Use of microbiota modulators for enhancing bone development and / or bone strength

Abstract

The present invention relates to the use of a microbiota modulator for enhancing bone development and / or bone strength wherein the microbiota modulator modulates the abundance of one or more bacterial classification populations in the intestinal tract of a subject, the classified population of one or more bacteria is selected from the group consisting of Enterobacter butyricum, Selicobacter sanguineus, Paraprevor claralatus and Amino Cercaceae.

Description

Technical Field

[0001] The present invention relates to the use of a microbiota modulator for enhancing bone development and / or bone strength in a subject. Background Art

[0002] Bone is a dynamic tissue that undergoes remodeling, a lifelong process involving resorption (the breakdown of old bone) and ossification, which is key to shaping the skeleton and repairing fractures. Ossification, or osteogenesis, is the process by which bone is formed by osteoblasts.

[0003] Bone growth during infancy is a key parameter regarding bone strength during human life. Ossification begins approximately six weeks after fertilization in the embryo. Prior to this time, the embryonic skeleton consists entirely of fibrous membranes and hyaline cartilage. Bone growth continues until approximately 25 years of age. Bones can grow in thickness throughout life, but after 25 years of age, ossification plays a major role in bone remodeling and repair.

[0004] Bone strength is the result of the combined effects of several parameters involved in bone growth. In this regard, not only factors such as age, gender, position in the body, mineral content, disease, etc. have an impact on bone strength, but also trabecular architecture (the form and orientation of trabeculae) and cortical microarchitecture (especially with regard to the redistribution of porosity) play an important role in bone health and strength.

[0005] Bone is composed of cortical bone (or compact bone) and trabecular bone (or cancellous bone). Cortical bone accounts for about 80% of the body's bone mass and has a lower surface area than trabecular bone due to its lower porosity. Trabecular bone is located at the ends of long bones and accounts for about 20% of the total mass of the skeleton.

[0006] Breastfeeding is considered an ideal source of nutrition and is the first choice for feeding infants up to at least 6 months of age. Therefore, human milk (HM) has long been considered a design model for infant formula (IF). Even though many improvements have been made in the nutrient composition of IF over the past few decades, there are still huge differences in composition and the functional benefits brought about by HM. Therefore, with regard to bone metabolism, for example, lower bone mineral density can be observed in infants fed with formula compared to infants fed with breast milk.

[0007] Therefore, new methods of enhancing bone development and / or bone strength are needed, especially in formula-fed infants. Summary of the invention

[0008] It has now been unexpectedly discovered that bone development and / or bone strength in a subject is associated with the abundance of Intestinimonas butyriciproducens, Turicibacter sanguinis, Paraprevotella clara, and Acidaminococcaceae in the subject's intestine. Specifically, it has been unexpectedly discovered that the abundance of Enterobacter butyriciproducens and Turicibacter sanguinis is positively correlated with bone outcomes, and that the abundance of Paraprevotella clara and Acidaminococcaceae is negatively correlated with bone outcomes.

[0009] In one aspect, the present invention provides a microbiota modulator for modulating the abundance of one or more bacterial taxa in the intestine of a subject, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0010] The microbiota modulator can increase the abundance of Enterobacter butyricum and / or Zurichia coli in the intestinal tract of the subject, and / or the microbiota modulator can reduce the abundance of Paraprevotella clara and / or Aminaminococcaceae in the intestinal tract of the subject. The microbiota modulator can increase the abundance of Enterobacter butyricum and Zurichia coli in the intestinal tract of the subject, and / or the microbiota modulator can reduce the abundance of Paraprevotella clara and Aminaminococcaceae in the intestinal tract of the subject.

[0011] In some embodiments, the microbiota modulator is Enterobacter butyricum and / or Zurichia coli. In some embodiments, the microbiota modulator is a probiotic comprising or consisting of Enterobacter butyricum and / or Zurichia coli. Enterobacter butyricum and / or Zurichia coli can be administered at a dose of 1E6 cfu / day to 1E10 cfu / day.

[0012] In some embodiments, the microbiota modulator is a mixture of oligosaccharides, the oligosaccharides comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide. In some embodiments, the microbiota modulator is a prebiotic, the prebiotic comprising or consisting of at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide.

[0013] In some embodiments, the microbiota regulator is a combination of a mixture of oligosaccharides and Enterobacter butyricum and / or Zurichia coli, wherein the mixture of oligosaccharides comprises at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide. The mixture of oligosaccharides and Enterobacter butyricum and / or Zurichia coli can be administered individually, simultaneously or sequentially. In some embodiments, the mixture of oligosaccharides and Enterobacter butyricum and / or Zurichia coli are administered simultaneously. In some embodiments, the microbiota regulator is a synbiotic comprising: (i) Enterobacter butyricum and / or Zurichia coli; and / or (ii) a mixture of oligosaccharides, wherein the mixture of oligosaccharides comprises at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide, or consists of them.

[0014] In some embodiments, the mixture of oligosaccharides includes at least one sialylated oligosaccharide. At least one sialylated oligosaccharide can be selected from the group consisting of: 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetrasaccharide b (LSTb), sialyllactose-N-tetrasaccharide c (LSTc), disialyl lactose-N-tetrasaccharide and their combination. In some embodiments, at least one sialylated oligosaccharide is selected from 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL) and their combination. In some embodiments, the mixture of oligosaccharides includes 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) or consists of them.

[0015] In a preferred embodiment, the mixture of oligosaccharides comprises at least one fucosylated oligosaccharide. The at least one fucosylated oligosaccharide may be selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL) (also known as 2',3-difucosyllactose (LDFT)), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II ...I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N In some embodiments, the at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'FL), difucosyllactose (diFL), and combinations thereof. In some embodiments, the mixture of oligosaccharides comprises or consists of 2'-fucosyllactose (2'FL) and difucosyllactose (diFL).

[0016] In a preferred embodiment, the mixture of oligosaccharides comprises at least one N-acetylated oligosaccharide. At least one N-acetylated oligosaccharide can be selected from the group consisting of: N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT) and a combination thereof. In some embodiments, at least one N-acetylated oligosaccharide is selected from lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT) and a combination thereof. In some embodiments, the mixture of oligosaccharides comprises lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) or is composed of them.

[0017] In a preferred embodiment, the mixture of oligosaccharides comprises or consists of 2'-fucosyllactose (2'FL), difucosyllactose (diFL), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT). In some embodiments, the mixture of oligosaccharides comprises or consists of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 2'-fucosyllactose (2'-FL), difucosyllactose (diFL), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0018] In some embodiments, the mixture of oligosaccharides comprises:

[0019] - 10% to 35% by weight, preferably 10% to 30% by weight, more preferably 10% to 25% by weight, of at least one sialylated oligosaccharide relative to the total weight of the oligosaccharide mixture;

[0020] - 30% to 80% by weight, preferably 40% to 80% by weight, more preferably 50% to 70% by weight of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture; and / or

[0021] 10 to 35 wt.-%, preferably 15 to 30 wt.-%, more preferably 15 to 20 wt.-%, of at least one N-acetylated oligosaccharide, relative to the total weight of the oligosaccharide mixture.

[0022] The microbiota modulator may be provided in any suitable form. The microbiota modulator may be in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement.

[0023] The microbiota modulator may be in the form of a nutritional composition, more preferably a synthetic nutritional composition.

[0024] In a preferred embodiment, the microbiota modulator is in the form of an infant formula. In this case, the infant formula may be a preterm infant formula, a human milk fortifier, a first infant formula, an follow-on formula, a baby food formula, an infant cereal formula or a growing-up milk.

[0025] The microbiota modulator may be in the form of a supplement. The supplement may be used, for example, for premature infants or children or adults.

[0026] In a preferred embodiment, the microbiota modulator is in a form for feeding premature infants, such as premature infant formula, human milk fortifier or supplement. The microbiota modulator can also be in a form for children or adults, such as yogurt or medical food, and pet food, especially young pet food.

[0027] Microbiota modulators may be used before the weaning period and / or during the weaning period and / or after the weaning period. Microbiota modulators may be used during hospitalization and / or after discharge.

[0028] In some embodiments, the microbiota modulator is in the form of a composition (e.g., an infant formula) comprising:

[0029] - 0.01 to 2% by weight, preferably 0.05 to 1.5% by weight, most preferably 0.07 to 1% by weight of at least one sialylated oligosaccharide;

[0030] - 0.05 to 3 wt. %, preferably 0.1 to 2 wt. %, most preferably 0.2 to 1.5 wt. % of at least one fucosylated oligosaccharide; and / or

[0031] - 0.01 to 1 wt.-%, preferably 0.03 to 0.6 wt.-%, most preferably 0.05 to 0.5 wt.-% of at least one N-acetylated oligosaccharide.

[0032] Weight % may refer to the total weight of the composition in powder form.

[0033] In one aspect, the present invention provides a microbiota modulator for enhancing bone development and / or bone strength in a subject, wherein the microbiota modulator modulates the abundance of one or more bacterial taxa in the intestine of the subject, the one or more bacterial taxa selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0034] In one aspect, the present invention provides a method for enhancing bone development and / or bone strength in a subject, the method comprising administering a therapeutically effective amount of a microbiota modulator to a subject in need thereof, wherein the microbiota modulator modulates the abundance of one or more bacterial taxa in the intestine of the subject, the one or more bacterial taxa selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0035] The microbiota modulator can be any microbiota modulator for modulating the abundance of one or more bacterial taxa in the intestinal tract of a subject as described herein, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0036] In some embodiments, the subject is a human or an animal. In preferred embodiments, the subject is a human. In some embodiments, the subject is a teenager, a teenager, a child, a toddler, or an infant. In preferred embodiments, the subject is an infant. In other embodiments, the subject is an adult.

[0037] Enhancing bone development and / or bone strength may include at least one of the following physiological processes: bone catch-up growth, bone mass gain, peak bone mass optimization, promoting bone formation, promoting bone anabolism, increasing bone mineral density and microarchitecture, regulating bone biomechanical properties, regulating the rate of bone formation and / or bone resorption, assisting bone regeneration during fracture healing, and regulating the bone resorption process.

[0038] The microbiota modulator may increase one or more bone parameters selected from the group consisting of bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct. BV), and bone ultimate force (FMax). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is shown that B. sanguinis is positively correlated with bone strength (biomechanics) and that HMO blends increase its relative abundance. (A) shows that the relative abundance of B. sanguinis is positively correlated with bone strength. (B) shows that HMO blend A, HMO blend B, and HMO blend C each increase the relative abundance of B. sanguinis compared to lactose.

[0040] Figure 2 P. clara is shown to be negatively correlated with bone microarchitecture (connectivity) and that HMO blends reduce its relative abundance. (A) shows that the relative abundance of P. clara is negatively correlated with trabecular architecture. (B) shows that HMO blend A, HMO blend B, and HMO blend C each reduce the relative abundance of P. clara compared to lactose. DETAILED DESCRIPTION

[0041] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will appreciate that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0042] Any reference to prior art documents in this specification cannot be regarded as an admission that such prior art is well-known technology or forms part of the common general knowledge in the field.All publications mentioned in the specification are incorporated herein by reference.

[0043] As used in this specification, the words "include", "comprising" and similar words should not be understood to have an exclusive or exhaustive meaning. In other words, they are intended to mean "including, but not limited to". The term "include / comprising" and similar words also include the term "consisting of..."

[0044] Unless otherwise indicated, the practice of the present invention will adopt conventional techniques within the capabilities of those of ordinary skill in the art. Such techniques are described in the literature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0045] Numerical ranges are inclusive of the numbers defining the range and all percentages disclosed herein are on a w / w basis unless otherwise stated.

[0046] Microbiome modulators

[0047] In one aspect, the present invention provides a microbiota modulator for modulating the abundance of one or more bacterial taxa in the intestine of a subject, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0048] As used herein, the term "microbiota modulator" or "intestinal microbiota modulator" may refer to any agent that modulates the intestinal microbiota of a subject. Examples of such agents include probiotics, prebiotics, postbiotics, antibiotics, and fecal microbiota transplants. Suitably, the microbiota modulator is in the form of a probiotic, a prebiotic, and / or a synbiotic. Suitably, the microbiota modulator of the present invention comprises or consists of a probiotic, a prebiotic, and / or a synbiotic.

[0049] In some embodiments, the microbiota regulator of the present invention is in the form of a probiotic. In some embodiments, the microbiota regulator of the present invention comprises or consists of a probiotic. As used herein, the term "probiotic" may refer to a component containing a sufficient number of live microorganisms to change the intestinal microbiota of a subject (see, e.g., Hill, C. et al., 2014. Nature reviews Gastroenterology & hepatology, 11 (8), p. 506).

[0050] In some embodiments, the microbiota regulator of the present invention is in the form of a prebiotic. In some embodiments, the microbiota regulator of the present invention comprises or consists of a prebiotic. As used herein, the term "prebiotic" may refer to a non-digestible component that benefits a subject by selectively stimulating the favorable growth and / or activity of one or more bacterial taxa (see, e.g., Gibson, GR et al., 2017. Exemplary prebiotics include oligosaccharides, such as human milk oligosaccharides.

[0051] In some embodiments, the microbiota regulator of the present invention is in the form of a synbiotic. In some embodiments, the microbiota regulator of the present invention comprises a synbiotic or consists of it. As used herein, the term "synbiotic" may refer to a component containing both probiotics and prebiotics (see, for example, Swanson, KS et al., 2020. Nature Reviews Gastroenterology & Hepatology, 17 (11), pp. 687-701).

[0052] Enterobacter butyricigenes and Haemophilus zurichiensis

[0053] The microbiota modulator may increase the abundance of Enterobacter butyricum and / or S. zurichiensis in the intestine of the subject.

[0054] In some embodiments, the microbiota modulator is Enterobacter butyricum and / or Zurichia coli. In some embodiments, the microbiota modulator comprises or consists of Enterobacter butyricum and / or Zurichia coli.

[0055] In some embodiments, the microbiota regulator increases the abundance of Enterobacter butyricum in the intestinal tract of the subject. In some embodiments, the microbiota regulator is Enterobacter butyricum. In some embodiments, the microbiota regulator comprises or consists of Enterobacter butyricum. Enterobacter butyricum is a species that produces butyric acid that is usually present in the intestinal tract (see, for example, Bui, TPN et al., 2016. Environmental microbiology reports, 8 (6), pp. 1024-1037). Any suitable strain of Enterobacter butyricum can be used. Enterobacter butyricum can be cultured in a conventional manner. For example, Enterobacter butyricum strain AF211 can be cultured in an anaerobic enhanced Clostridium medium at 37 ° C in a gas phase of N2 / CO2 (80: 20, v / v) (see Bui, TPN et al., 2016. Environmental microbiology reports, 8 (6), pp. 1024-1037).

[0056] In some embodiments, the microbiota modulator increases the abundance of Bacillus thuringiensis in the intestine of the subject. In some embodiments, the microbiota modulator is Bacillus thuringiensis. In some embodiments, the microbiota modulator comprises or consists of Bacillus thuringiensis. Bacillus thuringiensis is commonly found in the intestinal tract and is capable of interacting with compounds derived from the host (see, e.g., Cuív, PO et al., 2011. Journal of bacteriology, 193 (5), pp. 1288-1289). Any suitable strain of Bacillus thuringiensis can be used. Bacillus thuringiensis can be cultured in a conventional manner. For example, Bacillus thuringiensis strain MOL361 T It can be cultured on different standard media under anaerobic and aerobic conditions (see Bosshard, PP et al., 2002. International journal of systematic and evolutionary microbiology, 52(4), pp. 1263-1266).

[0057] The microbiota modulator may provide any suitable amount of Enterobacter butyricigenicus and / or Zürichia coli.

[0058] Suitably, Enterobacter butyricum and / or Zurichia coli are administered at a dose of at least 1E5 cfu / day, at least 1E6 cfu / day, at least 1E7 cfu / day, at least 1E8 cfu / day, at least 1E9 cfu / day or at least 1E10 cfu / day. In some embodiments, Enterobacter butyricum and / or Zurichia coli are administered at a dose of 1E5 cfu / day to 1E10 cfu / day or 1E6 cfu / day to 1E10 cfu / day.

[0059] Suitably, the microbiota modulator comprises or consists of Enterobacter butyricum and / or Zurichia coli in an amount of at least 1E4 cfu / ml, at least 1E5 cfu / ml, at least 1E6 cfu / ml, at least 1E7 cfu / ml, at least 1E8 cfu / ml or at least 1E9 cfu / ml cfu / ml. In some embodiments, the microbiota modulator comprises or consists of Enterobacter butyricum and / or Zurichia coli in an amount of 1E4 cfu / ml to 1E9 cfu / ml or 1E5 cfu / ml to 1E9 cfu / ml.

[0060] Suitably, the microbiota modulator comprises butyric acid-producing Enterobacter and / or Zurichia coli in an amount of at least 1E4 cfu / g, at least 1E5 cfu / g, at least 1E6 cfu / g, at least 1E7 cfu / g, at least 1E8 cfu / g or at least 1E9 cfu / g. In some embodiments, the microbiota modulator comprises or consists of butyric acid-producing Enterobacter and / or Zurichia coli in an amount of 1E4 cfu / g to 1E9 cfu / g or 1E5 cfu / g to 1E9 cfu / g.

[0061] Paraprevotella clara and Acidaminococcaceae

[0062] Microbiota modulators can reduce the abundance of Paraprevotella clara and / or Acidaminococcaceae in the intestines of subjects.

[0063] In some embodiments, the microbiota modulator reduces the abundance of Paraprevotella clara in the intestinal tract of the subject. Paraprevotella clara is a member of the Prevotellaceae family isolated from human feces (see, e.g., Morotomi, M. et al., 2009. International Journal of Systematic and Evolutionary Microbiology, 59(8), pp. 1895-1900).

[0064] In some embodiments, the microbiota regulator reduces the abundance of the Acidaminococcus family in the intestinal tract of the subject. The Acidaminococcus family is a genus of bacteria whose members can use amino acids as the sole energy source for growth and have been isolated from human intestinal samples (see, e.g., Ricaboni, D. et al., 2017. New Microbes and New Infections, 15, pp. 46-48). Species of the genus Acidaminococcus include Acidaminococcus intestini and Acidaminococcus fermentans.

[0065] Oligosaccharide mixture

[0066] In some embodiments, the microbiota modulator is a mixture of oligosaccharides. In some embodiments, the microbiota modulator comprises or consists of a mixture of oligosaccharides.

[0067] Oligosaccharides are one of the best-known prebiotics. The oligosaccharide may be any suitable oligosaccharide for modulating the abundance of one or more bacterial taxa in the intestinal tract of a subject, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0068] The oligosaccharide may be a human milk oligosaccharide (HMO). Many different types of HMO are found in human milk and are generally based on a combination of glucose, galactose, sialic acid (N-acetylneuraminic acid), fucose and / or N-acetylglucosamine with many different bonds between them. Almost all HMOs have a lactose molecule at their reducing end, and the terminal positions of the non-reducing end are occupied by sialic acid and / or fucose (if any). HMOs may be acidic (e.g., oligosaccharides containing charged sialic acid) or neutral (e.g., fucosylated oligosaccharides).

[0069] Suitably, the mixture of oligosaccharides may comprise at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide. In some embodiments, the mixture of oligosaccharides comprises or consists of at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide.

[0070] In some embodiments, the mixture of oligosaccharides comprises 10 to 35 wt%, preferably 10 to 30 wt%, more preferably 10 to 25 wt% of at least one sialylated oligosaccharide relative to the total weight of the oligosaccharide mixture.

[0071] In some embodiments, the mixture of oligosaccharides comprises 30 to 80 wt%, preferably 40 to 80 wt%, more preferably 50 to 70 wt% of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture.

[0072] In some embodiments, the mixture of oligosaccharides comprises 10 to 35 wt%, preferably 15 to 30 wt%, more preferably 15 to 20 wt% of at least one N-acetylated oligosaccharide relative to the total weight of the oligosaccharide mixture.

[0073] In some embodiments, the mixture of oligosaccharides comprises or consists of:

[0074] - 30% to 80% by weight, preferably 40% to 80% by weight, more preferably 50% to 70% by weight, of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture; and

[0075] 10 to 35 wt.-%, preferably 15 to 30 wt.-%, more preferably 15 to 20 wt.-%, of at least one N-acetylated oligosaccharide, relative to the total weight of the oligosaccharide mixture.

[0076] In some embodiments, the mixture of oligosaccharides comprises or consists of:

[0077] - 10% to 35% by weight, preferably 10% to 30% by weight, more preferably 10% to 25% by weight, of at least one sialylated oligosaccharide, relative to the total weight of the oligosaccharide mixture;

[0078] - 30% to 80% by weight, preferably 40% to 80% by weight, more preferably 50% to 70% by weight, of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture; and

[0079] 10 to 35 wt.-%, preferably 15 to 30 wt.-%, more preferably 15 to 20 wt.-%, of at least one N-acetylated oligosaccharide, relative to the total weight of the oligosaccharide mixture.

[0080] Oligosaccharides can be obtained by any suitable method. Suitable methods for synthesizing oligosaccharides are well known to those skilled in the art. For example, methods for preparing oligosaccharides by microbial fermentation, enzymatic methods, chemical synthesis or a combination of these techniques have been developed (see, for example, Zeuner et al., 2019. Molecules, 24 (11), p. 2033).

[0081] Sialyl oligosaccharides

[0082] In some embodiments, the microbiota modulator is a mixture of oligosaccharides comprising or consisting of at least one sialylated oligosaccharide. In some embodiments, the microbiota modulator comprises or consists of a mixture of oligosaccharides comprising or consisting of at least one sialylated oligosaccharide.

[0083] Non-limiting examples of sialylated oligosaccharides include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetraose b (LSTb), sialyllactose-N-tetraose c (LSTc), disialyllactose-N-tetraose, and combinations thereof.

[0084] In a preferred embodiment, the at least one sialylated oligosaccharide is selected from the group consisting of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), and combinations thereof.

[0085] As used herein, the term "3'-sialyllactose" (3'-SL, 3-SL, 3'SL or 3SL) refers to (6R)-5-acetylamino-3,5-dideoxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]-β-L-threo-hex-2-pyranosyl-(2->3)-β-D-galactopyranosyl-(1->4)-D-glucopyranose (IUPAC).

[0086] As used herein, the term "6'-sialyllactose" (6'-SL, 6-SL, 6'SL or 6SL) refers to (6R)-5-acetamido-3,5-dideoxy-6-[(1R,2R)-1,2,3-trihydroxypropyl]-β-L-threo-hex-2-pyranosyl-(2->6)-β-D-galactopyranosyl-(1->4)-D-glucopyranose (IUPAC).

[0087] Sialyl oligosaccharides can be obtained by any suitable method. For example, 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL) can be isolated from natural sources (such as animal milk) using chromatography or filtration techniques. Alternatively, sialyllactose can also be prepared by biotechnology means, by fermentation techniques based on enzymes (recombinant enzymes or natural enzymes), by chemical synthesis or by microbial fermentation techniques, using specific sialyltransferases or sialidases, neuraminidase. In the latter case, the microorganism can express its natural enzymes and substrates, or can be engineered to produce corresponding substrates and enzymes. Single microbial cultures or mixed cultures can be used. Sialyl oligosaccharides can be formed initially with acceptor substrates of any degree of polymerization (DP), starting from DP=1. Alternatively, sialyllactose can be produced by chemical synthesis from lactose and free N'-acetylneuraminic acid (sialic acid). Sialyl lactose can also be obtained commercially from, for example, Kyowa Hakko Kogyo, Japan or Gene Chem, Republic of Korea.

[0088] Fucosylated oligosaccharides

[0089] In some embodiments, the microbiota modulator is a mixture of oligosaccharides comprising or consisting of at least one fucosylated oligosaccharide. In some embodiments, the microbiota modulator comprises or consists of a mixture of oligosaccharides comprising or consisting of at least one fucosylated oligosaccharide.

[0090] Non-limiting examples of fucosylated oligosaccharides include: 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose (such as lactose-N-fucopentose I (LNFP-I), lactose-N-fucopentose II (LNFP-II), lactose-N-fucopentose III (LNFP-III), or lactose-N-fucopentose V (LNFP-V)), lactose-N-fucohexose, lactose-N-difucohexose I, lactose-neofucopentose V (LNFP-V), lactose-N-difucosylhexose-I (LNDFH-I), lactose-N-fucopentose II (LNFP-II), lactose-N-fucopentose III (LNFP-III), or lactose-N-fucopentose V (LNFP-V)). N-neodifucosyllactose-I (LNnDFH), fucosyllactose-N-hexose, fucosyllactose-N-neohexose (such as, fucosyllactose-N-neohexose I, fucosyllactose-N-neohexose II), monofucosyllactose-N-hexose III (MFNLH-III), difucosyllactose-N-hexose I, difucosyllactose-N-neohexose, difucosyllactose-N-neohexose I, difucosyllactose-N-neohexose II, difucosyllactose-N-hexose-a (DFLNHa), fucosyl-p-lactose-N-hexose, trifucosyl-p-lactose-N-hexose I and combinations thereof.

[0091] In a preferred embodiment, the at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'FL), difucosyllactose (diFL), and combinations thereof.

[0092] Fucosylated oligosaccharides can be obtained by any suitable method. For example, 2'FL can be prepared by biotechnological means using specific fucosyltransferases and / or fucosidases, by using enzyme-based (recombinant or natural enzyme) fermentation techniques or microbial fermentation techniques. In the latter case, the microorganism can express its natural enzymes and substrates, or can be engineered to produce the corresponding substrates and enzymes. Alternatively, 2'FL can be produced by chemical synthesis from lactose and free fucose. diFL can be synthesized by enzymes, biotechnology and / or chemical methods.

[0093] N-acetylated oligosaccharides

[0094] In some embodiments, the microbiota modulator is a mixture of oligosaccharides comprising or consisting of at least one N-acetylated oligosaccharide. In some embodiments, the microbiota modulator comprises or consists of a mixture of oligosaccharides comprising or consisting of at least one N-acetylated oligosaccharide.

[0095] Suitably, at least one N-acetylated oligosaccharide is selected from the group consisting of: N-acetyl-glucosamine, N-acetyl-galactosamine and combinations thereof. Non-limiting examples of N-acetylated oligosaccharides include: LNT (lactose-N-tetraose), p-lactose-N-neohexose (p-LNnH), LNnT (lactose-N-neotetraose) and any combination thereof. Other examples are lactose-N-hexose, lactose-N-neohexose, p-lactose-N-hexose, p-lactose-N-neohexose, lactose-N-octose, lactose-N-neooctose, iso-lactose-N-octose, p-lactose-N-octose and lactose-N-decasaccharide.

[0096] In a preferred embodiment, the at least one N-acetylated oligosaccharide is selected from the group consisting of lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and combinations thereof.

[0097] N-acetylated oligosaccharides can be obtained by any suitable method. For example, LNnT can be chemically synthesized by enzymatic transfer of sugar units from donor moieties to acceptor moieties using glycosyltransferases. Alternatively, LNnT can be prepared by chemically converting free or oligosaccharide (e.g., lactulose)-bound ketone-hexose (e.g., fructose) into N-acetylhexosamine or oligosaccharides containing N-acetylhexosamine. LNT can be synthesized by enzymes, biotechnology and / or chemical methods.

[0098] Administration of oligosaccharides

[0099] The oligosaccharides may be administered in any suitable form and in any suitable amount.

[0100] For example, for infant formula or growing-up milk, the skilled person may base the amount of HMOs (e.g., 2'FL, diFL, LNT, LNnT, 3SL and / or 6SL) on the amounts found in human breast milk, produced by a well-nourished mother for an infant or child of the same age. In human breast milk, such amounts may fall within the following ranges: diFL: 100 mg / L-500 mg / L, LNT: 50 mg / L-300 mg / L, LNnT: 200 mg / L-2000 mg / L, 2'FL: 500 mg / L-3000 mg / L, 3'SL: 100 mg / L-400 mg / L, 6'SL: 50 mg / L-750 mg / L. However, they may exceed this compared to human breast milk, depending on, for example, the bioavailability of the HMOs from the infant formula.

[0101] As a guide, for example, for infant formula or growing-up milk, fucosylated oligosaccharides (e.g., 2'FL and / or diFL) may be present in the nutritional composition in a total amount of 0.1 g / L to 4 g / L of the composition, e.g., 0.1 g / L to 3.5 g / L of the composition (e.g., 0.15 g / L to 3 g / L, 0.2 g / L to 2.5 g / L, 0.3 g / L to 2 g / L, 0.4 g / L to 2 g / L, 0.5 g / L to 2 g / L of the composition) (the concentration may refer, for example, to the concentration after reconstitution of the composition with water). In a specific embodiment, the nutritional composition comprises 200 mg to 1800 mg of total fucosylated oligosaccharides per liter of the nutritional composition.

[0102] As a guide, for example, for infant formula or growing-up milk, sialylated oligosaccharides (e.g., 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL)) may be present in the nutritional composition at a concentration of 50 mg / L to 750 mg / L, e.g., 50 mg / L to 500 mg / L, e.g., 100 mg / L to 300 mg / L, 100 mg / L to 400 mg / L of the nutritional composition. In a particular embodiment, the nutritional composition comprises 120 mg to 400 mg of total sialylated oligosaccharides per liter of the nutritional composition.

[0103] If the nutritional composition comprises 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), it may be particularly advantageous that the 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are contained in the nutritional composition in a weight ratio of 10:1 to 1:10, such as 10:1 to 2:1, 8:1 to 3:1, between 6:1 to 3:1, 5:1 to 3:1, 5:1 to 4:1 or 1:2 to 1.5:1.

[0104] As a guide, for example for infant formula or growing-up milk, the N-acetylated oligosaccharides (e.g., LNT and / or LNnT) may be present in the nutritional composition in a total amount of 0.05 g / L to 0.5 g / L of the composition, for example 0.1 g / L to 0.5 g / L, 0.2 g / L to 0.4 g / L or 0.3 g / L of the composition, for example 0.35 g / L of the composition.

[0105] Suitably, the microbiota modulator (e.g. in the form of a nutritional composition such as an infant formula) may comprise:

[0106] - a total amount of 0.05 g / L to 0.75 g / L, 0.05 g / L to 0.5 g / L, 0.1 g / L to 0.3 g / L or 0.1 mg / L to 0.4 mg / L of at least one sialylated oligosaccharide;

[0107] - a total amount of 0.1 g / L to 4 g / L, 0.1 g / L to 3.5 g / L, 0.15 g / L to 3 g / L, 0.2 g / L to 2.5 g / L, 0.3 g / L to 2 g / L, 0.4 g / L to 2 g / L or 0.5 g / L to 2 g / L of at least one fucosylated oligosaccharide; and / or

[0108] - a total amount of 0.05 g / L to 0.5 g / L, 0.1 g / L to 0.5 g / L or 0.2 g / L to 0.4 g / L of at least one N-acetylated oligosaccharide.

[0109] Suitably, the microbiota modulator (e.g. in the form of a nutritional composition such as an infant formula) may comprise:

[0110] - 0.01 to 2% by weight, preferably 0.05 to 1.5% by weight, most preferably 0.07 to 1% by weight of at least one sialylated oligosaccharide;

[0111] - 0.05 to 3 wt. %, preferably 0.1 to 2 wt. %, most preferably 0.2 to 1.5 wt. % of at least one fucosylated oligosaccharide; and / or

[0112] - 0.01 to 1 wt.-%, preferably 0.03 to 0.6 wt.-%, most preferably 0.05 to 0.5 wt.-% of at least one N-acetylated oligosaccharide.

[0113] Weight % may refer to the total dry weight of the composition (eg, the total weight of the composition in powder form).

[0114] Composition

[0115] Suitably, the microbiota modulator may be in the form of a composition.The composition may comprise any therapeutically effective amount of the microbiota modulator.

[0116] In some embodiments, the microbiota modulator is in the form of a nutritional composition, an infant formula, a medical food product for clinical nutrition, or a supplement.

[0117] In some embodiments, the microbiota modulator is in the form of a nutritional composition. As used herein, a "nutritional composition" may refer to a composition that provides nutrients to a subject. Such nutritional compositions are typically administered orally or intravenously, and they typically include a lipid or fat source and a protein source.

[0118] In some embodiments, the microbiota modulator is in the form of a synthetic nutritional composition. As used herein, "synthetic nutritional composition" may mean a mixture obtained by chemical and / or biological methods, the chemical properties of which may be the same as the mixture naturally present in mammalian milk (i.e., the synthetic composition is not breast milk).

[0119] In some embodiments, the microbiota modulator is in the form of a medical food product for clinical nutrition. As used herein, "medical food products for clinical nutrition" may also be referred to as "foods for special medical purposes (FSMP)", and refer to specialized foods designed to help meet the nutritional or dietary needs of subjects with a disease, disorder or medical condition who are temporarily or permanently unable to obtain adequate nutritional intake from normal foods or by modifying their normal diet.

[0120] The composition can be any type of composition into which a microbiota modulator can be incorporated, such as a composition in the form of a food or beverage product, an animal feed product, a nutritional supplement for humans or animals, or a pharmaceutical composition. The composition can be in solid (e.g., powder), liquid, or semi-liquid form. In some embodiments, the microbiota modulator is in the form of a food composition, a pet food composition, a beverage, a nutritional formula, a nutritional supplement, or a nutraceutical.

[0121] Food and beverage products include all products intended for oral consumption by humans for the purpose of providing nutrition and / or pleasure. It can be, for example, a nutritional composition, such as for infants and / or young children, or for individuals who need special nutrition due to poor health, or for the elderly. Examples of food and beverage products include dairy products, such as dairy products or yogurt, soups, sauces, sweet and salty snacks, powdered beverages and cereal products.

[0122] The composition can also be in the form of an animal food product or a nutritional supplement for an animal. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc.

[0123] In a preferred embodiment, the microbiota modulator is in the form of an infant formula. In this case, the infant formula may be a preterm infant formula, a human milk fortifier, a first infant formula, an follow-on formula, a baby food formula, an infant cereal formula or a growing-up milk.

[0124] As used herein, the term "infant formula" refers to a food intended for specific nutritional use for infants during the first few months of life and which satisfies the nutritional requirements of such persons in itself (in accordance with Article 2(c) of Council Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formula and follow-on formula). It also refers to a nutritional composition intended for infants and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and Special Infants (including foods for special medical purposes). The expression "infant formula" covers both "first stage infant formula" and "second stage infant formula" or "follow-on formula".

[0125] In some embodiments, the microbiota modulator is in the form of a first infant formula.Generally speaking, a "first infant formula" is intended for use as a breast milk substitute for infants from birth.

[0126] In some embodiments, the microbiota modulator is in the form of an follow-on formula. A "second step infant formula" or "follow-on formula" can be given starting from the 6th month. It can constitute the main liquid element in the gradually diversified diet of such people.

[0127] In some embodiments, the microbiota modulator is in the form of a preterm infant formula.As used herein, the term "preterm infant formula" refers to an infant formula intended for premature infants.

[0128] In some embodiments, the microbiota modulator is in the form of a milk fortifier. As used herein, the term "milk fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk (for human milk fortifiers, it is human milk) or infant formula. It is used to increase the calories, protein, minerals and vitamins in breast milk fed to premature infants or infants with low birth weight. The term "breast milk" should be understood as mother's milk or mother's colostrum, or milk of a milk donor or colostrum of a milk donor.

[0129] In some embodiments, the microbiota modulator is in the form of a baby food formula.As used herein, the term "baby food formula" refers to a food material intended for specific nutritional use by infants or children, such as toddlers, during the first few years of life.

[0130] In some embodiments, the microbiota modulator is in the form of a growing-up milk. As used herein, the term "growing-up milk" (or GUM) refers to a milk formula food product provided after one year. It is typically a dairy beverage suitable for the specific nutritional needs of young children.

[0131] In some embodiments, the microbiota modulator is in the form of an infant cereal composition.As used herein, the term "infant cereal composition" refers to a food material intended for specific nutritional use by infants or children, such as toddlers, during the first few years of life.

[0132] In another specific embodiment, the microbiota modulator is in the form of a fortifier. The fortifier may be a breast milk fortifier or a formula fortifier, such as an infant formula fortifier. Therefore, when the infant or toddler is premature, the fortifier is a particularly advantageous embodiment.

[0133] In another specific embodiment, the microbiota modulator is in the form of a supplement. As used herein, a "supplement" or "dietary supplement" can be used to supplement a subject's nutrition (it is usually used as such, but it can also be added to any type of composition intended to be ingested by a subject).

[0134] When the composition is a supplement, it can be provided in unit dosage form. Supplements are generally present in the form of liquids, gels, powders or tablets or capsules. Powder supplements generally encompass supplements to be dissolved in water or sprayed on food or beverages. Such supplements are intended to provide additional nutrition and / or health benefits to the subject consuming it. Supplements can be used to provide nutrients and / or health benefits to humans and animals, as defined above. Supplements include, for example, powder supplements added to breast milk, for example, for premature or low birth weight infants.

[0135] In another specific embodiment, the microbiota modulator is in the form of a pharmaceutical product. Pharmaceutical products include, for example, drops, syrups, powders, tablets or capsule products intended to treat or prevent adverse medical conditions in individuals in need thereof.

[0136] The nutritional composition of the present invention, especially infant formula, typically contains a protein source, a carbohydrate source and a lipid source. However, in some embodiments, especially if the nutritional composition of the present invention is a supplement or fortifier, only lipid (or lipid source) may be present.

[0137] The nutritional composition according to the present invention may contain a protein source. The amount of protein may be 1.6 g / 100 kcal to 3 g / 100 kcal. In some embodiments, particularly when the composition is used for premature infants / young children, the amount of protein may be 2.4 g / 100 kcal to 4 g / 100 kcal or higher than 3.6 g / 100 kcal. In some other embodiments, the amount of protein may be less than 2.0 g / 100 kcal, for example between 1.8 g / 100 kcal and 2 g / 100 kcal, or less than 1.8 g / 100 kcal.

[0138] Protein sources based on, for example, whey, casein and mixtures thereof can be used, as well as plant-based (e.g., soy-based) protein sources. With regard to the whey protein of interest, the protein source can be based on acid whey or sweet whey or a mixture thereof, and can contain alpha-lactalbumin and beta-lactoglobulin in any desired proportion. In some embodiments, the protein source is mainly whey (i.e., more than 50% of the protein comes from whey protein, such as 60%> or 70%>). The protein can be intact or hydrolyzed, or a mixture of intact protein and hydrolyzed protein. The so-called term "intact" means that the main part of the protein is intact, i.e., the molecular structure has not changed, for example, at least 80% of the protein has not changed, such as at least 85% of the protein has not changed, preferably at least 90% of the protein has not changed, even more preferably at least 95% of the protein has not changed, such as at least 98% of the protein has not changed. In a specific embodiment, 100% of the protein has not changed.

[0139] The term "hydrolyzed" means in the context of the present invention that a protein has been hydrolyzed or broken down into its constituent amino acids.

[0140] The protein may be fully hydrolyzed or partially hydrolyzed. If hydrolyzed protein is desired, a hydrolysis process may be performed as desired and as known in the art. For example, a whey protein hydrolyzate may be prepared by enzymatic hydrolysis of the whey fraction in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This makes it possible to reduce the degree of lysine blockage from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source.

[0141] In a specific embodiment, the protein of the composition is hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein may be 2 to 20, 8 to 40, or 20 to 60, or 20 to 80, or greater than 10, 20, 40, 60, 80 or 90. For example, a nutritional composition containing a hydrolyzate with a degree of hydrolysis of less than about 15% is available from Nestle Company under the trademark Commercially available.

[0142] At least 70%, 80%, 85%, 90%, 95% or 97% of the protein may be hydrolyzed. In a particular embodiment, 100% of the protein is hydrolyzed.

[0143] In a particular embodiment, the protein of the composition is a plant-based protein.

[0144] The nutritional composition according to the invention may contain a carbohydrate source. This is particularly preferred in case the nutritional composition according to the invention is an infant formula. In this case, any carbohydrate source commonly present in infant formulas may be used, such as lactose, sucrose, sucrose, maltodextrin, starch and mixtures thereof, but one of the preferred carbohydrate sources for infant formulas is lactose.

[0145] The nutritional composition 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. It may be particularly beneficial if the composition includes fat in an amount of 25 to 30 g / 100 g of the dry weight of the composition. Non-limiting examples of essential fatty acids include: linoleic acid (LA), alpha-linolenic acid (ALA). The composition of the present invention may also contain gangliosides, monosialoganglioside-3 (GM3) and disialoganglioside 3 (GD3) and combinations thereof.

[0146] The nutritional composition of the present invention may also contain all vitamins and minerals that are considered to be necessary for daily diet and in significant amounts of nutrition. The minimum requirements of certain vitamins and minerals have been determined. Examples of minerals, vitamins and other nutrients optionally present in the composition of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, vitamin E, vitamin K1, vitamin K2, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine and L-carnitine. Minerals are usually added in the form of salts. The presence and amount of specific minerals and other vitamins will vary according to the target group. If necessary, the nutritional composition of the present invention may contain emulsifiers and stabilizers, such as soybeans, lecithin, citric acid monoglyceride and citric acid diglyceride, etc.

[0147] The composition of the present invention may also contain other substances that may have a beneficial effect, especially on bone health or bone development, such as lactoferrin, osteopontin, TGFβ, slgA, glutamine, nucleotides, nucleosides, etc.

[0148] Preparation of composition

[0149] The composition according to the present invention may be prepared by any known or other suitable means. For example, a nutritional composition (e.g., an infant formula) may be completed by blending a protein source with a carbohydrate source and a lipid source in suitable proportions. If an emulsifier is used, it may be included at this stage. Vitamins and minerals may be added at this stage, but may also be added later to avoid thermal degradation. Water (preferably reverse osmosis water or deionized water) may then be added and mixed to form a liquid mixture. The mixing temperature is preferably room temperature, but may also be higher. The liquid mixture may then be heat treated to reduce the bacterial load. The mixture may then be homogenized.

[0150] If it is desired to produce a powdered composition, the homogenized mixture is dried in a suitable drying apparatus, such as a spray dryer or a freeze dryer, and converted into a powder.

[0151] Methods for preparing infant and toddler formulas are based on the concept that the product must be nutritionally adequate and microbiologically safe to eat. Therefore, steps to eliminate or limit microbial growth are essential for the production method. The processing technology for each specific formula belongs to the manufacturer, but generally speaking, for powder products, it involves preserving oil-in-water (o / w) emulsions by dehydration, or for ready-to-eat or concentrated liquid products, it involves sterilization. Powdered infant formulas can be produced by various methods, such as dry mixing of dehydrated ingredients to form a uniform formula, or hydrating and wet mixing of a mixture of macro ingredients (such as fat, protein and carbohydrate ingredients), and then evaporating the resulting mixture and spray drying. A combination of the above two methods can be used, wherein a basic powder is first prepared by wet mixing and spray drying of all or some macro ingredients, and then dry mixing of the remaining ingredients (including carbohydrates, minerals and vitamins and other trace nutrients) to obtain the final formula. Liquid formulas are provided in ready-to-eat form or as concentrated liquids, the latter requiring dilution with water, usually 1:1. The manufacturing methods used for these products are similar to those used to make reconstituted milk.

[0152] If it is desired to produce a liquid infant formula, the homogenised mixture can be filled into suitable containers, preferably under aseptic conditions. However, the liquid composition can also be cooked in a container, and suitable equipment for carrying out such filling and cooking is commercially available.

[0153] Enhance bone development and / or bone strength

[0154] The present inventors have shown that bone development and / or bone strength of a subject is associated with the abundance of Enterobacter butyricum, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae in the subject's intestine. Specifically, the abundance of Enterobacter butyricum and Zurichia coli is positively correlated with bone outcome, and the abundance of Paraprevotella clara and Acidaminococcaceae is negatively correlated with bone outcome.

[0155] The microbiota modulators of the present invention modulate the abundance of one or more bacterial taxa in the intestinal tract of a subject, thereby enhancing bone development and / or bone strength in the subject, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara and Acidaminococcaceae.

[0156] In one aspect, the invention provides a microbiota modulator according to the invention for use in enhancing bone development and / or bone strength in a subject.

[0157] In one aspect, the invention provides a method for enhancing bone development and / or bone strength in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a microbiota modulator according to the invention.

[0158] In the context of the present invention, the term "enhancing bone development and / or bone strength" may specifically refer to one or more of the following physiological processes: bone catch-up growth, bone mass acquisition, peak bone mass optimization, promotion of bone formation, promotion of bone anabolism, increase in bone mineral density and microarchitecture, regulation of bone biomechanical properties, regulation of the rate of bone formation and / or bone resorption, assisting bone regeneration during fracture healing, and regulating the bone resorption process.

[0159] The microbiota modulators of the present invention can enhance bone development. As used herein, "promoting bone development" can refer to supporting normal bone metabolism, for example, in childhood and adolescence, and / or homeostasis. During childhood and adolescence, bones are shaped by a process known as modeling, which allows new bone to form in one location and old bone to be removed from another location within the same bone. Bone remodeling is a lifelong process in which mature bone tissue is removed from the skeleton and new bone tissue is formed. Supporting normal bone metabolism and / or homeostasis can refer to supporting normal shaping and / or remodeling of bones. Supporting normal bone metabolism and / or homeostasis can produce normal bone anatomy and physiological state.

[0160] The microbiota regulator of the present invention can enhance bone growth and / or strength. As used herein, "promoting bone growth and / or strength" may refer to supporting normal bone growth and / or strength, for example, in childhood and adolescence. Supporting normal bone growth and / or strength may lead to normal bone anatomy and physiology. Suitable methods and parameters for determining bone growth and bone strength will be known to the technician (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469 (8), pp. 2128-2138). Suitably, one or more bone parameters selected from the following may be used to determine normal bone growth and / or strength: trabecular bone volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), inner and outer diameters, anterior and posterior diameters, bone limit force (FMax) and bone stiffness. In some embodiments, one or more bone parameters selected from the following are used to determine normal bone growth and / or strength: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV) and bone ultimate force (FMax). Suitable methods for determining these parameters are available to those skilled in the art.

[0161] The microbiota regulators of the present invention can promote catch-up growth in subjects with growth retardation. Neonatal bone maturation can be a predictor of height increase in children born less than gestational age during the first year of life (see, e.g., Pepe, G. et al., 2020. Frontiers in Endocrinology, 11, p. 147). Suitable methods and parameters for determining catch-up growth are known to those skilled in the art. Appropriately, height velocity can be used to determine catch-up growth.

[0162] The microbiota modulators of the present invention can enhance bone healing. As used herein, "promoting bone healing" can refer to supporting normal bone healing, such as healing after a fracture. Fractures are one of the most common injuries to the musculoskeletal system. Although fracture treatments have improved significantly in recent decades, most of all fractures still show delayed healing and complications including non-union. Therefore, supporting normal bone healing can, for example, prevent delayed healing and / or non-union. Increasing age may increase the risk of delayed healing or non-union. The microbiota modulators of the present invention can prevent and / or reduce the frequency and / or incidence and / or severity and / or duration of fractures.

[0163] The microbiota regulator of the present invention can increase the abundance of Enterobacter butyricum and / or Bacillus zurichiensis in the intestinal tract of the subject, and / or reduce the abundance of Paraprevotella clara and / or Acidaminococcaceae in the intestinal tract of the subject, thereby enhancing the bone development and / or bone strength of the subject.

[0164] The microbiota modulator of the present invention can increase the abundance of Enterobacter butyricum and / or Bacillus zurichiensis in the intestine of the subject, thereby enhancing the bone development and / or bone strength of the subject.

[0165] The microbiota modulators of the present invention can increase the abundance of Enterobacter butyricum in the intestinal tract of a subject, thereby enhancing the subject's bone development and / or bone strength. The inventors have shown that the abundance of Enterobacter butyricum in the intestinal tract of a subject is particularly positively correlated with bone microarchitecture, especially trabecular structure. In some embodiments, the microbiota modulators of the present invention increase the abundance of Enterobacter butyricum in the intestinal tract of a subject, thereby enhancing the development of bone microarchitecture, especially trabecular structure.

[0166] The microbiota modulators of the present invention can increase the abundance of Bacillus zurichiensis in the intestinal tract of a subject, thereby enhancing bone development and / or bone strength in the subject. The inventors have shown that the abundance of Bacillus zurichiensis in the intestinal tract of a subject is particularly positively correlated with bone strength. In some embodiments, the microbiota modulators of the present invention increase the abundance of Bacillus zurichiensis in the intestinal tract of a subject, thereby enhancing bone strength.

[0167] The microbiota modulator of the present invention can reduce the abundance of Paraprevotella clara and / or Acidaminococcaceae in the intestinal tract of a subject, thereby enhancing bone development and / or bone strength of the subject.

[0168] The microbiota modulators of the present invention can reduce the abundance of Paraprevotella clara in the intestinal tract of a subject, thereby enhancing bone development and / or bone strength in the subject. The inventors have shown that the abundance of Paraprevotella clara in the intestinal tract of a subject is particularly negatively correlated with bone microarchitecture. In some embodiments, the microbiota modulators of the present invention reduce the abundance of Paraprevotella clara in the intestinal tract of a subject, thereby enhancing the development of bone microarchitecture, especially trabecular structure.

[0169] The microbiota modulators of the present invention can reduce the abundance of the Acidaminococcaceae in the intestinal tract of a subject, thereby enhancing bone development and / or bone strength in the subject. The inventors have shown that the abundance of the Acidaminococcaceae in the intestinal tract of a subject is particularly negatively correlated with bone strength. In some embodiments, the microbiota modulators of the present invention reduce the abundance of the Acidaminococcaceae in the intestinal tract of a subject, thereby enhancing bone strength.

[0170] The "abundance" of a bacterial taxon in the intestinal tract of a subject can be determined by any suitable method (see, e.g., Tang, Q. et al., 2020. Frontiers in cellular and infection microbiology, 10, p. 151). Suitably, the abundance of a bacterial taxon can be obtained from or can be obtained from a stool sample. The abundance can be a relative abundance and / or an absolute abundance. Preferably, the abundance is a relative abundance, for example, the abundance can be calculated relative to the total abundance of bacteria in the intestinal tract of a subject.

[0171] Subjects and route of administration

[0172] The subject can be any suitable subject. Suitably, the subject can be a mammal. In preferred embodiments, the subject is a human. In other embodiments, the subject is an animal, preferably wherein the animal is a pet. The pet can be an animal selected from dogs, cats, birds, fish, rodents (such as mice, rats and guinea pigs, rabbits) and the like.

[0173] The present invention is particularly suitable for infants and young children who are at risk of bone disease, have a family history of bone disease, or have experienced at least one, preferably several, fracture episodes. The present invention is also particularly suitable for infants and young children who are born prematurely or have a low birth weight or experience intrauterine growth retardation, or infants and young children who suffer growth retardation due to malnutrition or experience diseases (such as Crohn's disease and / or celiac disease and / or cancer), or infants and young children treated with drugs (such as chemotherapeutic drugs and / or corticosteroids) that cause malabsorption, anorexia, and / or metabolic bone diseases. The present invention is particularly preferably used for infants and children who are born prematurely or have a low birth weight or experience intrauterine growth retardation or suffer from intrauterine malnutrition or suffer from growth delay.

[0174] In some embodiments, the subject is a teenager, a teenager, a child, or an infant. The term "juvenile" may refer to an individual who has not yet reached adulthood. The term "teenager" may refer to an individual from the beginning of puberty to adulthood. The term "child" may refer to an individual between the birth stage and puberty.

[0175] In some embodiments, the subject is an infant, a toddler, or a young child. The term "infant" may refer to a subject whose age is about 0 to about 1 year old. The term "toddler" may refer to a subject whose age is about 1 to about 3 years old. The term "young child" may refer to a subject whose age is about 3 to about 5 years old.

[0176] In some embodiments, the subject is premature or has a low birth weight or experiences intrauterine growth retardation. The term "preterm infant" may refer to an infant whose gestational age is less than 37 weeks at birth. The term "low birth weight infant" may refer to an infant whose live birth weight is less than 2,500 g.

[0177] In some embodiments, the subject suffers and / or is suffering from growth retardation. The definition of growth retardation may refer to a "height for age" value that is less than two standard deviations of the median value of the WHO child growth standards (see, e.g., De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pp. 12-26). In some embodiments, the subject suffers and / or is suffering from growth retardation. The term "growth retardation" can describe a slower weight gain pattern than expected for age and gender in infants, children, and other adolescents (see, e.g., King, C. and Davis, T., 2010. European journal of clinical nutrition, 64 (1), pp. S11-S13). In some embodiments, the subject suffers and / or is suffering from growth retardation and / or growth retardation due to malnutrition or experiencing diseases such as anorexia, Crohn's disease and / or celiac disease. In some embodiments, the subject suffers and / or is suffering from growth retardation and / or growth reduction due to treatment with drugs that cause malabsorption, anorexia, and / or metabolic bone disease, such as chemotherapeutic drugs and / or corticosteroids.

[0178] The present invention may also be applied to adolescents or adults who are at risk of bone disease or who have experienced at least one, preferably several, episodes of bone fracture, or who were born prematurely or with low birth weight or who experienced intrauterine growth retardation, or who suffer growth retardation due to malnutrition or who experience disease (such as Crohn's disease and / or celiac disease and / or cancer), or who are treated with drugs that cause malabsorption, anorexia and / or metabolic bone disease (such as chemotherapeutic drugs and / or corticosteroids), or who suffer growth delay during infancy and / or childhood (including adolescence) due to disease or malnutrition or drug use.

[0179] In some embodiments, the subject is a teenager or an adult. In some embodiments, the subject is an adult, preferably wherein the subject is an elderly person. In some embodiments, the subject is at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. The subject may have one or more fractures. The subject may have one or more delayed unions and / or one or more nonunions, or may be at risk of these occurring.

[0180] The microbiota modulator can be administered by any suitable method known to the skilled person. For example, the microbiota modulator can be administered by oral and / or enteral administration. In some embodiments, the microbiota modulator is administered orally.

[0181] Example

[0182] The present invention is further described with reference to the following examples. It should be understood that the invention as claimed is not intended to be limited in any way by these examples.

[0183] Example 1

[0184] Female Göttingen miniature pigs ( Minipigs (n=48, Ellegaard, Denmark) were randomized and fed with milk replacers containing a blend of sialylated (2-HMO: 3'SL and 6'SL; 0.68 g / L, hereinafter referred to as "HMO blend A"), neutral (4-HMO: LNnT, LNT, 2'FL and di-FL; 4 g / L, hereinafter referred to as "HMO blend B"), sialylated or neutral (6HMO: 4 g / L, hereinafter referred to as "HMO blend C") human milk oligosaccharides (HMOs), or lactose (C: 4 g / L, hereinafter referred to as "M") from 10 days of age to 11 weeks of age (weaning). From weaning, piglets were provided with an equal controlled amount of a mild-western normal chow diet (13% protein, 14% fat, 33% cho, 4.4 M Cals GE / kg) until 48 weeks of age with ad libitum access to drinking water. The piglets were kept with the sows until weaning and were subsequently fed the same diet. The miniature pigs were sacrificed at 12 months of age (adult equivalence). The tibia was excised directly and stored at -20°C. Feces were collected at the same time points and stored at -20°C.

[0185] 1) Measurement of bone mineral density (BMD)

[0186] BMD is measured by dual energy X-ray absorptiometry (or DXA). BMD corresponds to bone mineral content divided by the surface area of ​​the tissue analyzed and is expressed in g / cm 2 The bones of miniature pigs are within the range of human children's bone size. Therefore, we used a DXA scanner with human parameters to evaluate the BMD of miniature pig tibiae. The equipment used: LunariDXA (GE Healthcare) located in the VCLB metabolic unit, software: EnCORE 2011 (version 13.60.003, GE Healthcare). BMD analysis has been performed for all tibiae and different regions of interest (proximal tibiae, mid-shaft tibiae, and distal tibiae) as previously described in the literature (Bonnet N et al. Bone. 2005; 37 (5): 622-33).

[0187] 2) Analysis of trabecular and cortical bone microstructure

[0188] Trabecular and cortical microarchitecture were evaluated using micro-computed tomography (μCT UCT40, Scanco Medical AG, Basserdorf Switzerland) in the proximal metaphysis and mid-shaft diaphysis of the tibia, respectively, as previously described in the literature (Bonnet N et al. J Bone Miner Res. 2017; doi: 10: 1002). Briefly, trabecular and cortical bone areas were evaluated using isotropic 12 μm voxels.

[0189] For the tibial trabecular region, 200 tomographic images obtained from 100 tomographic images under the proximal growth plate were analyzed to eliminate the main cancellous tissue. The tibial cortical structure was evaluated using 50 consecutive CT tomographic images (600 μm) located at the mid-shaft of the tibia. Morphological variables were calculated from the binarized images using a direct three-dimensional technique that does not rely on prior assumptions about the underlying structure (Bonnet N et al., MedPhys 2009; 36(4): 1286-97).

[0190] For trabecular bone area, bone volume fraction (BV / TV) was estimated. For cortical bone at the midshaft of the femur and tibia, cortical bone volume (Ct.BV, mm 3 ).

[0191] 3) Bone biomechanical analysis

[0192] To test the biomechanical properties of the bone, axial compression tests were performed on the tibia. Load was applied in compression mode at a nominal deformation rate of 2 mm / min until fracture. The load-displacement curve was recorded during the test. The ultimate load and failure energy were determined.

[0193] 4) Microbiome analysis

[0194] Microbiome composition (taxon abundance) from fecal samples was analyzed and statistical correlations with bone outcomes were performed using Spearman correlations to highlight correlations between taxon abundance and bone phenotypes. A pig / minipig microbiome gene catalog containing 6,601,931 genes was used to identify bacterial taxa. Libraries were sequenced using 2×150 bp paired-end sequencing on the Illumina platform. The approach is based on the concept of metagenomic species.

[0195] 5) Results

[0196] Bacterial genus analysis showed that Enterobacter, Zurichia, and Paraprevotella were among the top 10 bacterial genera associated with different bone outcomes. Specifically, Zurichia was positively correlated with tibia mineral density, cortical structure, and bone strength, and Enterobacter and Paraprevotella were associated with trabecular structure. The results are shown in the table below.

[0197]

[0198] Bacterial taxa analysis showed that Enterobacter butyricum, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae each belonged to the top 10 bacterial taxa associated with different bone outcomes. Specifically: (i) Enterobacter butyricum was positively correlated with trabecular structure; (ii) Zurichia coli was positively correlated with bone strength; (iii) Paraprevotella clara was negatively correlated with trabecular structure; and (iv) Acidaminococcaceae was negatively correlated with bone strength. The results are shown in the table below.

[0199]

[0200] Further analysis confirmed that B. zurichiensis was positively correlated with bone strength (r = 0.37, p < 0.01) (see Figure 1 A). Bacillus thuringiensis was also positively correlated with BMD (r=0.26, p<0.05), trabecular structure (r=0.28, p<0.05), and cortical structure (r=0.36, p<0.01). Each of HMO blend A, HMO blend B, and HMO blend C increased the relative abundance of Bacillus thuringiensis in miniature pigs fed with milk replacers and reached a similar level compared to naturally fed subjects (see Figure 1 B).

[0201] Further analysis confirmed that P. clara was negatively correlated with trabecular structure (r = -0.54, p < 0.0001) (see Figure 2 A). P. clara was also negatively correlated with BMD (r = -0.31, p < 0.05) and bone strength (r = -0.29, p < 0.05). Each of HMO Blend A, HMO Blend B, and HMO Blend C reduced the relative abundance of P. clara in miniature pigs fed milk replacers and reached a similar level of relative abundance compared to naturally fed subjects (see Figure 2 B).

[0202] Implementation

[0203] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paragraphs).

[0204] 1. A microbiota modulator for enhancing bone development and / or bone strength in a subject, wherein the microbiota modulator modulates the abundance of one or more bacterial taxa in the intestinal tract of the subject, the one or more bacterial taxa selected from the group consisting of Enterobacter butyricigenicus, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0205] 2. A method for enhancing bone development and / or bone strength in a subject, the method comprising administering a therapeutically effective amount of a microbiota modulator to a subject in need thereof, wherein the microbiota modulator modulates the abundance of one or more bacterial taxa in the intestine of the subject, the one or more bacterial taxa selected from Enterobacter butyricum, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0206] 3. A microbiota modulator for use according to paragraph 1 or a method according to paragraph 2, wherein the microbiota modulator is Enterobacter butyricum and / or Bacillus zurichiensis.

[0207] 4. A microbiota modulator for use according to paragraph 1 or a method according to paragraph 2, wherein the microbiota modulator is a mixture of oligosaccharides comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide.

[0208] 5. A microbiota modulator for use according to paragraph 1 or a method according to paragraph 2, wherein the microbiota modulator is a combination comprising a mixture of oligosaccharides and Enterobacter butyricum and / or Zurichia coli, wherein the mixture of oligosaccharides comprises at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide, and wherein the mixture of oligosaccharides and Enterobacter butyricum and / or Zurichia coli are administered separately, simultaneously or sequentially.

[0209] 6. A microbiota modulator or method for use according to paragraph 3 or paragraph 5, wherein

[0210] The butyric acid-producing Enterobacter and / or the blood Zurichia coli were grown at a rate of 1E6 cfu / day to 1E10 cfu / day.

[0211] Daily dose administration.

[0212] 7. The microbiota modulator or method for use according to any one of paragraphs 4 to 6, wherein the at least one sialylated oligosaccharide is selected from the group consisting of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetrasaccharide b (LSTb), sialyllactose-N-tetrasaccharide c (LSTc), disialyl lactose-

[0213] N-tetrasaccharides and combinations thereof.

[0214] 8. A microbiota modulator or method for use according to any one of paragraphs 4 to 7, wherein the at least one sialylated oligosaccharide is selected from 3'-sialyllactose (3'-

[0215] SL), 6'-sialyllactose (6'-SL), and combinations thereof.

[0216] 9. The microbiota modulator or method for use according to any one of paragraphs 4 to 8, wherein the mixture comprises 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0217] 10. The microbiota modulator or method for use according to any one of paragraphs 4 to 9, wherein the at least one fucosylated oligosaccharide is selected from the group consisting of:

[0218] 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), lactose-neofucopentose V (LNnFP-V), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neodifucosylhexose (LNnDFH), monofucosyllactose-N-hexose-III (MFNLH-III), difucosyllactose-N-hexose-a (DFLNHa) and combinations thereof.

[0219] 11. The microbiota modulator or method for use according to any one of paragraphs 4 to 10, wherein the at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'FL), difucosyllactose (diFL) and combinations thereof.

[0220] 12. The microbiota modulator or method for use according to any one of paragraphs 4 to 11, wherein the mixture comprises 2'-fucosyllactose (2'FL) and difucosyllactose (diFL).

[0221] 13. The microbiota modulator or method for use according to any one of paragraphs 4 to 12, wherein the at least one N-acetylated oligosaccharide is selected from the group consisting of:

[0222] N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), and combinations thereof.

[0223] 14. The microbiota modulator or method for use according to any one of paragraphs 4 to 13, wherein the at least one N-acetylated oligosaccharide is selected from lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) and combinations thereof.

[0224] 15. A microbiota modulator or method for use according to any one of paragraphs 4 to 14, wherein the mixture comprises lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

[0225] 16. A microbiota modulator or method for use according to any one of paragraphs 4 to 15, wherein the mixture comprises or consists of 2'-fucosyllactose (2'FL), difucosyllactose (diFL), lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), optionally wherein the mixture comprises 3'-sialyllactose (3'-

[0226] The invention relates to a novel oligonucleotide comprising: a ...

[0227] 17. A microbiota modulator for use according to any one of paragraphs 4 to 16 or

[0228] The method, wherein the oligosaccharide mixture comprises:

[0229] - 10% to 35% by weight, preferably 10% to 30% by weight, more preferably 10% to 25% by weight, of at least one sialylated oligosaccharide relative to the total weight of the oligosaccharide mixture;

[0230] - 30% to 80% by weight, preferably 40% to 80% by weight, more preferably 50% to 70% by weight of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture; and / or

[0231] 10 to 35 wt.-%, preferably 15 to 30 wt.-%, more preferably 15 to 20 wt.-%, of at least one N-acetylated oligosaccharide, relative to the total weight of the oligosaccharide mixture.

[0232] 18. A microbiota modulator or method for use according to any preceding paragraph, wherein

[0233] The microbiota modulator is in the form of a nutritional composition, a medical food product for clinical nutrition, or a supplement.

[0234] 19. A microbiota modulator or method for use according to any preceding paragraph, wherein

[0235] The microbiota modulator is in the form of an infant formula.

[0236] 20. A microbiota modulator or method for use according to any preceding paragraph, wherein

[0237] The microbiota modulator is in the form of a preterm infant formula, a human milk fortifier, a follow-on infant formula, a baby food formula, an infant cereal formula, or a growing-up milk.

[0238] 21. The microbiota modulator or method for use according to any one of paragraphs 18 to 20, wherein the composition comprises Enterobacter butyricum and / or Zurichia coli in an amount of 1E5 cfu / g to 1E9 cfu / g.

[0239] 22. A microbiota modulator for use according to any one of paragraphs 18 to 21 or

[0240] The method, wherein the composition comprises:

[0241] - 0.01% to 2% by weight, preferably 0.05% to 1.5% by weight, most preferably 0.07% to 1% by weight, of at least one sialylated oligosaccharide relative to the total weight of the composition in powder form;

[0242] - 0.05 to 3 wt.-%, preferably 0.1 to 2 wt.-%, most preferably 0.2 to 1.5 wt.-% of at least one fucosylated oligosaccharide relative to the total weight of the composition in powder form; and / or

[0243] - 0.01% to 1% by weight, preferably 0.03% to 0.6% by weight, most preferably 0.05% to 0.5% by weight of at least one N-acetylated oligosaccharide relative to the total weight of the composition in powder form.

[0244] 23. A microbiota modulator or method for use according to any of the preceding paragraphs, wherein the subject is a teenager, adolescent, child, toddler or infant, optionally wherein the subject suffers from growth retardation and / or growth reduction.

[0245] 24. A microbiota modulator or method for use according to any one of paragraphs 1 to 22, wherein the subject is an adult, optionally wherein the subject is elderly and / or at risk of delayed healing and / or non-healing.

[0246] 25. A microbiota modulator or method for use according to any one of the preceding paragraphs, wherein the subject is a human or an animal, preferably wherein the subject is a human.

[0247] 26. A microbiota modulator or method for use according to any of the preceding paragraphs, wherein the microbiota modulator increases the abundance of Enterobacter butyricigenicus and / or Bacillus zurichiensis in the intestinal tract of the subject, and / or the microbiota modulator reduces the abundance of Paraprevotella clara and / or Acidaminococcaceae in the intestinal tract of the subject.

[0248] 27. A microbiota modulator or method for use according to any of the preceding paragraphs, wherein enhancing bone development and / or bone strength comprises at least one of the following physiological processes: bone catch-up growth, bone mass gain, peak bone mass optimization, promoting bone formation, promoting bone anabolism, increasing bone mineral density and microarchitecture, regulating bone biomechanical properties, regulating the rate of bone formation and / or bone resorption, assisting bone regeneration during fracture healing, and regulating the bone resorption process.

[0249] 28. A microbiota modulator or method for use according to any of the preceding paragraphs, wherein the microbiota modulator increases one or more bone parameters selected from the group consisting of bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV) and bone ultimate force (FMax).

[0250] 29. Use of a microbiota modulator for modulating the abundance of one or more bacterial taxa in the intestine of a subject, wherein the one or more bacterial taxa are selected from Enterobacter butyricigenes, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

[0251] 30. The use according to paragraph 29, wherein the microbiota modulator is as defined in any one of paragraphs 3 to 26.

[0252] 31. A microbiota modulator comprising Enterobacter butyricum and / or Zurichia coli.

[0253] 32. The microbiota modulator according to paragraph 31, wherein the microbiota modulator is a probiotic or a synbiotic.

[0254] 33. A microbiota modulator according to paragraph 31 or paragraph 32, wherein the microbiota modulator is defined according to any one of paragraphs 5 to 22.

[0255] Although the present invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. In addition, if known equivalents exist for specific features, such equivalents should be incorporated as if explicitly mentioned in this specification.

Claims

1. A microbiota modulator for enhancing bone development and / or bone strength in a subject, wherein the microbiota modulator modulates the abundance of one or more bacterial taxa in the intestinal tract of the subject, the one or more bacterial taxa selected from the group consisting of Enterobacter butyricigenicus, Zurichia coli, Paraprevotella clara, and Acidaminococcaceae.

2. The microbiota modulator for use according to claim 1, wherein the microbiota modulator is Enterobacter butyricum and / or Bacillus zurichiensis.

3. A microbiota modulator for use according to claim 1, wherein the microbiota modulator is an oligosaccharide mixture comprising at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide.

4. The microbiota modulator for use according to claim 1, wherein the microbiota modulator is a combination comprising an oligosaccharide mixture and Enterobacter butyricum and / or Zurichia coli, wherein the oligosaccharide mixture comprises at least one sialylated oligosaccharide, at least one fucosylated oligosaccharide and / or at least one N-acetylated oligosaccharide, and wherein the oligosaccharide mixture and Enterobacter butyricum and / or Zurichia coli are administered separately, simultaneously or sequentially.

5. A microbiota regulator for use according to claim 3 or claim 4, wherein: (a) the at least one sialylated oligosaccharide is selected from the group consisting of: 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), sialyllactose-N-tetrasaccharide b (LSTb), sialyllactose-N-tetrasaccharide c (LSTc), disialyllactose-N-tetrasaccharide and combinations thereof, preferably wherein the at least one sialylated oligosaccharide is selected from the group consisting of 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL) and combinations thereof; (b) the at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (diFL), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V), lactose-neofucopentose-V (LNFP-VV), lactose-N-fucopentose-V (LNFP-VV), lactose-N-fucopentose-V (LNFP-VV), lactose-N-fucopentose-V (LNFP-VV), lactose-N-fucopentose-I (LNFP-I), lactose-N-fucopentose-II (LNFP-II), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-V (LNFP-V ...III (LNFP-III), lactose-N-fucopentose-V (LNFP-VV), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose-III (LNFP-III), lactose-N-fucopentose ), lactose-N-difucosylhexose-I (LNDFH-1), lactose-N-neodifucosylhexose (LNnDFH), monofucosyllactose-N-hexose-III (MFNLH-III), difucosyllactose-N-hexose-a (DFLNHa), and combinations thereof, preferably wherein the at least one fucosylated oligosaccharide is selected from the group consisting of 2'-fucosyllactose (2'FL), difucosyllactose (diFL), and combinations thereof; and / or (c) the at least one N-acetylated oligosaccharide is selected from the group consisting of N-acetyl-glucosamine, N-acetyl-galactosamine, lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT) and combinations thereof, preferably wherein the at least one N-acetylated oligosaccharide is selected from the group consisting of lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT) and combinations thereof.

6. A microbiota regulator for use according to any one of claims 3 to 5, wherein the oligosaccharide mixture comprises: (a) sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL); (b) 2'-fucosyllactose (2'FL) and difucosyllactose (diFL); and / or (c) lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT); preferably wherein the oligosaccharide mixture comprises 2'-fucosyllactose (2'FL), difucosyllactose (diFL), lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT) or consists of them.

7. A microbiota regulator for use according to any one of claims 3 to 6, wherein the oligosaccharide mixture comprises: (a) 10% to 35% by weight, preferably 10% to 30% by weight, more preferably 10% to 25% by weight, of at least one sialylated oligosaccharide, relative to the total weight of the oligosaccharide mixture; (b) 30% to 80% by weight, preferably 40% to 80% by weight, more preferably 50% to 70% by weight, of at least one fucosylated oligosaccharide relative to the total weight of the oligosaccharide mixture; and / or (c) 10 to 35 wt %, preferably 15 to 30 wt %, more preferably 15 to 20 wt % of at least one N-acetylated oligosaccharide, relative to the total weight of the oligosaccharide mixture.

8. The microbiota modulator for use according to any one of claims 1 to 7, wherein the microbiota modulator is in the form of a nutritional composition, a medical food product for clinical nutrition or a supplement.

9. A microbiota modulator for use according to any one of claims 1 to 8, wherein the microbiota modulator is in the form of an infant formula, preferably wherein the microbiota modulator is in the form of a preterm infant formula, a human milk fortifier, a first infant formula, a follow-on formula, a baby food formula, an infant cereal formula or a growing-up milk.

10. A microbiota regulator for use according to claim 8 or claim 9, wherein the composition comprises: (a) 0.01% to 2% by weight, preferably 0.05% to 1.5% by weight, most preferably 0.07% to 1% by weight, of at least one sialylated oligosaccharide relative to the total weight of the composition in powder form; (b) 0.05 to 3 wt. %, preferably 0.1 to 2 wt. %, most preferably 0.2 to 1.5 wt. % of at least one fucosylated oligosaccharide relative to the total weight of the composition in powder form; and / or (c) 0.01 to 1 wt. %, preferably 0.03 to 0.6 wt. %, most preferably 0.05 to 0.5 wt. % of at least one N-acetylated oligosaccharide relative to the total weight of the composition in powder form.

11. A microbiota modulator for use according to any one of the preceding claims, wherein the subject is a human.

12. The microbiota modulator for use according to claim 11, wherein the subject is a child, a toddler or an infant.

13. A microbiota modulator for use according to any one of the preceding claims, wherein the microbiota modulator increases the abundance of Enterobacter butyricigenicus and / or Bacillus zurichiensis in the intestinal tract of the subject, and / or the microbiota modulator reduces the abundance of Paraprevotella clara and / or Acidaminococcaceae in the intestinal tract of the subject.

14. A microbiota modulator for use according to any one of the preceding claims, wherein the microbiota modulator increases one or more bone parameters selected from the group consisting of bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV) and bone ultimate force (FMax).

15. A microbiota modulator comprising Enterobacter butyricum and / or Zurichia coli.

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