Med. 2020; see above).

[0038] As used herein, “growth inhibition” may refer to inhibition of growth in height or length of an infant or young child.

[0039] Using age-specific growth indicators such as body length / height, it is also possible to identify children whose growth is stunted (short stature) due to long-term malnutrition or recurring illness.

[0040] Appropriately, growth inhibition can be defined as a decrease in the age-specific body length z-score (LAZ) or a decrease in LAZ over time. LAZ may also be called the age-specific height z-score (HAZ).

[0041] Children who are growing normally generally tend to follow a growth curve parallel to the median and the z-score line (see Figure 5). Most children grow on a "track," i.e., on or between the z-score lines and roughly parallel to the median. This track may be located below or above the median. When interpreting a growth chart, attention should be paid to the following situations, as they may indicate a problem or suggest a risk: the child's growth curve crosses the z-score line, the child's growth curve has a steep slope or decline, or the child's growth curve remains flat (stagnant), i.e., there is no increase in weight or body length / height.

[0042] Stunting can be defined as a static outcome. For example, stunting can be defined as age-specific body length / height being less than -2. For example, stunting can be defined as age-specific height being less than -2 standard deviations from the median of the World Health Organization (WHO) child growth curves (https: / / sdgdata.gov.uk / 2-2-1 / #:~:text=Definitions,(WHO)%20Child%20Growth%20Standards).

[0043] Growth retardation can be defined as a dynamic change, such as a change in the LAZ (Level-Axial Scale) assessed over time (e.g., 0–36 months, 6–36 months, or 6–24 months). Infants whose LAZ score decreases over time may be defined as having growth retardation.

[0044] The dynamic change in growth is sometimes called "growth velocity" or "height velocity." Growth velocity or height velocity can be defined as shown in equation (1).

[0045]

number

[0046] Malnutrition results from reduced food intake and / or disease. Malnutrition is associated with an increased risk of medical complications and infections, an increased risk of death from disease and infection, and micronutrient deficiencies. Non-specific examples of micronutrient deficiencies associated with malnutrition include iron deficiency, iodine deficiency, and vitamin D deficiency. The most common method for assessing malnutrition, especially in infants and young children, is anthropometric measurement. Malnutrition is usually diagnosed by one of three methods: measuring the subject's weight and height, measuring the subject's middle upper arm circumference (MUAC), or checking for edema in the subject's lower limbs or feet. Malnutrition can be classified into two types: severe acute malnutrition (SAM) and moderate acute malnutrition (MAM). A subject is classified as SAM if their height-specific weight Z-score (WHZ) is lower than 3 standard deviations (-3s.d.) from the median of the World Health Organization (WHO) standard growth curve. Subjects with a WHZ (Waist-to-Head Zest) of -2s.d. to -3s.d. from the median of the WHO standard growth curve are classified as having moderate acute malnutrition (MAM). For subjects aged approximately 6 months to 5 years, a MUAC (Mutually Measured Amount of Head Circumference) of less than 12.5 cm also indicates moderate acute malnutrition. Finally, the presence of edema in both feet and lower extremities is a sign of SAM (Severe Acute Malnutrition). WHO standard growth curves are available from the WHO. For example, see World Health Organization Department of Nutrition for Health and Development: WHO child growth standards growth velocity based on weight, length and head circumference: methods and development; World Health Organization, 2009 or current edition.

[0047] Appropriately, infants or toddlers may be SAM. Appropriately, infants or toddlers may be MAM.

[0048] Individuals at risk of growth stunting may be those living in areas with limited or no access to comprehensive nutritional food, and / or those living in areas experiencing disease outbreaks. Individuals at risk of growth stunting may also be malnourished.

[0049] Treating individuals at risk of growth inhibition may reduce or prevent the occurrence of growth inhibition in those individuals.

[0050] The term "prevention" in relation to growth inhibition may refer to reducing or eliminating growth inhibition.

[0051] For example, prevention of growth inhibition may be a reduction in the incidence or occurrence of growth inhibition in the treated subject; if growth inhibition occurs, a reduction in the incidence, duration, and / or severity of growth inhibition; or a combination thereof. With respect to each embodiment, the amount of reduction in the treated subject may be about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% compared to the untreated subject.

[0052] Formula for infants or toddlers Appropriately, an infant or toddler may be under approximately 60 months of age. For example, an infant or toddler may be under 48 months of age, under 36 months of age, or under 24 months of age.

[0053] Appropriately, infants or toddlers may be approximately 1–60 months old, 1–48 months old, 2–60 months old, 2–48 months old, 2–36 months old, or 4–36 months old, 6–36 months old, or 6–24 months old.

[0054] For example, an infant or toddler may be at least about 6 months old, at least about 10 months old, at least about 12 months old, at least about 14 months old, at least about 16 months old, at least about 20 months old, or at least about 24 months old.

[0055] Appropriately, infants or toddlers may be approximately 6 to 60 months old, approximately 6 to 48 months old, approximately 6 to 36 months old, or approximately 6 to 24 months old.

[0056] An infant may be a child under 12 months of age. A toddler may be a child between 1 and 5 years of age, or between 1 and 3 years of age.

[0057] The subjects may be mammals. Preferably, the subjects are humans. Unless otherwise specified, the ages referred to herein refer to human subjects.

[0058] composition The composition may be suitable for or appropriately administered to infants or toddlers in any suitable form, for example, as a nutritional composition in dosage units (e.g., tablets, capsules, powder sachets, etc.). The composition may be in powder, semi-liquid, or liquid form. The composition may be added to nutritional compositions, infant formulas, food compositions, supplements for infants or toddlers, baby food, follow-up formulas, growing-up milk, infant or toddler cereals, or fortifiers. In some embodiments, the composition of the present invention is an infant formula, baby food, infant or toddler cereal, growing-up milk, or a supplement or fortifier that may be intended for infants or toddlers.

[0059] The terms "complementary feeding period," "complementary period," "transitional period," "transitional feeding period," and "weaning period" are interchangeable and refer to the period in an infant's or toddler's diet during which either breast milk or formula is replaced by another food. Infants and toddlers typically gradually change or transition from an exclusive milk-feeding diet (either breast milk or formula) to a mixed diet including milk and / or solid foods. The transition period varies from infant to toddler, but is typically between approximately 4 months and 18 months of age, for example, between approximately 6 months and 18 months of age, although in some cases it may extend to approximately 24 months or longer. In humans, the weaning period typically begins between 4 and 6 months of age and is considered complete, typically around 24 months of age, when the infant or toddler is no longer given breast milk or infant formula. In some embodiments, the weaning period is 4 to 24 months.

[0060] More appropriately, the composition is a meal composition or a nutritional composition.

[0061] The terms "dietary composition" or "nutritional composition" refer to any composition or formula of any kind that provides nutritional benefits to an individual and is safe for human or animal consumption. Such nutritional compositions may be in solid (e.g., powder) or semi-solid or liquid form and may contain one or more major nutrients, micronutrients, food additives, water, etc. For example, a nutritional composition may contain the following major nutrients: protein sources, lipid sources, carbohydrate sources, and any combination thereof. Furthermore, a nutritional composition may contain the following micronutrients: vitamins, minerals, fiber, phytochemicals, antioxidants, prebiotics, probiotics, and any combination thereof. The composition may also contain food additives such as stabilizers (if provided in solid form) or emulsifiers (if provided in liquid form). The amounts of various raw materials can be expressed in grams per 100g of composition on a dry weight basis if they are solids, such as powders, and in concentration as grams per 1L of composition if they are liquids (the latter also includes liquid compositions obtained after reconstituting powders with liquids, such as milk or water, and also includes, for example, reconstituted infant or toddler formulas or follow-on / follow-up formulas, or infant or toddler cereal products, or other formulas designed for infant or toddler nutrition). Generally, nutritional compositions can be formulated for enteral, oral, parenteral, or intravenous administration and typically contain one or more nutrients selected from lipid or fat sources, protein sources, and carbohydrate sources. Preferably, nutritional compositions are for oral administration.

[0062] In certain embodiments, the composition of the present invention is a "synthetic nutritional composition." The expression "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means.

[0063] As used herein, the expression “Infant Formula” refers to a food intended for specific nutritional supplementation purposes for infants during the first month of life, which, by itself, satisfies the nutritional requirements for infants in this category (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 for Infant Formulas and Follow-on Formulas). This formula is intended for infants or toddlers and also refers to nutritional compositions as defined in Codex Alimentarius (Codex STAN72-1981), and special infant foods (including foods for specific medical purposes). The expression “Infant Formula” encompasses both “Infant Starter Formulas” and “Follow-up Formulas” or “Follow-on Formulas.”

[0064] A "follow-up formula" or "follow-on formula" is typically given from six months of age onward. Such formulas constitute the main liquid component in the increasingly diverse diet of infants in this category.

[0065] The term "infant food" refers to food intended for specific nutritional purposes during the first year of life for infants or toddlers.

[0066] The phrase "cereal composition for infants or toddlers" refers to food intended for specific nutritional purposes during the first year of life for infants or toddlers.

[0067] The term "growing-up milk" (or GUM) generally refers to a milk beverage for young children or children that is fortified with vitamins and minerals.

[0068] A “fortifier” may be a liquid or solid nutritional composition suitable for fortifying or mixing with human breast milk, infant or toddler formula, or growing-up milk. Therefore, a fortifier can be administered after being dissolved in human breast milk, after being dissolved in infant or toddler formula, after being dissolved in growing-up milk, or after being dissolved in human breast milk fortified with other nutrients, or it can be administered as a standalone composition. When administered as a standalone composition, a milk fortifier may also be identified as a “supplement.”

[0069] The nutritional composition may include a protein source. The protein content can be 1.6 to 3 g / 100 kcal.

[0070] In addition to soy-based protein sources, protein sources based on whey, casein, and mixtures thereof may be used. With regard to whey protein, the protein source may be based on acidic whey, sweet whey, or mixtures thereof, and may contain α-lactalbumin and β-lactoglobulin in any desired proportion.

[0071] In some embodiments, the primary protein source is whey (i.e., more than 50%, for example more than 60% or more than 70% of the protein is derived from whey protein). The protein may be in its intact state, hydrolyzed state, or a mixture of intact and hydrolyzed proteins. The term “intact” means that the main portion of the protein is intact, i.e., its molecular structure remains unchanged, for example, at least 80% of the protein remains unchanged, for example at least 85% of the protein remains unchanged, preferably at least 90% of the protein remains unchanged, and more preferably at least 95% of the protein remains unchanged, for example at least 98% of the protein remains unchanged. In certain embodiments, the protein is not changed at all.

[0072] In a particular embodiment, the protein in the nutritional composition is hydrolyzed, completely hydrolyzed, or partially hydrolyzed. The degree of hydrolysis (DH) of the protein can be 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, greater than 20, greater than 40, greater than 60, greater than 80, or greater than 90. Alternatively, the protein component can be replaced with a mixture or synthetic amino acids, for example, for premature or low birth weight infants.

[0073] In the context of this invention, the term "hydrolyzed" means that a protein has been hydrolyzed, or broken down into its constituent amino acids. The protein may be fully hydrolyzed or partially hydrolyzed. For example, for infants or young children who are considered to be at risk of developing milk allergies, it may be desirable to supply partially hydrolyzed protein (degree of hydrolysis 2-20%). When hydrolyzed protein is required, the hydrolysis process may be carried out as desired, as is known in the art. For example, hydrolyzed whey protein can be prepared by enzymatically hydrolyzing a whey fraction in one or more steps. It has been found that when the whey fraction used as a raw material is substantially lactose-free, the lysine blackage that the protein undergoes during the hydrolysis process is significantly reduced. This can reduce the degree of lysine blackage from about 15% by weight of total lysine to less than 10% by weight of lysine, and for example, about 7% by weight of lysine significantly improves the nutritional value of the protein source.

[0074] In one embodiment of the present invention, at least 70% of the protein is hydrolyzed, for example, at least 80% of the protein is hydrolyzed, for example, at least 85% of the protein is hydrolyzed, or at least 90%, 95%, or 98% of the protein is hydrolyzed. In a particular embodiment, 100% of the protein is hydrolyzed.

[0075] The nutritional composition may contain a carbohydrate source. This is particularly preferred when the nutritional composition is an infant formula. In this case, any carbohydrate source conventionally found in infant formulas, such as lactose, sucrose, saccharose, maltodextrin, starch, and mixtures thereof, may be used, but one of the preferred carbohydrate sources is lactose.

[0076] Nutritional compositions may contain a lipid source. This is particularly appropriate if the nutritional composition is an infant formula. In this case, the lipid source may be any lipid or fat suitable for use in an infant formula. Some suitable fat sources include palm oil, structured triglyceride oil, high-oleic sunflower oil and high-oleic safflower oil, and medium-chain triglyceride oil. Essential fatty acids linoleic acid and alpha-linolenic acid may also be added, as may small amounts of oil such as fish oil or microbial oil rich in preformed arachidonic acid and docosahexaenoic acid. The ratio of n-6 fatty acids to n-3 fatty acids in the fat source may be about 5:1 to about 15:1, for example, about 8:1 to about 10:1.

[0077] Nutritional compositions may also contain vitamins and minerals considered essential for daily diet in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins, and other nutrients that may be optionally included in the compositions of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amount of specific minerals and other vitamins will vary depending on the target population. If necessary, the nutritional compositions of the present invention may also contain emulsifiers and stabilizers, such as soy, lecithin, and mono- and diglyceride citrate esters.

[0078] The nutritional composition may also optionally contain other substances that may have beneficial effects, such as lactoferrin, nucleotides, and nucleosides.

[0079] Nutritional compositions may be prepared in any preferred form. The following examples illustrate such compositions.

[0080] For example, formulas such as infant formula are prepared by blending protein sources, carbohydrate sources, and fat sources together in appropriate proportions. If used, emulsifiers can be added at this stage. Vitamins and minerals may be added at this stage, but are usually added later to avoid thermal decomposition. Any lipophilic vitamins and emulsifiers can be dissolved in the fat source before blending. Then, water, preferably reverse-osmotic water, can be mixed to form a liquid mixture. The water temperature should be in the range of about 50°C to about 80°C, as appropriate, to aid in the dispersion of the components. A commercially available liquefaction device can also be used to form the liquid mixture.

[0081] Next, the liquid mixture is homogenized.

[0082] The liquid mixture may then be subjected to a heat treatment to reduce the bacterial load, for example, by rapidly heating the liquid mixture to a temperature in the range of approximately 80°C to 150°C for approximately 5 seconds to approximately 5 minutes. This heating may be carried out by steam injection, by autoclave, or by heat exchanger, for example, by plate heat exchanger.

[0083] Next, the liquid mixture can be cooled to approximately 60°C to 85°C, for example, by flash cooling. Then, the liquid mixture may be homogenized again, for example, in two stages: in the first stage to approximately 10 MPa to 30 MPa, and in the second stage to approximately 2 MPa to 10 MPa. The homogenized mixture may then be further cooled, and any heat-sensitive components, such as vitamins and minerals, may be added. The pH and solids content of the homogenized mixture are usually adjusted at this point.

[0084] If the final product is a powder, the homogenized mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer to convert it into a powder. The moisture content of the powder should be less than approximately 5% by weight. The mixture may be spray-dried or freeze-dried.

[0085] If a liquid composition is preferred, the homogenized mixture may be sterilized and then aseptically filled into a suitable container, or it may be filled into a container first and then retorted.

[0086] The nutritional composition can be given, for example, immediately after the birth of an infant. The nutritional composition of the present invention can also be given to an infant or toddler during the first week, or the second week, or the third week, or the first month, or the second month, or the third month, or the fourth month, or the sixth month, or the eighth month, or the tenth month, or the first year, or the second year or older. In some particularly advantageous embodiments of the present invention, the composition is given (or administered) to the infant or toddler from about six months of age. For example, the composition may be administered from about six months, about ten months, about twelve months, about fourteen months, about sixteen months, about twenty months, about twenty-four months, or about thirty-six months of age.

[0087] Ideally, the composition should be given (or administered) to infants or toddlers from about 10 months of age.

[0088] The composition may be appropriately administered to infants or toddlers aged approximately 6 to 60 months, approximately 6 to 48 months, or approximately 6 to 36 months. The composition may also be appropriately administered to infants or toddlers aged approximately 10 to 60 months, approximately 10 to 48 months, or approximately 10 to 36 months.

[0089] In one embodiment, the nutritional composition is given to the infant or toddler as a composition to supplement breast milk. In some embodiments, the infant or toddler receives breast milk for at least two weeks, one month, two months, four months, or six months after birth. In one embodiment, the nutritional composition of the present invention is given to the infant or toddler after breastfeeding for such a period, or together with breastfeeding for such a period. In another embodiment, the nutritional composition is given to the infant or toddler alone or as the primary nutritional composition for at least one period, for example, after one month, two months, or four months after birth, or for at least one month, two months, four months, or six months.

[0090] The composition may appropriately include probiotics containing Bifidobacterium pseudocatenulatum.

[0091] The composition may appropriately contain probiotics, including Streptococcus thermophilus.

[0092] The term "probiotics" refers to microbial cell preparations or components that have beneficial effects on the health or well-being of the host (Salminen S, Ouwehand A, Benno Y. et al. "Probiotics: how should they be defined." Trends Food Sci. Technol. 1999:10 107-10; Hill C, et al. Nat Rev Gastroenterol Hepato. 2014). Microbial cells, in a broad sense, include bacteria or yeast.

[0093] Bifidobacterium pseudocatenulatum is present in the composition in, for example, about 10 per gram of composition by dry weight. 3 ~10 12 CFU probiotic strain, fucare10 7 ~10 12 cfu, for example 10 8 ~10 10It may be included in the amount of the probiotic strain of cfu. In one embodiment, Bifidobacterium pseudocatenulatum is viable. In some other embodiments, both viable Bifidobacterium pseudocatenulatum and inactivated Bifidobacterium pseudocatenulatum may be present.

[0094] Streptococcus thermophilus may be included in the composition, for example, at about 10 3 ~10 12 cfu of the probiotic strain, more preferably 10 7 ~10 12 cfu, for example 10 8 ~10 10 cfu of the probiotic strain. In one embodiment, Streptococcus thermophilus is viable. In some other embodiments, both viable Streptococcus thermophilus and inactivated Streptococcus thermophilus may be present.

[0095] The term "cfu" should be understood as colony forming unit.

[0096] Appropriately, the present invention may include the use of a combination of a nutritional composition or dietary composition described herein and a probiotic composition described herein, for example, a probiotic composition comprising Bifidobacterium pseudocatenulatum and / or Streptococcus thermophilus. Appropriately, the present invention may include synbiotics or the use of synbiotics. As used herein, synbiotics may refer to a mixture comprising a microorganism and a substrate selectively utilized by the microorganism, preferably a combination that provides health benefits to the host (e.g., Swanson KS, et al. Nat Rev Gastroenterol Hepatol. 2020). For example, the synbiotics of the present invention comprises an HMO as a prebiotic and B. pseudocatenulatum and / or S. thermophilus as a probiotic. Appropriately, the synbiotics of the present invention comprises an HMO as a prebiotic and B. pseudocatenulatum as a probiotic.

[0097] The compositions for use according to the present invention may be administered by any suitable method. Preferably, the compositions are for oral administration. Therefore, it is preferable that the compositions be administered orally.

[0098] In some embodiments, the compositions according to the present invention may be used before and / or during weaning. The nutritional compositions may be administered (or given or fed) at the appropriate age and duration.

[0099] When a combination (for example, a combination of prebiotics and probiotics as described herein) is administered to a target, the combination may be administered separately, simultaneously, or sequentially.

[0100] The composition may appropriately be Nan Pelargon®.

[0101] Bifidobacterium pseudocatenulatum Bifidobacterium is a genus of anaerobic bacteria that are Gram-positive, non-motile, and often have a branched structure. Bifidobacterium is widely distributed in the gastrointestinal tract and constitutes one of the major bacterial genera in the mammalian gastrointestinal microbiome.

[0102] The genus Bifidobacterium possesses a unique fructose-6-phosphate phosphoketolase pathway used for fermenting carbohydrates. Much metabolic research on Bifidobacterium has focused on oligosaccharide metabolism because it is available in habitats where these non-carbohydrate nutrients are limited. It is clear that Bifidobacterium lineages associated with infants have evolved the ability to ferment milk oligosaccharides, while species associated with adults utilize plant oligosaccharides, which aligns with what they encounter in their respective environments.

[0103] The reference strain for Bifidobacterium pseudocatenulatum is ATCC 27919. The reference genome for Bifidobacterium pseudocatenulatum is provided by GenBank assembly accession number: GCF_020541885.1.

[0104] Appropriately, Bifidobacterium pseudocatenulatum may contain 16S rRNA gene sequences with a 99% cutoff identity value and 80% minimum query and target coverage when compared with the 16S rRNA sequences of ATCC 27919 and / or GCF_020541885.1 using BLASTn. Appropriate comparisons can be performed using known methods, for example, as described by Maturana and Cardenasm (Front Microbiol. 2021; 660920).

[0105] Appropriately, Bifidobacterium pseudocatenulatum may have at least 95% ANI (mean nucleotide identity), at least 0.99 TETRA (tetranucleotide frequency), and / or at least 95% AAI (mean amino acid identity) for whole-genome datasets compared with ATCC 27919 and / or GCF_020541885.1. Appropriately, Bifidobacterium pseudocatenulatum microorganisms may have at least 95% ANI (mean nucleotide identity), at least 0.99 TETRA (tetranucleotide frequency), and at least 95% AAI (mean amino acid identity) for whole-genome datasets compared with ATCC 27919 and / or GCF_020541885.1. Appropriate comparisons can be performed using known methods, for example, as described by Maturana and Cardenasm (above).

[0106] Appropriately, Bifidobacterium pseudocatenulatum can be identified using metagenomics. A suitable metagenomics method may be performed, for example, using shotgun sequencing data. Metagenomics can also favorably enable the estimation of the relative abundance of the organism. Suitable metagenomics methods are known in the art, including, for example, MetaPhLaN 3.0 (Beghini et al.; eLife 2021;10:e65088; see https: / / huttenhower.sph.harvard.edu / metaphlan).

[0107] In some embodiments, Bifidobacterium pseudocatenulatum is isolated from humans.

[0108] More precisely, "to increase Bifidobacterium pseudocatenulatum" means to increase the absolute or relative number of Bifidobacterium pseudocatenulatum in the gut microbiota. For example, prebiotics can assist or support the growth and / or survival of microorganisms. Alternatively, the probiotic composition for use in this invention contains Bifidobacterium pseudocatenulatum, thereby increasing the number of Bifidobacterium pseudocatenulatum in the gut microbiota.

[0109] The abundance of Bifidobacterium pseudocatenulatum in the gut microbiota can be measured, for example, by evaluating the relative abundance of Bifidobacterium pseudocatenulatum in a sample derived from the target organism using the metagenomics method described herein.

[0110] The level of Bifidobacterium pseudocatenulatum may be compared to a baseline value determined before administration of the compositions described herein. The baseline value may be determined before the first administration of the compositions described herein, or after the first administration of the compositions described herein, but before subsequent administrations.

[0111] The methods described herein are typically performed on the outside of a human or animal body, for example, on a sample obtained in advance from the subject to be tested. Preferably, the sample is a fecal sample.

[0112] For example, the composition may increase the amount of Bifidobacterium pseudocatenulatum by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, or 100 times compared to the amount of Bifidobacterium pseudocatenulatum before administration of the composition.

[0113] Bifidobacterium pseudocatenulatum has been reported to enhance bone mass density (BMD) by reducing bone resorption and increasing bone formation (Fernandez-Murga et al.; Bone; 2020; 141; 115580), and to improve hyperleptinemia and restore leptin signaling in obese mice (Agusti et al.; Mol. Neurobiol. 55(6), 5337-5352).

[0114] The present invention provides the microorganism Bifidobacterium pseudocatenulatum for use in preventing and / or treating growth retardation in infants or young children. Thus, Bifidobacterium pseudocatenulatum may be provided as a probiotic as defined herein.

[0115] Suitablely, Bifidobacterium pseudocatenulatum may be provided in a composition as defined herein. The composition may be for use in the prevention and / or treatment of growth retardation in infants or young children. The composition may further comprise prebiotics such as the prebiotics described herein.

[0116] Streptococcus thermophilus The present invention may include the use of Streptococcus thermophilus as a probiotic or in compositions described herein.

[0117] The present invention may further include combinations of using Streptococcus thermophilus as a probiotic or in compositions described herein.

[0118] Streptococcus thermophilus is a Gram-positive bacterium, a facultative anaerobic bacterium belonging to the Viridans group. It is negative for cytochrome, oxidase, and catalase tests, and positive for alpha-hemolytic activity tests. Streptococcus thermophilus is non-motile and does not form endospores. It is also classified as a lactic acid bacterium.

[0119] The reference strain for Streptococcus thermophilus is ATCC 19258. The reference genome for Streptococcus thermophilus is provided by GenBank assembly accession number: GCA_903886475.1.

[0120] Appropriately, Streptococcus thermophilus may contain 16S rRNA gene sequences that, when compared with the 16S rRNA sequences of ATCC 19258 and / or GCA_903886475.1 using BLASTn, exhibit a 99% cutoff identity value and 80% minimum query and target coverage. Appropriate comparisons can be performed using known methods, such as those described in Maturana and Cardenasm (Front Microbiol. 2021;660920).

[0121] Appropriately, Streptococcus thermophilus may have at least 95% ANI (mean nucleotide identity), at least 0.99 TETRA (tetranucleotide frequency), and / or at least 95% AAI (mean amino acid identity) for whole-genome datasets compared with ATCC 19258 and / or GCA_903886475. Appropriately, Streptococcus thermophilus microorganisms may have at least 95% ANI (mean nucleotide identity), at least 0.99 TETRA (tetranucleotide frequency), and at least 95% AAI (mean amino acid identity) for whole-genome datasets compared with ATCC 19258 and / or GCA_903886475. Appropriate comparisons can be performed using known methods, for example, those described by Maturana and Cardenasm (above).

[0122] Appropriately, Streptococcus thermophilus can be identified using metagenomics. A suitable metagenomics method may be performed, for example, using shotgun sequencing data. Metagenomics can also favorably enable the estimation of the relative abundance of the organism. Suitable metagenomics methods are publicly known in the art, including, for example, MetaPhLaN 3.0 (Beghini et al.; eLife 2021;10:e65088; see https: / / huttenhower.sph.harvard.edu / metaphlan).

[0123] In some embodiments, Streptococcus thermophilus is isolated from humans.

[0124] More precisely, "to promote the growth of Streptococcus thermophilus" means to increase the absolute or relative number of Streptococcus thermophilus in the gut microbiota. For example, prebiotics can assist or support the growth and / or survival of microorganisms. Alternatively, the probiotic composition for use in this invention contains Streptococcus thermophilus, thereby increasing the number of Streptococcus thermophilus in the gut microbiota.

[0125] The abundance of Streptococcus thermophilus in the gut microbiota can be measured, for example, by evaluating the relative abundance of Streptococcus thermophilus in a sample derived from the target using the metagenomics method described herein.

[0126] The level of Streptococcus thermophilus may be compared to a baseline value determined before administration of the compositions described herein. The baseline value may be determined before the first administration of the compositions described herein, or after the first administration of the compositions described herein, but before subsequent administrations.

[0127] For example, the composition may increase the amount of Streptococcus thermophilus by at least 1.5, 2, 3, 4, 5, 10, 50, or 100 times compared to the amount of Streptococcus thermophilus before administration of the composition.

[0128] Prebiotics The composition may appropriately include prebiotics.

[0129] The term "prebiotics" refers to indigestible carbohydrates that have a beneficial effect on the host by selectively stimulating the growth and / or activity of healthy bacteria in the human colon (Gibson GR, et al. Nat Rev Gastroenterol Hepatol. 2017).

[0130] Preferably, the prebiotics are provided in the form of dietary fiber. For example, the dietary fiber may be prebiotic fiber.

[0131] Preferably, prebiotics may be included in raw materials, such as dietary ingredients.

[0132] Such raw materials may be selected from the group consisting of human milk oligosaccharides (HMOs), refined polysaccharides or refined oligosaccharides, dietary fiber components, semi-refined food ingredients, raw food ingredients, food additives, and semi-refined or refined peptidoglycans.

[0133] More accurately, prebiotics are HMOs.

[0134] Suitablely, the prebiotic composition includes prebiotics (e.g., contained in the raw materials or fiber) that promote the growth of Bifidobacterium pseudocatenulatum in the gut microbiota. Suitablely, the prebiotic composition includes prebiotics (e.g., contained in the raw materials or fiber) that promote the growth of Streptococcus thermophilus in the gut microbiota.

[0135] Prebiotics or compositions may be provided as fermented dairy products (e.g., yogurt). Fermented dairy products such as yogurt have been shown to increase the abundance of S. thermophilus in the gut microbiota (see, for example, Pasoli et al.; Nat Comm; 2000; 11(1); 2610; Oyarzun et al; Comput Struct Biotechnol J; 2022; 5(2); 1632-1641; Yazdi et al.; Journal of Functional Foods; 2022; 105089).

[0136] The composition may contain oligosaccharides (e.g., human milk oligosaccharides) and / or at least one type of fiber and / or at least one type of its precursor. The oligosaccharides and / or fibers and / or their precursors may be selected from a list including galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin, xylooligosaccharides (XOS), polydextrose, and any combination thereof. These components may typically be present in amounts of 0 to 10% by weight of the composition. In certain embodiments, the nutritional composition may also contain at least one type of BMO (milk oligosaccharide).

[0137] The present invention provides prebiotics for use in preventing and / or treating growth retardation in infants or young children. Preferably, the prebiotics promote the growth of B. pseudocatenulatum in the gut microbiota of infants or young children. Preferably, the prebiotics promote the growth of S. thermophilus in the gut microbiota of infants or young children.

[0138] Appropriately, prebiotics promote the growth of B. pseudocatenulatum in the gut microbiota of infants or young children to prevent and / or treat growth retardation in infants or young children. Appropriately, prebiotics promote the growth of S. thermophilus in the gut microbiota of infants or young children to prevent and / or treat growth retardation in infants or young children.

[0139] Appropriately, prebiotics may be provided in compositions as defined herein. These compositions may be used, for example, to prevent and / or treat growth retardation in infants or young children by promoting the growth of B. pseudocatenulatum and / or S. thermophilus in the gut microbiota of the infant or young child. The compositions may further comprise probiotics such as the B. pseudocatenulatum and / or S. thermophilus microorganisms described herein.

[0140] Human milk oligosaccharides (HMOs) Appropriately, prebiotics can be HMOs.

[0141] The term "HMO" or "HMO(plural)" refers to human milk oligosaccharides. These carbohydrates are highly resistant to enzymatic hydrolysis, suggesting that HMOs may exhibit important functions not directly related to their calorie value. In particular, they have been shown to play an essential role in the early development of infants and young children, such as the maturation of the immune system. Many types of HMOs are found in human breast milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose, and / or N-acetylglucosamine, with a wide variety of bonds between them, resulting in a very large number of oligosaccharide types in human breast milk, with over 130 structures identified to date. Almost all of these oligosaccharides have a lactose residue at the reducing end, and the non-reducing terminal position is occupied by sialic acid and / or fucose (if present). Depending on the presence of fucose and sialic acid in the oligosaccharide structure, HMOs can be classified as non-fucosylated molecules (neutral), or as fucosylated molecules (neutral) and sialylated molecules (acidic) and non-sialylated molecules, respectively.

[0142] Appropriately, the term "able to metabolize HMOs" may mean that Bifidobacterium pseudocatenulatum encodes at least one CAZyme that can utilize HMOs. For example, a CAZyme can catalyze the hydrolysis of glycosidic bonds within HMOs. Appropriately, Bifidobacterium pseudocatenulatum may encode at least one, at least two, at least three, at least four, or at least five CAZymes that can utilize HMOs. Appropriately, the term "able to metabolize HMOs" may mean that HMOs can promote the growth and / or survival of Bifidobacterium pseudocatenulatum (for example, when added to an anaerobic culture of Bifidobacterium pseudocatenulatum). B. The growth and / or survival of Bifidobacterium pseudocatenulatum can be measured by measuring the abundance of 16S rDNA, which can be measured, for example, using PCR.

[0143] HMOs that can promote the growth and / or survival of Bifidobacterium pseudocatenulatum may increase the number of Bifidobacterium pseudocatenulatum in anaerobic cultures by at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, or at least 100% compared to the number of Bifidobacterium pseudocatenulatum in control anaerobic cultures without HMOs. Appropriately, HMOs that can promote the growth and / or survival of Bifidobacterium pseudocatenulatum may increase the number of Bifidobacterium pseudocatenulatum in anaerobic cultures by a statistically significant amount (e.g., p-value < 0.05 as determined by one-way ANOVA) compared to the number of Bifidobacterium pseudocatenulatum in control anaerobic cultures without HMOs.

[0144] The above disclosures referring to Bifidobacterium pseudocatenulatum may be equally applicable to Streptococcus thermophilus.

[0145] The term "fucosylated oligosaccharide" refers to oligosaccharides that contain fucose residues. Such oligosaccharides are neutral in nature. Some examples include 2'-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II, and any combination thereof. Fucosylated oligosaccharides are the largest fraction in human breast milk, with 2'-FL accounting for up to 30% of all HMOs. Fucosylated oligosaccharides are thought to reduce the risk of infection and inflammation, and to reduce inflammatory responses by enhancing the growth and metabolic activity of certain symbiotic microorganisms.

[0146] The term "N-acetylated oligosaccharide" encompasses both "N-acetyl-lactosamine" and "oligosaccharides containing N-acetyl-lactosamine." These are neutral oligosaccharides that have an N-acetyl-lactosamine residue. Appropriate examples include LNT (lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), DSLNT (dicialyllacto-N-tetraose), and any combination thereof. Other examples include lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, iso-lacto-N-octaose, para-lacto-N-octaose, and lacto-N-decaose.

[0147] The phrases "at least one type of fucosylated oligosaccharide" and "at least one type of N-acetylated oligosaccharide" mean "at least one type of fucosylated oligosaccharide" and "at least one type of N-acetylated oligosaccharide."

[0148] The term "sialylated oligosaccharide" refers to oligosaccharides that have a charged sialic acid residue. Sialylated oligosaccharides are acidic. Some examples include 3'-sialyl lactose (3-SL), 6'-sialyl lactose (6-SL), and sialyl lact-N-tetraose (Lst-, e.g., Lst-a, Lst-b, or Lst-c).

[0149] HMOs include fucosylated oligosaccharides (i.e., oligosaccharides having fucose residues; e.g., 2'-fucosyllactose (2-FL), 3-fucosyllactose (3-FL), difucosyllactose (DiFL), lacto-N-fucopentaose (e.g., lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II, and any combination thereof), N-acetylated oligosaccharides (e.g., LNT (lacto-N-tetraose)). These may be para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose), DSLNT (diciallacto-N-tetraose), lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, isolact-N-octaose, para-lacto-N-octaose, and lacto-N-decaose, and any combination thereof), and / or sialylated oligosaccharides (e.g., 3'-sialyl lactose (3-SL), 6'-sialyl lactose (6-SL), or Lst (sialyllacto-N-tetraose), Lst-a, Lst-b, or Lst-c)).

[0150] Appropriately, prebiotics may include HMOs selected from the group consisting of 2'-FL, di-FL, 6'-SL, and LNnT, and any combination thereof. The HMO may be a combination of 2'-FL and di-FL. The HMO may be a combination of 6'-SL and LNnT.

[0151] HMOs can be provided in combination with galactooligosaccharides (GOS).

[0152] The prebiotics may include at least one prebiotic oligosaccharide selected from the group consisting of 2'-O-fucosyl lactose (2'FL), 3'-O-fucosyl lactose (3FL), lacto-difucotetraose / difucosyl lactose (DFL), 3'-O-sialyl lactose (3-SL), 6'-O-sialyl lactose (6-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), or any combination thereof.

[0153] The prebiotics may include at least one prebiotic oligosaccharide selected from the group consisting of 3FL, 3'SL, LNnT, lacto-N-fucopentaose (LNFP I), LNFP II, LNFP III, sialyl-lacto-N-tetraose (LST)b, LSTc, dicialyl lacto-N-tetraose (DSLNT), fucosyl lacto-N-hexaose (FLNH), difucosyl lacto-N-hexaose (DFLNH), and dicialyl lacto-N-hexaose (DSLNH), or any combination thereof.

[0154] The prebiotics may include 34% to 85% by weight of 2'-FL, 10% to 40% by weight of LNT, 4% to 14% by weight of DFL, and a combination of 9% to 31% by weight of 3-SL and 6-SL.

[0155] In some embodiments, prebiotics are 26% to 65% by weight, preferably 32% to 54% by weight of 2'-FL, LNT in an amount of 10% to 40% by weight, preferably about 11% to about 20% by weight, 4% to 14% by weight, preferably 4% to 8% by weight of DFL, A combination of 3'-SL and 6'-SL in an amount of 9% to 31% by weight, preferably 8% to 22% by weight, It contains 12% to 38% by weight, preferably 17% to 31% by weight, of 3-FL.

[0156] The prebiotics may contain 2'-FL in amounts of 0.001 g / L to 12 g / L, preferably 0.002 g / L to 10 g / L, and more preferably 0.005 g / L to 5 g / L.

[0157] The prebiotics may contain 0.001 g / L to 5 g / L of DFL, preferably 0.002 g / L to 4 g / L of DFL, and more preferably 4 g / L to 3 g / L of DFL.

[0158] The prebiotics may contain LNT in a concentration of 0.01 g / L to 6 g / L, preferably 0.025 g / L to 5 g / L, and more preferably 0.05 g / L to 1 g / L.

[0159] The prebiotics may contain 6'-SL in concentrations of 0.001 g / L to 2 g / L, preferably 0.002 g / L to 1.5 g / L, and more preferably 0.005 g / L to 1 g / L.

[0160] The prebiotics may contain 3'-SL at a concentration of 0.01 g / L to 2 g / L, preferably 0.025 g / L to 1.5 g / L, and more preferably 0.05 g / L to 1 g / L.

[0161] The prebiotics may contain 0.01 g / L to 7 g / L of 3-FL, preferably 0.025 g / L to 6 g / L of 3-FL, and more preferably 0.05 g / L to 5 g / L of 3-FL.

[0162] Suitablely, the oligosaccharide mixture comprises or consists of 2'-fucosyl lactose (2'FL), difucosyl lactose (diFL), lacto-N-tetraose (LNT), and lacto-N-neotetraose. In some embodiments, the oligosaccharide mixture comprises or consists of 3'-sialyl lactose (3'-SL), 6'-sialyl lactose (6'-SL), 2'-fucosyl lactose (2'FL), difucosyl lactose (diFL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT).

[0163] In some embodiments, the mixture of oligosaccharides is At least one sialylated oligosaccharide in an amount of 10-35% by weight, preferably 10-30% by weight, and more preferably 10-25% by weight, relative to the total weight of the oligosaccharide mixture. At least one fucosylated oligosaccharide in an amount of 30-80% by weight, preferably 40-80% by weight, more preferably 50-70% by weight, relative to the total weight of the oligosaccharide mixture, and / or The oligosaccharide mixture contains at least one N-acetylated oligosaccharide in an amount of 10-35% by weight, preferably 15-30% by weight, and more preferably 15-20% by weight, relative to the total weight of the oligosaccharide mixture.

[0164] The present invention provides HMOs or combinations of HMOs for use in preventing and / or treating growth retardation in infants or young children. Preferably, the HMOs increase B. pseudocatenulatum and / or S. thermophilus in the gut microbiota of infants or young children.

[0165] Appropriately, HMOs may be provided in compositions as defined herein. These compositions may be intended for use in preventing and / or treating growth retardation in infants or young children. The compositions may further comprise probiotics such as the B. pseudocatenulatum microorganism described herein.

[0166] HMOs have been shown to increase the abundance of B. pseudocatenulatum in the infant microbiome (see, e.g., Cheema et al.; Int J Mol Sci; 2022; 23(5): 2804 and this example). Levels of various HMOs in breast milk have also been reported to be associated with infant growth, including body length / height (Samuel TM, et al. Sci Rep. 2022).

[0167] Microbiota and microbiome The term "intestinal microbiota" can refer to the composition of microorganisms (including bacteria, archaea, and fungi) that inhabit the digestive tract.

[0168] The term "gut microbiome" can encompass both the "gut microflora" and their "theatre of activity," which may include their structural elements (nucleic acids, proteins, lipids, polysaccharides), metabolites (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).

[0169] Therefore, in this invention, the term "intestinal microbiome" can be used interchangeably with the term "intestinal microflora."

[0170] method The present invention further provides a method for predicting or evaluating whether an infant or toddler is at risk of growth retardation, comprising measuring the level of Bifidobacterium pseudocatenulatum in one or more samples obtained from the infant or toddler.

[0171] While not bound by theory, the inventors concluded that infants or young children with low levels (e.g., abundance and / or activity) of Bifidobacterium pseudocatenulatum in their microbiome are at increased risk of growth inhibition.

[0172] Levels of Bifidobacterium pseudocatenulatum may be compared to reference levels, which indicate the predicted risk of growth retardation in infants or children. The term reference level is synonymous with “control level” and broadly includes data used by those skilled in the art to facilitate the accurate interpretation of technical data.

[0173] Reference values ​​may be based on the values ​​(e.g., mean) of Bifidobacterium pseudocatenulatum in a population of infants and / or toddlers who are at risk of growth retardation or are known to have growth retardation. Reference values ​​may be based on the values ​​(e.g., mean) of Bifidobacterium pseudocatenulatum in a population of infants and / or toddlers who are not at risk of growth retardation or are known not to have growth retardation. Reference values ​​may be based on the values ​​(e.g., mean) of Bifidobacterium pseudocatenulatum in a population of infants and / or toddlers who are not at risk of growth retardation or are known to have growth retardation.

[0174] The reference level may be matched to the age of the test sample.

[0175] Appropriately, infants or toddlers may be approximately 1–60 months old, 1–48 months old, 2–60 months old, 2–48 months old, 2–36 months old, or 4–36 months old, 6–36 months old, or 6–24 months old.

[0176] Preferably, the infant or toddler may be at least 10 months old.

[0177] For example, an infant or toddler may be at least about 10 months old, at least about 12 months old, at least about 14 months old, at least about 16 months old, at least about 20 months old, or at least about 24 months old.

[0178] Preferably, the infant or toddler may be approximately 10 to 48 months old, approximately 10 to 36 months old, approximately 10 to 24 months old, or approximately 10 to 18 months old.

[0179] This method is typically performed on the outside of a human or animal body, for example, on a sample obtained in advance from the subject to be tested. Preferably, the sample is a fecal sample.

[0180] Appropriately, this method provides that a difference between the level of Bifidobacterium pseudocatenulatum in a test sample and a reference level indicates a risk of growth inhibition. Appropriately, this method may provide that a difference between the level of Bifidobacterium pseudocatenulatum in a test sample and a reference level indicates an increased risk of growth inhibition.

[0181] For example, a difference of 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, or 100 times between the level measured in the test sample and the reference level may indicate an increased risk of growth inhibition.

[0182] Appropriately, decreased levels of Bifidobacterium pseudocatenulatum are associated with an increased risk of growth retardation. Appropriately, infants or young children with decreased levels of Bifidobacterium pseudocatenulatum are identified as being at risk of growth retardation.

[0183] For example, levels of Bifidobacterium pseudocatenulatum measured in a test sample that are 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 50 times, or 100 times lower than the reference level may indicate an increased risk of growth inhibition.

[0184] Appropriately, the method of the present invention further includes combining the level of Bifidobacterium pseudocatenulatum with one or more anthropometric measurements.

[0185] Appropriately, infants or young children assessed to be at risk of growth retardation using the method of the present invention may be treated with compositions according to the present invention for reducing the risk of developing growth retardation and / or preventing growth retardation.

[0186] Appropriately, the method of the present invention may include measuring the level of Streptococcus thermophilus as a substitute for Bifidobacterium pseudocatenulatum. Appropriately, the method of the present invention may include measuring the levels of both Bifidobacterium pseudocatenulatum and Streptococcus thermophilus.

[0187] The method of the present invention may be performed on one or more samples obtained from a subject. For example, the method of the present invention may be performed using a first sample obtained at a given point in time and a second sample obtained at a time interval after the first sample was obtained. The method of the present invention may be performed two or more times on samples obtained from the same subject over a period of time. For example, samples may be obtained repeatedly once a month, once a year, or once every two years. [Examples]

[0188] Next, the present invention will be further described by examples, which are provided to help those skilled in the art to carry out the present invention and are not intended to limit the scope of the present invention.

[0189] Example 1: Tests on microbiome and health The microhealth cohort is described by Vidal et al. (https: / / www.medrxiv.org / content / 10.1101 / 19000505v1) and is registered as NCT02361164 on clinicaltrials.gov.

[0190] In short, we collected information on anthropometric measurements, diarrhea and acute respiratory infections (ARI), medication use including antibiotics, breastfeeding status, weaning, nasopharyngeal and fecal pathogen and microbiome profiles, and secretory status (FUT2, FUT3) from n=220 infants or toddlers. Fecal samples for microbiome analysis were collected at birth, 2 months, 6 months, 10 months, 15 months, 18 months, and 24 months of age. Growth outcomes were recorded at birth, 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 15 months, 18 months, and 24 months of age. See Figure 1 for an overview.

[0191] Example 2: Definition of Reference Population and Growth-Deficit Population Based on WHO guidelines, stunted growth was defined by (i) dynamic changes or (ii) static outcomes.

[0192] For dynamic changes, the change in age-specific body length z-score (LAZ) was evaluated over time (6 to 24 months). The inventors identified three patterns: a small change in the LAZ score over time (negligible slope), a decrease in the LAZ score over time (negative slope), and an improvement in the LAZ score over time (positive slope). Infants whose LAZ score change over time, calculated as a slope, was <-0.0485434516523868 were classified as "underdeveloped" (n=48). Infants whose LAZ score change over time, calculated as a slope, was >0.00495716034271725 were designated as the reference population (n=48). In infants aged 6–24 months, the microbiome of infants in the first quartile with delayed growth was compared to that of infants in the fourth quartile with improved growth, based on changes in body length z-score (LAZ).

[0193] For static outcomes, the LAZ score was evaluated at 24 months of age, and "stunted growth" was defined as length_for_age_24m < -2, while length_for_age_24m ≥ -2 was defined as the reference population.

[0194] Example 3: Differences in the microbiome between a stunted population and a control population. Regarding dynamic changes, microbiome data was obtained only for samples corresponding to 6–24 months. First, bacterial species with a relative abundance of less than 0.01% on average were excluded. Thus, 168 out of approximately 750 species remained after this step. Next, using the nearZeroVar function of mixOmics Rpackage, with parameters -freqCut=95 / 05 and uniqueCut=20, features with near-zero variance were removed. After this step, 78 species remained (see Figure 2).

[0195] As defined above, to determine whether there were differences in the overall microbiome between two groups, the "reference" group and the "underdeveloped" group, the inventors used the machine learning algorithm sPLS-DA (Le Cao KA, et al. BMC Bioinformatics. 2011). Feature selection to find bacteria showing differences was performed in M-fold mode with 10 divisions and 10 iterations, using centroid distance measures to maintain a balanced error rate between the two classes. To identify time intervals with differing feature abundance in the metagenomic longitudinal study, the inventors used the R package MetaLonDA (Metwally AA, et al. Microbiome. 2018). MetaLonDA was run in "screening mode" with 100 permutations. The hits thus identified were confirmed by rerunning MetaLonDA with the recommended 1000 permutations.

[0196] For static outcomes, the same technical methods as described above were used to identify bacteria that were significantly different between the two groups over time (length_for_age_24m: <-2 is defined as "poor development"; ≥-2 is defined as "reference") (see Figures 3 and 4).

[0197] Multiple algorithms were used to identify common hits associated with dynamic changes in age-specific body length z-scores (LAZ) (6–24 months) and static measures of laz <-2 or ≥-2 (at 24 months). Based on multiple pieces of evidence, B. pseudocatenulatum and S. thermophilus were identified as bacterial signatures of inadequate height profiles up to 24 months. Example 4: Effects of galactooligosaccharides and HMOs on B. pseudocatenulatum in the infant microbiome Using ExVivo's D-SIFR® technology, the gut microbiota of three 3-year-old children was simulated, and samples were tested using combinations of galactooligosaccharides (GOS), HMO1 (2'FL and diFL), and / or HMO2 (LNnT and 6'SL).

[0198] Treatment with GOS, HMO1, and / or HMO2 has been found to have a significant bifidogenic effect, including increasing the levels of B. pseudocatenulatum. For example, treatment with HMO(+ / -GOS) increases the level of B. pseudocatenulatum by at least twofold. Materials and methods Fecal samples were collected according to procedures approved by the Ethics Committee of Ghent University Hospital.

[0199] Colonic fermentation of test products by the intestinal microbiota in fecal samples was evaluated 24 hours after inoculation.

[0200] Repeated measures ANOVA analysis was performed to statistically evaluate the therapeutic effects on basic fermentation parameters, cell count, microbial diversity, and the microbial composition (at the phylum level) of samples from three infants (based on paired t-tests, thus considering that the values ​​are comparisons between samples from a given identical donor). The statistical significance of possible therapeutic effects was tested by the Benjamini-Hochberg post-hoc test.

[0201] For quantitative shallow shotgun sequencing, a standardized Illumina library was prepared during DNA extraction, followed by 3M total DNA sequencing.

[0202] All publications referenced in the above specification are incorporated herein by reference. Various modifications and changes to the methods, compositions and uses disclosed in connection with the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the form disclosed for carrying out the present invention will be obvious to those skilled in the art and are intended to fall within the scope of the following claims.

[0203] Embodiment Various preferred features and embodiments of the present invention are described below with reference to the numbered paragraphs.

[0204] 1. A method for identifying infants or toddlers at risk of growth retardation, comprising measuring the amount of Bifidobacterium pseudocatenulatum and / or Streptococcus thermophilus present in one or more samples obtained from the infant.

[0205] 2.B. The method described in paragraph 1, in which infants or toddlers with reduced levels of pseudocatenulatum are identified as being at risk of growth retardation.

[0206] 3. The method according to paragraph 1 or 2, further comprising measuring the amount of Bifidobacterium pseudocatenulatum and Streptococcus thermophilus present in one or more samples obtained from the infant.

[0207] 4. Infants or toddlers with reduced levels of S. thermophilus are identified as being at risk of growth retardation, as described in paragraph 3.

[0208] 5. The method according to any one of paragraphs 1 to 4, wherein the growth inhibition is related to malnutrition.

[0209] 6. The method according to any one of paragraphs 1 to 5, wherein the growth inhibition is inhibition of height or body length growth.

[0210] 7. The method according to paragraph 6, wherein the inhibition of height or body length growth is defined as a decrease over time in the age-specific body length z-score (LAZ) or a decrease over time in the LAZ, or a decrease over time in the age-specific height z-score (HAZ) or a decrease over time in the HAZ.

[0211] 8. A composition for use in preventing and / or treating growth retardation in an infant or young child, wherein the composition promotes the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota of the infant or young child.

[0212] 9. The composition for use as described in paragraph 8, wherein the composition promotes the growth of B. pseudocatenulatum in the intestinal microbiota of the infant or young child, and optionally the composition contains B. pseudocatenulatum microorganisms.

[0213] 10. The composition for use as described in paragraph 8 or 9, wherein the composition increases S. thermophilus in the intestinal microbiota of the infant or toddler, and optionally the composition contains S. thermophilus microorganisms.

[0214] 11. The composition for use as described in paragraph 9 or 10, wherein the composition is administered in combination with a prebiotic.

[0215] 12. The composition for use as described in paragraph 8, wherein the composition comprises a prebiotic.

[0216] 13. The composition for use as described in paragraph 12, wherein the composition is administered in combination with B. pseudocatenulatum and / or the microorganism Streptococcus thermophilus.

[0217] 14. A combination of B. pseudocatenulatum and / or Streptococcus thermophilus microorganisms and prebiotics for use in preventing and / or treating growth retardation in infants or young children.

[0218] 15. A composition or combination for use according to any one of paragraphs 11 to 14, wherein the prebiotic is in the form of a dietary composition or a nutritional composition.

[0219] 16. A composition or combination for use according to any one of paragraphs 11 to 15, wherein the prebiotic comprises human milk oligosaccharides (HMOs).

[0220] 17. Human milk oligosaccharides (HMOs) or combinations of HMOs for use in preventing and / or treating growth retardation in infants or young children, wherein the HMOs or combinations of HMOs promote the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota of the infant or young child.

[0221] 18. The composition or combination for use described in paragraph 16, or the HMO or combination of HMOs for use described in paragraph 17, wherein the HMO is selected from the group consisting of 2'-FL, 3-FL, di-FL, 3'-SL, 6'-SL, LNT, and LNnT, and any combination thereof, and preferably the HMO is (i) a combination of 2'-FL and di-FL, or (ii) a combination of 6'-SL and LNnT.

[0222] 19. Microorganism B. pseudocatenulatum for use in the prevention and / or treatment of growth retardation in infants or young children.

[0223] 20. Compositions, combinations, HMOs, or microorganisms of B. pseudocatenulatum and / or Streptococcus thermophilus for use according to any one of paragraphs 8 to 19, wherein the infant or toddler is assessed to have reduced levels of B. pseudocatenulatum in the intestinal microbiota.

[0224] 21. Compositions, combinations, HMOs, or microorganisms of B. pseudocatenulatum and / or Streptococcus thermophilus for use as described in any one of paragraphs 8 to 19, for which the infant or toddler is assessed to be at risk of growth inhibition by the method described in any one of paragraphs 1 to 7.

[0225] 22. Compositions, combinations, HMOs, or microorganisms of B. pseudocatenulatum and / or Streptococcus thermophilus for use as described in any one of paragraphs 8 to 21, wherein the growth inhibition is associated with malnutrition.

[0226] 23. Compositions, combinations, HMOs, or microorganisms for use according to any one of paragraphs 8 to 22, wherein the growth inhibition is inhibition of height or body length growth.

[0227] 24. Compositions, combinations, HMOs, or microorganisms for use described in paragraph 23, in which the inhibition of height or body length growth is defined as a decrease over time in the age-specific body length z-score (LAZ) or LAZ, or a decrease over time in the age-specific height z-score (HAZ) or HAZ.

[0228] 25. Compositions, combinations, HMOs, or microorganisms of B. pseudocatenulatum and / or Streptococcus thermophilus for use as described in any one of paragraphs 8 to 24, which enhance bone development and / or bone strength in the subject.

[0229] 26. A method for preventing and / or treating growth retardation in an infant or young child, wherein the method comprises administering to the infant or young child a composition that promotes the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota.

[0230] 27. Use of compositions to regulate the abundance of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestines of infants or young children.

[0231] 28. The use according to paragraph 27, wherein the composition is a composition defined in any one of paragraphs 6 to 18.

[0232] 29. Use of HMOs or combinations of HMOs to regulate the abundance of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestines of infants or young children.

[0233] 31. The method, composition for use, combination for use, or use described in any one of paragraphs 1 to 30, wherein the infant or toddler is less than approximately 60 months of age, preferably less than 36 months of age, preferably less than 24 months of age, preferably between approximately 6 months and approximately 24 months of age.

[0234] 32. The method, composition for use, combination for use, or use according to any one of paragraphs 1 to 31, wherein the microorganism is B. pseudocatenulatum.

[0235] 33.B. A probiotic composition containing Pseudocatenulatum.

[0236] 34. The probiotic composition according to paragraph 31, wherein the probiotic composition further comprises S. thermophilus.

[0237] 35.B. A synbiotic composition containing Pseudocatenulatum and prebiotics.

[0238] 36. The synbiotic composition according to paragraph 33, wherein the prebiotic promotes the growth of B. pseudocatenulatum in the intestinal microbiota of an infant or young child.

[0239] 37. The synbiotic composition according to paragraph 35 or 36, wherein the prebiotic is as defined in any one of paragraphs 15 to 18.

Claims

1. A method for identifying infants or young children at risk of growth retardation, comprising measuring the amount of Bifidobacterium pseudocatenulatum and / or Streptococcus thermophilus in one or more samples obtained from the infant.

2. B. The method according to claim 1, wherein an infant or toddler with reduced levels of pseudocatenulatum is identified as being at risk of growth retardation.

3. The method according to claim 1 or 2, wherein the growth inhibition is an inhibition of height or body length growth, and optionally, the growth inhibition of height or body length is defined as a decrease in age-specific body length z-score (LAZ) or a decrease in LAZ over time, or a decrease in age-specific height z-score (HAZ) or a decrease in HAZ over time.

4. A composition for use in preventing and / or treating growth retardation in infants or young children, wherein the composition promotes the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota of the infant or young child.

5. The composition for use according to claim 4, wherein the composition promotes the growth of B. pseudocatenulatum in the intestinal microbiota of the infant or toddler, and optionally the composition comprises B. pseudocatenulatum microorganisms.

6. The composition for use according to claim 4, wherein the composition comprises a prebiotic.

7. A combination of B. pseudocatenulatum and / or Streptococcus thermophilus microorganisms and prebiotics for use in preventing and / or treating growth retardation in infants or young children.

8. The composition or combination for use according to claim 6 or 7, wherein the prebiotic is in the form of a dietary composition or a nutritional composition.

9. The composition or combination for use according to claim 8, wherein the prebiotic comprises human milk oligosaccharide (HMO).

10. Human milk oligosaccharides (HMOs) or combinations of HMOs for use in preventing and / or treating growth retardation in infants or young children, wherein the HMOs or combinations of HMOs promote the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota of the infant or young child.

11. B. Pseudocatenulatum and / or Streptococcus thermophilus microorganisms for use in preventing and / or treating growth retardation in infants or young children.

12. A composition, combination, HMO, or microorganism B. pseudocatenulatum and / or Streptococcus thermophilus for use according to any one of claims 4 to 11, wherein the infant or toddler is assessed to be at risk of growth inhibition by the method according to any one of claims 1 to 3.

13. A composition, combination, HMO, or microorganism for use according to any one of claims 4 to 12, wherein the growth inhibition is inhibition of height or body length growth, and optionally, the growth inhibition of height or body length growth is defined as a decrease in age-specific body length z-score (LAZ) or a decrease in LAZ over time, or a decrease in age-specific height z-score (HAZ) or a decrease in HAZ over time.

14. A composition, combination, HMO, or microorganism for use according to any one of claims 4 to 13, wherein bone development and / or bone strength are enhanced in the subject.

15. A synbiotic composition comprising B. pseudocatenulatum and / or Streptococcus thermophilus and a prebiotic, wherein the prebiotic is an HMO that promotes the growth of B. pseudocatenulatum and / or Streptococcus thermophilus in the intestinal microbiota of infants or young children.

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