Lactoferrin for enhancing bone strength and / or bone growth

Abstract

The invention relates to human lactoferrin for use in enhancing bone strength and / or bone growth in a subject.

Description

[0001] LACTOFERRIN FOR ENHANCING BONE STRENGTH AND / OR BONE GROWTH FIELD OF THE INVENTION

[0002] The invention relates to lactoferrin, and compositions comprising the same, for use in enhancing bone strength and / or bone growth.

[0003] BACKGROUND TO THE INVENTION

[0004] Lactoferrin, also known as lactotransferrin, is a globular multifunctional glycoprotein found in milk and other bodily fluids. In milk, lactoferrin helps to protect infants from infections. It is a member of the transferrin family and plays a crucial role in the innate immune system, mainly at mucosa. It acts as a natural defense mechanism against bacteria, viruses, and fungi by binding to iron and preventing their growth and replication. Lactoferrin also stimulates the production of immune cells, such as lymphocytes and macrophages, which are essential for fighting off infections. Hence, lactoferrin is known for its antimicrobial, anti-inflammatory, and immunomodulatory properties.

[0005] In addition, lactoferrin is a protein that plays a crucial role in regulating iron levels in the body. It binds ferric iron tightly and retains it even at low pH, giving it antimicrobial and antioxidant properties. Lactoferrin's structure consists of two lobes, each binding a single iron ion. The iron binding sites are highly conserved and favorable for iron binding. The protein undergoes conformational changes when binding and releasing iron.

[0006] The growth and development of the human skeleton requires an adequate supply of many different nutritional factors. Classical nutrient deficiencies are associated with stunting (e.g. energy, protein, Zn), rickets (e.g. vitamin D) and other bone abnormalities (e.g. Cu, Zn, vitamin C). There is evidence to suggest that peak bone mass and later fracture risk are influenced by the pattern of growth and nutritional exposures in childhood. However, it is challenging to define dietary reference values using bone health as a criterion, and the question of what type of diet constitutes the best support for optimal bone growth and development remains open (see e.g. Prentice, A., et al., 2006. Proceedings of the Nutrition Society, 65(4), pp.348-360). Several approaches may be taken to improve the intake of bone strength and growth-limiting nutrients, including administration of micronutrient supplements, fortification of food with micronutrients or improved dietary intake. However, particularly in populations where dietary quality is poor, deficiencies can co-occur, in which case bone strength and growth may be affected.

[0007] Accordingly, there is a need for new nutritional interventions to enhance bone strength and / or bone growth. SUMMARY OF THE INVENTION

[0008] The inventors have found that lactoferrin, in particular at certain iron saturation levels and from certain origins and / or in combination with other agents, allows for enhancing the efficacy of bone mineralisation, and thus bone strength and growth. In particular, the inventors determined a higher efficacy of bone mineralisation in in vitro model systems when lactoferrin is from human origin (e.g. compared to bovine lactoferrin). The efficacy is even higher with human lactoferrin with a low iron saturation level. Bone mineralisation (and thus, for example, bone strength) was determined to be improved using MC3T3 pre-osteoblasts through the measurement of Alkaline Phosphatase (ALP) enzymatic activity, which is a marker of osteoblast differentiation. These findings thus provide for methods for enhancing bone strength and growth in a subject, including subjects that are early in life, adults, or who are aging.

[0009] In one aspect, the invention provides human lactoferrin for use in enhancing bone strength and / or bone growth in a subject.

[0010] In another aspect, the invention provides use of human lactoferrin for enhancing bone strength and / or bone growth in a subject. The use may be non-therapeutic use, for example use in nutrition.

[0011] In another aspect, the invention provides a method of enhancing bone strength and / or bone growth in a subject comprising administering human lactoferrin to the subject.

[0012] In another aspect, the invention provides use of human lactoferrin for the manufacture of a composition (preferably a nutritional composition) for enhancing bone strength and / or bone growth in a subject.

[0013] In another aspect, the invention provides use of human lactoferrin for the manufacture of a medical food product for enhancing bone strength and / or bone growth in a subject.

[0014] Preferably, the use is for enhancing bone strength in the subject.

[0015] The source of the lactoferrin is not particularly limited. In some embodiments, the lactoferrin is recombinant human lactoferrin, isolated human lactoferrin, or a combination thereof. In preferred embodiments, the lactoferrin is recombinant human lactoferrin.

[0016] In some embodiments, the lactoferrin is iron-depleted lactoferrin, partially iron-saturated lactoferrin, or iron-saturated lactoferrin. In preferred embodiments, the lactoferrin is partially iron-saturated lactoferrin. In some embodiments, about 50% to 100% of the metal ion-binding sites present in the lactoferrin population have iron bound. In some embodiments, about 50% to 100% of the metal ion-binding sites present in the lactoferrin population do not have iron bound.

[0017] In preferred embodiments, about 40% to 60% of the metal ion-binding sites present in the lactoferrin population have iron bound.

[0018] In some embodiments, the lactoferrin is in the form of a composition, for example comprising one or more further components, such as components described herein.

[0019] In some embodiments, the lactoferrin is in the form of a nutritional composition. In some embodiments, the nutritional composition is selected from: an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement. In some embodiments, the nutritional composition is an infant formula. In some embodiments, the nutritional composition is a supplement. In some embodiments, the supplement is a pediatric supplement.

[0020] In some embodiments, the nutritional composition further comprises a short chain fatty acid (SCFA) precursor or SCFA-originating ingredient. Preferably, the SCFA precursor or SCFA-originating ingredient comprises a fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS) or a HMO. In preferred embodiments, the SCFA precursor or SCFA-originating ingredient comprises a HMO. In particularly preferred embodiments, the SCFA precursor or SCFA-originating ingredient comprises a HMO and a probiotic (preferably B. infant is).

[0021] In some embodiments, the nutritional composition comprises about 0.03 g to about 5 g lactoferrin per L or about 0.02 g to about 4 g lactoferrin per 100g, preferably wherein the nutritional composition comprises about 0.05 g to about 2.5 g lactoferrin per L or about 0.04 g to about 2 g lactoferrin per 100g.

[0022] In some embodiments, the nutritional composition comprises about 0.1 g to about 5 g lactoferrin per L or about 0.1 g to about 4 g lactoferrin per 100g, preferably wherein the nutritional composition comprises about 0.1 g to about 2.5 g lactoferrin per L or about 0.1 g to about 2 g lactoferrin per 100g.

[0023] In preferred embodiments, the nutritional composition further comprises vitamin C.

[0024] In some embodiments, the nutritional composition further comprises the HMO LNFP-I.

[0025] In some embodiments, the nutritional composition further comprises a HMO mixture comprising or consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lacto-N-neotetraose (LNnT), and / or 3-fucosyllactose (3FL), and optionally lacto-N-fucopentaose I (LNFP-I).

[0026] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

[0027] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0028] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0029] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0030] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0031] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-fucopentaose I (LNFP-I).

[0032] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0033] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT)

[0034] In some embodiments, the nutritional composition further comprises 2'-fucosyllactose (2FL). In some embodiments, the subject is human. In some embodiments, the subject is an infant, a young child, a child, or an adult. In some embodiments, the subject is an infant.

[0035] In some embodiments, the subject suffered from and / or is suffering from stunted growth and / or faltering growth.

[0036] In some embodiments, the subject was born preterm or with low-birth weight or experienced intra-uterine growth retardation. In some embodiments, the lactoferrin enhances bone mineralisation.

[0037] In some embodiments, the lactoferrin promotes osteoblast mineralisation and / or osteoblast differentiation.

[0038] In some embodiments, the lactoferrin improves one or more bone parameter selected from: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax).

[0039] In some embodiments, the lactoferrin promotes catch-up growth, preferably wherein catch-up growth is determined using height velocity.

[0040] In some embodiments, the use or method according to the invention is therapeutic.

[0041] In some embodiments, the use or method according to the invention is non-therapeutic.

[0042] BRIEF DESCRIPTION OF THE FIGURES

[0043] Figure 1: Effect on Alkaline Phosphatase (ALP) enzymatic activity (a marker of osteoblast differentiation and thus bone mineralisation) by human and bovine lactoferrin (LF).

[0044] Figure 2: Effect on Alkaline Phosphatase (ALP) enzymatic activity (a marker of osteoblast differentiation and thus bone mineralisation) by human and bovine lactoferrin (LF) and a shortchain fatty acids (SOFA) cocktail and / or 3-hydroxybutyric acid (3HB).

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] Definitions

[0047] As used herein, the following terms have the following meanings.

[0048] The term "subject" may, for example, refer to an infant, young child, child, an infant small for gestational age (SGA) or a preterm infant.

[0049] The term "infant" means a child under the age of 12 months.

[0050] The expression "young child" means a child aged between one and three years, also called toddler.

[0051] The term “child” means a child aged between three and twelve years. Preferably, the term “child” means a child aged between three and six years.

[0052] A "preterm" or "premature" subject means an infant or young child who was not born at term. Generally it refers to an infant or young child born prior 36 weeks of gestation. By the expression "small for gestational age" or "SGA" it is referred to an infant or young child who is smaller in size than normal for their gestational age at birth, most commonly defined as a weight below the 10th percentile for the gestational age. In some embodiments, SGA may be associated with intrauterine growth restriction (IUGR), which refers to a condition in which a foetus is unable to achieve its potential size.

[0053] By the expression “low birth weight”, it should be understood as any body weight under 2500g at birth.

[0054] The expression "nutritional composition" means a composition which nourishes a subject. This nutritional composition is usually to be taken orally or intravenously. It may include a lipid or fat source, a carbohydrate source and / or a protein source. In a particular embodiment the nutritional composition is a ready-to-drink composition such as a ready-to-drink formula. In a particular embodiment, the nutritional composition of the present invention is a "synthetic nutritional composition". The expression "synthetic nutritional composition" means a mixture obtained by chemical and / or biological means, which can be chemically identical to the mixture naturally occurring in mammalian milks (i.e. the synthetic nutritional composition is not breast milk).

[0055] The expression "infant formula" as used herein refers to a foodstuff intended for particular nutritional use by infants during the first months of life and satisfying by itself the nutritional requirements of this category of person (Article 2(c) of the European Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant formulae and follow-on formulae). It also refers to a nutritional composition intended for infants and as defined in Codex Alimentarius (Codex STAN 72-1981) and Infant Specialities (incl. Food for Special Medical Purpose).

[0056] The expression "infant formula" encompasses both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0057] A "follow-up formula" or "follow-on formula" is given from the 6th month onwards and includes “growing-up milk”. It constitutes the principal liquid element in the progressively diversified diet of this category of person.

[0058] The expression “growing-up milk” (or “GUM”) refers to a milk-based drink generally with added vitamins and minerals, that is intended for young children or children.

[0059] The expression "baby food" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life. The expression "infant cereal composition" means a foodstuff intended for particular nutritional use by infants or young children during the first years of life.

[0060] The term "fortifier" refers to liquid or solid nutritional compositions suitable for mixing with breast milk or infant formula.

[0061] The expression “weaning period” means the period during which the mother's milk is substituted by other food in the diet of an infant or young child.

[0062] The "mother's milk" should be understood as the breast milk or the colostrum of the mother. An “oligosaccharide” is a saccharide polymer containing a small number (typically three to ten) of simple sugars (monosaccharides).

[0063] The term "HMO" or "HMOs" refers to human milk oligosaccharide(s). These carbohydrates are resistant to enzymatic hydrolysis by digestive enzymes (e.g. pancreatic and / or brush border), indicating that they may display functions not directly related to their caloric value. It has especially been illustrated that they play a vital role in the early development of infants and young children, such as the maturation of the immune system. Many different kinds of HMOs are found in the human milk. Each individual oligosaccharide is based on a combination of glucose, galactose, sialic acid (N- acetylneuraminic acid), fucose and / or N-acetylglucosamine with many and varied linkages between them, thus accounting for the enormous number of different oligosaccharides in human milk - over 130 such structures have been identified so far. Almost all of them have a lactose moiety at their reducing end while sialic acid and / or fucose (when present) occupy terminal positions at the non-reducing ends. The HMOs can be acidic (e.g. charged sialic acid containing oligosaccharide) or neutral (e.g. fucosylated oligosaccharide). Some examples of HMOs are the fucosylated oligosaccharides, the N-acetylated oligosaccharides and / or the sialylated oligosaccharides.

[0064] A "fucosylated oligosaccharide" is an oligosaccharide having a fucose residue. It has a neutral nature. Some examples are LNFP-I (lacto-N-fucopentaose I), 2’-FL (2' fucosyllactose), 3-FL (3-fucosyllactose).

[0065] The expressions “fucosylated oligosaccharides comprising an alpha-1, 2-fucosyl-epitope” and “2-fucosylated oligosaccharides” encompass fucosylated oligosaccharides with a certain homology of form since they contain an alpha-1, 2'-fucosyl-epitope, therefore a certain homology of function can be expected.

[0066] The expression “N-acetylated oligosaccharide(s)” encompasses both “N-acetyl-lactosamine” and “oligosaccharide(s) containing N-acetyl-lactosamine”. They are neutral oligosaccharides having an N-acetyl-lactosamine residue. Suitable examples are LNT (lacto- N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT (lacto-N-neotetraose) and any combinations thereof. Other examples are lacto-N-hexaose, lacto-N-neohexaose, para- lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N- neooctaose, iso- lacto-N-octaose, para- lacto-N-octaose and lacto-N-decaose.

[0067] A "sialylated oligosaccharide" is a charged sialic acid containing oligosaccharide, i.e. an oligosaccharide having a sialic acid residue. It has an acidic nature. Some examples are 3’-SL (3’-sialyllactose) and 6’-SL (6’-sialyllactose). The expressions "sialylated oligosaccharide" and "sialyllactose (SL)" can be used interchangeably. The trisaccharide sialyllactose consists of lactose at the reducing terminus and one sialic acid residue at the non-reducing end via an alpha-2,3 binding or alpha-2,6 binding, resulting in 3'-SL and 6'-SL, respectively.

[0068] A "precursor of HMO" is a key compound that intervenes in the manufacture of HMO, such as sialic acid and / or fucose.

[0069] The term “GOS” as used herein means “Galacto-oligosaccharide". Galacto-oligosaccharides (GOS) as used herein typically consist of p-linked galactose moieties with galactose or glucose at the reducing end. Such GOS contains p-(1— >2), p-(1— >3), p-(1— >4), or p-(1— >6) linked galactose moieties and may have a degree of polymerization (DP) of 3-8 galactose units. The term GOS is therefore preferably referred to as oligosaccharide(s) comprising at least three galactose units, more preferably as oligosaccharide(s) comprising at least four galactose units, preferably having a degree of polymerization (DP) of 3-8 galactose units.

[0070] The nutritional composition of the present invention can be in solid form (e.g. powder) or in liquid form. The amount of the various ingredients (e.g. the oligosaccharides) can be expressed in g / 100g of composition on a dry weight basis when it is in a solid form, e.g. a powder, or as a concentration in g / L of the composition when it refers to a liquid form (this latter also encompasses liquid composition that may be obtained from a powder after reconstitution in a liquid such as milk, water, e.g. a reconstituted infant formula or a follow-on / follow-up formula or a growing-up milk or an infant cereal product or any other formulation designed for infant nutrition).

[0071] The expressions “infants / young children fed exclusively with human breast milk”, “infants or young children exclusively breast fed”, “exclusive breast fed infants or young children’and “breast-fed infants / young children” can be used interchangeably. They refer to infants or young children fed with a great majority (i.e. at least 90%, or at least 95%, or at least 99%) or all (100%) of nutrients and / or energy originating from human breast milk. The expression “conventional nutritional composition” refers to standard synthetic nutritional compositions such as infant formula, follow-up milks or growing-up milks already found in the market.

[0072] The terms “microbial”, “microflora” and “microbiota” can be used interchangeably.

[0073] The expressions “microbiota in the gut”, “microbiota of the gut”, “gut microbiota” and “intestinal microbiota” can be used interchangeably.

[0074] By the expressions “preventing” or “prevention”, it is meant avoiding that a physical state, a condition or their consequences occurs and / or decreasing its incidence (i.e. reduction of the frequency). Prevention also encompasses delay or prevention of the onset of the symptoms of the disease, disorder or condition. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.

[0075] By the expressions “treating” or “treatment”, it is meant a decrease of the duration and / or of the severity of a physical state, a condition or their consequences (e.g. a decrease or elimination of symptoms of the condition). Treatment also encompasses to reduce, alleviate or eliminate one or more symptoms associated with the disease, disorder or condition which is being treated and / or to slow down, reduce or block the progression of the disease, disorder or condition which is being treated.

[0076] The prevention and / or the treatment of a physical state, a condition or their consequences can occur during the treatment (i.e. during the administration of the composition of the present invention, either immediately after the start of the administration or some time after, e.g. some days or weeks after the start). But it can also encompass the prevention and / or the treatment later in life. The term “later in life” encompasses the effect after the termination of the intervention or treatment. The effect “later in life” can be from 1 week to several months, or even years, for example from 2 to 4 weeks, from 2 to 6 weeks, from 2 to 8 weeks, from 1 to 6 months or from 2 to 12 months. Suitably, the effect “later in life” can be from 12 months to 12 years, such as from 2 years to 10 years, or from 4 years to 5 years, after the termination of the intervention or treatment. Suitably, the effect “later in life” lasts until the subject is at least 5 years of age, such as at least 10 years of age, at least 20 years of age or at least 30 years of age.

[0077] The term “prebiotic” means non-digestible carbohydrates that beneficially affect the host by selectively stimulating the growth and / or the activity of healthy bacteria such as bifidobacteria in the colon of humans (Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995;125:1401-12). The term “probiotic” means microbial cell preparations or components of microbial cells with a beneficial effect on the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. etal. “Probiotics: how should they be defined” Trends Food Sci. Technol. 1999:10 107-10). The microbial cells are generally bacteria or yeasts.

[0078] The term “synbiotic” may refer to a component that contains both probiotics and prebiotics, or a live microbe and a substrate that is selectively utilized by the co-administered live microbe (see e.g. Swanson, K.S., et al., 2020. Nature Reviews Gastroenterology & Hepatology, 17(11), pp.687-701).

[0079] The term “cfu” should be understood as colony-forming unit.

[0080] All percentages are by weight unless otherwise stated.

[0081] All weights expressed in g per 100g of composition are dry weight unless otherwise stated. The term “SCFA” means short chain fatty acid(s).

[0082] The expression “increasing SCFA production” means that the amount of systemic and / or colonic SCFA, is higher in an individual fed with the nutritional composition according to the present invention in comparison with a standard. The SCFA production may be measured by techniques known by the skilled person such as by Gas-Liquid Chromatography.

[0083] In the present context, the term “gastrointestinal tract” includes the mouth, pharynx, oesophagus, stomach, small intestine, large intestine, rectum and anus. The term “intestine” includes the small intestine, the large intestine and rectum.

[0084] In the present context, the term “respiratory tract” refers to the passage formed by the nose, nasal cavity, pharynx, larynx, trachea, bronchi and the lungs through which air passes during breathing.

[0085] The term "lactoferrin" as used herein is intended to mean any native, synthetic or recombinant lactoferrin molecule or fragment thereof that includes at least one metal ion binding site including but not limited to sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or human lactoferrin and any metal ion binding fragment thereof including but not limited to N-lobe and C-lobe fragments. Full length native lactoferrin has two metal ion-binding sites and the N-lobe and C-lobe lactoferrin fragments each have one metal ion-binding site. Preferably, the metal ion binding fragment thereof for use according to the present invention comprises two metal ion-binding sites. In the present context, the term “recombinant lactoferrin” refers to a lactoferrin that is produced in a cell, e.g. recombinant host cell, of a different species or type as compared to the species or type of cell that produces lactoferrin in nature, or that is produced in a cell, e.g. e.g. recombinant host cell, at a level at which it is not produced in nature. In other words, it relates to lactoferrin which can be found inherently in mammalian milk (e.g. cow’s milk or human milk), but which is produced recombinantly (e.g., that is produced by a recombinant host cell).

[0086] In addition, in the context of the invention, the terms "comprising" or "comprises" do not exclude other possible elements. The composition of the present invention, including the many embodiments described herein, can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise depending on the needs.

[0087] Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. The invention will now be described in further details. It is noted that the various aspects, features, examples and embodiments described in the present application may be compatible and / or combined together any combination thereof.

[0088] Lactoferrin

[0089] In one aspect, the invention provides human lactoferrin for use in enhancing bone strength and / or bone growth in a subject. In another aspect, the invention provides use of human lactoferrin for enhancing bone strength and / or bone growth in a subject.

[0090] Lactoferrin, highly conserved in the various species, is a glycoprotein with a molecular weight of approximately 80 kDa and three potential glycosylation sites. It belongs to the transferrin family and is also known as lactotransferrin. Lactoferrin can reversibly bind two atoms of iron (in particular, Fe(lll)) per molecule with high affinity, even at acid pH values, which occur at infection sites. Lactoferrin consists of two halves, designated as N-lobe and C-lobe, each of which contains one ion-binding site. While the N-lobe of lactoferrin has been extensively studied and is known for its enhanced antimicrobial effect, the C-lobe of lactoferrin mediates various therapeutic functions.

[0091] Lactoferrin was originally isolated from milk, but is also found in other bodily fluids including tears, saliva, vaginal fluids, semen, nasal and bronchial secretions, bile, gastrointestinal fluids, urine. Lactoferrin is the second most abundant protein in human milk and is particularly abundant in human colostrum (approximately 6 g / L) and mature milk (approximately 2 g / L). Lactoferrin shows many biological functions for infants such as regulation of iron absorption in the bowel, immune response, antioxidant, anticarcinogenic, anti-inflammatory properties, and protection against microbial infection. Lactoferrin has anti-microbial activity against a range of pathogens including Gram positive and Gram negative bacteria, yeasts and fungi. The anti-microbial effect of lactoferrin is based on its capability of binding iron which is essential for the growth of the pathogens. Lactoferrin also inhibits the replication of several viruses and increases the susceptibility of some bacteria to antibiotics and lysozyme.

[0092] The present invention relates to lactoferrin obtainable from any source. Lactoferrin may be purified, e.g., from milk or may be produced recombinantly, or by a mixture of the two types. Suitably, currently commercially available lactoferrins may be used in the practice of the present invention, such as for example human lactoferrin.

[0093] The lactoferrin source may be an aqueous composition or milk or a derivative thereof. In one embodiment, the lactoferrin source is selected from the group consisting of a substantially pure lactoferrin composition, a crude lactoferrin composition, human milk.

[0094] Suitably, the lactoferrin source may be any human milk fraction or processing stream. Lactoferrin may also be obtained from sources such as recombined or fresh whole milk, recombined or fresh skim milk, reconstituted whole or skim milk powder, skim milk concentrate, skim milk retentate, concentrated milk, buttermilk, ultrafiltered milk retentate, milk protein concentrate (MPC), milk protein isolate (MPI), calcium depleted milk protein concentrate (MPC), low fat milk, low fat milk protein concentrate (MPC), mammalian colostrum (such as goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or human colostrum), a colostrum fraction, colostrum protein concentrate (CPC), colostrum whey, whey, whey protein isolate (WPI), whey protein concentrate (WPC), sweet whey, lactic acid whey, mineral acid whey, salt whey, reconstituted whey powder, any milk or colostrum processing stream comprising whey proteins, the retentate or permeate comprising whey proteins obtained by ultrafiltration or microfiltration of any milk or colostrum processing stream, or the breakthrough or adsorbed fraction comprising whey proteins obtained by chromatographic separation of any milk or colostrum processing stream.

[0095] In a preferred embodiment, the lactoferrin source is human milk or colostrum, or a human milk or colostrum fraction or processing stream as described herein.

[0096] Methods for obtaining lactoferrin, including lactoferrin at various iron saturation levels, are known in the art. For example, W02006 / 132553 A1, W02007 / 043900 A1 and W02007 / 065482 A1 describe methods for the production of lactoferrin. Suitably, any method for obtaining lactoferrin which is known in the art may be used to provide lactoferrin for use in accordance with the present invention.

[0097] Lactoferrin can be produced in large quantities recombinantly. Recombinant lactoferrin and lactoferrin metal ion binding fragments can be produced in cells including bacterial, yeast, animal and plant cells. Preferably, recombinant lactoferrin is produced in yeast cells. Production of lactoferrin recombinantly in various cell types is reported in W02023 / 007468 A1 and WO2024 / 036227 A2. Preferably, recombinant lactoferrin (for example, recombinant human lactoferrin) for use according to the present invention is produced as disclosed in W02023 / 007468 A1 or WO2024 / 036227 A2.

[0098] In one embodiment, the lactoferrin is recombinant human lactoferrin, or a metal ion binding fragment thereof.

[0099] In a preferred embodiment, the lactoferrin is recombinant human lactoferrin or a metal ion binding fragment thereof.

[0100] In a preferred embodiment, the lactoferrin is recombinant human lactoferrin.

[0101] In one embodiment, the lactoferrin (for example, recombinant human lactoferrin) is iron-depleted lactoferrin.

[0102] In one embodiment, the lactoferrin (for example, recombinant human lactoferrin) is iron saturated.

[0103] In a preferred embodiment, the lactoferrin (for example, recombinant human lactoferrin) is partially iron saturated.

[0104] Full length lactoferrin has two metal-ion binding sites and so can bind metal ions, for example iron, in a stoichiometric ratio of 2 metal ions per lactoferrin molecule. Therefore, there are three different forms of lactoferrin molecule depending on the iron saturation level: apolactoferrin (iron free), monoferric form (one ferric iron), and hololactoferrin (bound to two ferric irons) (Esmat A. et al., Journal of Food Research; Vol. 2, No. 4; 2013). It should be understood that there may be metal ion-exchange between lactoferrin molecules within a population.

[0105] In one embodiment, “iron-depleted lactoferrin” refers to a population of lactoferrin molecules, each lactoferrin molecule or metal ion binding fragment thereof within the population comprising two metal ion binding sites, where less than about 10% (such as about 9%) of the metal ion-binding sites present in the population have iron bound. Preferably, about 5% or less of the metal ion-binding sites present in the population have iron bound. Suitably, about 0% of the metal ion-binding sites present in the population have iron bound.

[0106] In one embodiment, “partially iron saturated lactoferrin” refers to a population of lactoferrin molecules, each lactoferrin molecule or metal ion binding fragment thereof within the population comprising two metal ion binding sites, where at least about 10% of the metal ionbinding sites present in the population have iron bound. Suitably, from about 10 to about 79% of the metal ion-binding sites present in the population have iron bound. Preferably, at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75% of the metal ion-binding sites present in the population have iron bound. Preferably, about 40% or more of the metal ionbinding sites present in the population have iron bound, such as 40% to 79% or 40% to 60%.

[0107] In one embodiment, “iron saturated lactoferrin” refers to a population of lactoferrin molecules, each lactoferrin molecule or metal ion binding fragment thereof within the population comprising two metal ion binding sites, where at least about 80% of the metal ion-binding sites present in the population have iron bound. Preferably, about 85% or more or at least about 85% of the metal ion-binding sites present in the population have iron bound. Suitably, at least about 90, 95, 99 or 100% of the metal ion-binding sites present in the population have iron bound.

[0108] In some embodiments, about 0% to about 10% of the metal ion-binding sites present in the lactoferrin population have iron bound.

[0109] In some embodiments, about 10% to about 79% of the metal ion-binding sites present in the lactoferrin population have iron bound. In some preferred embodiments, at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75% of the metal ion-binding sites present in the population have iron bound. In some embodiments, about 40% to 100% of the metal ionbinding sites present in the lactoferrin population have iron bound. In some embodiments, about 50% to 100% of the metal ion-binding sites present in the lactoferrin population have iron bound.

[0110] In some preferred embodiments, at least about 80%, such as at least about 85%, of the metal ion-binding sites present in the lactoferrin population have iron bound. In some embodiments, at least about 90, 95, 99 or 100% of the metal ion-binding sites present in the population have iron bound.

[0111] Methods for determining the percentage of metal ion-binding sites present in the population of lactoferrin molecules are known in the art. Any suitable method known in the art for determining the percentage of metal ion-binding sites present in the population of lactoferrin molecules may be used in the practice of the present invention. Suitably, the percentage of metal ion-binding sites present in the population of lactoferrin molecules having iron bound may be determined by spectrophotometric analysis (Brock & Azabe, 1976; Bates et al, 1967; Bates et al, 1973).

[0112] Suitably, the percentage of metal ion-binding sites present in the population of lactoferrin molecules having iron bound may be determined using a high-throughput method (Majka, G. et al., Anal Bioanal Chem 405, 5191-5200 (2013)).

[0113] Suitably, the percentage of metal ion-binding sites present in the population of lactoferrin molecules having iron bound may be determined by first determining the amount of iron in the sample, then determining the amount of lactoferrin present in the sample, followed by calculating the percentage of metal ion-binding sites present in the population of lactoferrin molecules having iron bound. For example, the amount of iron in the sample may be determined by sample dissolution in media (such as ultrapure water or a buffer solution), sample mineralization (such as acidic microwave assisted reaction), and analysis in inductively coupled plasma-optical emission spectroscopy (ICPOES) or inductively coupled plasma mass spectrometry (ICPMS). For example, the amount of lactoferrin present in the sample may be provided in the product certificate for commercially available sources of lactoferrin or may be determined experimentally using methods known in the art for protein quantification.

[0114] Typical lactoferrin enriched compositions may comprise lactoferrin in an amount of at least 1.6 g / L.

[0115] For example, the composition of the present invention may contain lactoferrin in a concentration of at least 0.75% (w / w), preferably at least 1 % (w / w). In one embodiment, the composition is to be administered in an amount corresponding to an ingestion of at least 0.25g lactoferrin, preferably at least 0.5 g lactoferrin more preferably at least 1 g lactoferrin per day per kg body weight.

[0116] The composition may also be consumed in an amount corresponding to at least 200mg lactoferrin / kg body weight / day intake for the subject.

[0117] Lactoferrin may be present in the composition in a concentration of at least 0.01 g per 100 kcal, preferably of at least 0.1 g per 100 kcal. For example, lactoferrin may be present in the composition in the range of about 0.01 g - 100 g, preferably about 0.1 g - 50 g, more preferably about 2 g - 25 g per 100 kcal of the composition. In some embodiments, the composition or combination comprises about 0.03 g to about 10 g human lactoferrin per L. In some preferred embodiments, the composition or combination comprises about 0.03 g to about 5 g human lactoferrin per L or about 0.02 g / 100g to about 4 g / 100g (g human lactoferrin per 100g of powder). In some preferred embodiments, the composition or combination comprises about 0.05 g to about 2.5 g human lactoferrin per L or about 0.04 g / 100g to about 2 g / 100g (g human lactoferrin per 100g of powder). In some embodiments, the composition or combination comprises about 0.1 g to about 10 g lactoferrin per L. In some preferred embodiments, the composition or combination comprises about 0.1 g to about 5 g lactoferrin per L or about 0.1 g / 100g to about 4 g / 100g (g lactoferrin per 100g of powder). In some preferred embodiments, the composition or combination comprises about 0.1 g to about 2.5 g lactoferrin per L or about 0.1 g / 100g to about 2 g / 100g (g lactoferrin per 100g of powder). In some embodiments, the composition or combination comprises about 1.5 g to about 4 g human lactoferrin per L.

[0118] Methods for enhancing bone strength and / or bone growth

[0119] The present inventors have shown that the lactoferrin of the present invention may be used to enhance bone strength and / or bone growth in a subject.

[0120] Within the context of the present invention, the term “enhancing bone strength and / or bone growth” may refer to, in particular, one or more of the following physiological processes: promotion of bone mineralization, increase of bone mineral density and micro-architecture, determination of bone biomechanical properties, modulation the ratio of bone formation and / or bone resorption, bone catch-up growth, bone mass acquisition, optimization of peak bone mass, and promotion of bone formation, promotion of bone anabolism.

[0121] In one aspect, the invention provides human lactoferrin for use in enhancing bone strength and / or bone growth in a subject.

[0122] In another aspect, the invention provides use of human lactoferrin for enhancing bone strength and / or bone growth in a subject. The use may be, for example, use in nutrition.

[0123] In another aspect, the invention provides a method of enhancing bone strength and / or bone growth in a subject comprising administering human lactoferrin to the subject.

[0124] As used herein, “promoting bone strength and / or growth” may refer to the support of normal bone strength and / or growth, for example during childhood and adolescence. Supporting normal bone strength and / or growth may result in normal bone anatomy and physiology. Suitable methods and parameters to determine bone strength and bone growth will be known to the skilled person (see e.g. Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469(8), pp.2128-2138). Suitably, normal bone strength and / or growth may be determined using one or more bone parameter selected from: trabecular bone volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medio-lateral diameter, antero-posterior diameter, bone ultimate force (FMax), and bone stiffness. In some embodiments, normal bone strength and / or growth is determined using one or more bone parameter selected from: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax). Suitable methods to determine these parameters will be available to the skilled person.

[0125] Subject

[0126] In some embodiments, the subject is a mammal, such as a human.

[0127] In some embodiments, the composition according to the invention is for use in infants, young children or children.

[0128] In one embodiment, the subject is an infant. In one embodiment, the subject is a young child. In one embodiment, the subject is a child.

[0129] The composition according to the invention may be for use in infants or young children. It is particularly adapted for infants under 6 months of age.

[0130] The infants, young children or children may be born term or preterm. In a particular embodiment, the nutritional composition of the invention is for use in infants, young children or children that were born preterm. Preterm infants may be at increased risk of poor nutrient utilization, impaired lean body mass growth, fat accumulation in the visceral area and metabolic disease later in life. Thus, in one embodiment the nutritional composition of the invention is for use in preterm infants.

[0131] In one embodiment, the subject is an infant or a young child that was born small for gestational age or low birth weight.

[0132] Infants or young children with low birth weight may or may not be preterm, and similarly, infants or young children who are small for gestational age may or may not be preterm.

[0133] The nutritional composition of the present invention may also be used in an infant or a young child that was born by C-section or that was vaginally delivered.

[0134] In some embodiments, the subject is born by C-section and / or preterm; and / or small for gestational age (SGA); and / or with a low, very low, or extremely low weight at birth (LWB, VLBW, or ELBW); and / or that experienced intra-uterine growth retardation (IIIGR). All infants and young children can benefit from the invention as all of them are or can be, at a certain age, susceptible to acquiring an unbalanced intestinal / gut microbiota.

[0135] In some advantageous embodiments of the invention, the nutritional composition is for use infants or young children having a fragile or unbalanced microbiota or dysbiosis of microbiota, such as preterm infants, infants born by Caesarean-section, infants born small for gestational age or with low birth weight, hospitalized infants / young children, infants / young children treated or having been treated by antibiotics and / or infants / young children suffering or having suffered from gut infection and / or gut inflammation.

[0136] It is indeed foreseen that the composition of the invention may be even more beneficial to infants born with possibly impaired gut microbiota or fragile infants / young children (such as prematurely born infants and / or infants born by C-section). It is also foreseen that the composition of the invention may be even more beneficial to infants / young children exhibiting intestinal disorders (such as diarrhea, infections or colic), especially after birth, for example, during the first 4 weeks after birth.

[0137] In embodiments of the invention, the infants born prematurely or born by caesarean section or born small for gestational age or with low birth weight, or exhibiting unbalanced or abnormal gut microbiota or suffering or having suffered from gut infection and / or gut inflammation, are targeted by the composition of the present invention, and especially when the infants are 0-6 months of age. Without being bound by the theory, it is believed that younger infants benefit even more from the composition of the invention, especially when the infants have (or are at risk of having) an unbalanced intestinal microbiota and / or have a fragile health condition (as exemplified by the conditions cited above).

[0138] The nutritional composition can be administered (or given or fed) at an age and for a period that depends on the needs.

[0139] In one embodiment, the infants or young children are 0-36 months of age, such as 0-12 months or 0-6 months of age. It is foreseen that the composition of the invention may be even more beneficial to infants just after birth (0-4 weeks or 0-8 weeks) as their intestinal tract may be more fragile.

[0140] The mammal to be treated is preferably a human being, but the mammal may also be nonhuman mammal, such as a non-human mammal selected from the group consisting of pig, cow, horse, dog, cat, goat, sheep and rabbit.

[0141] In other embodiments, the subject is a juvenile animal, preferably wherein the animal is a pet. A pet may be a mammal such as dogs or cats, or rodents such as mice, rats, and guinea pigs, rabbits, etc. In some embodiments, the pet is a small dog breed.

[0142] The term “juvenile” may refer to an individual that has not yet reached adulthood.

[0143] In some embodiments the nutritional composition according to the invention can be for use before and / or during the weaning period.

[0144] In some embodiments the nutritional composition according to the invention is for use in a subject at risk and / or in need.

[0145] The subject at risk and / or in need may be bottle-fed and / or formula-fed.

[0146] In one embodiment the composition of the invention is given to the subject as a supplementary composition to the mother's milk. In some embodiments the subject receives the mother's milk during at least the first 2 weeks, first 1, 2, 4, or 6 months. In one embodiment the nutritional composition of the invention is given to the subject after such period of mother's nutrition, or is given together with such period of mother's milk nutrition. In another embodiment the composition is given to the subject as the sole or primary nutritional composition during at least one period of time, e.g. after the 1st, 2ndor 4thmonth of life, during at least 1 , 2, 4 or 6 months. In one embodiment the nutritional composition of the invention is a complete nutritional composition (fulfilling all or most of the nutritional needs of the subject). In another embodiment the nutrition composition of the invention is a supplement or a fortifier intended for example to supplement human milk or to supplement an infant formula or a follow- on formula.

[0147] Composition

[0148] In some embodiments, the human lactoferrin is in the form of a composition (preferably a nutritional composition). The composition may include further components, for example components as described herein.

[0149] Nutritional composition

[0150] In some embodiments, the composition of the invention is in the form of a nutritional composition.

[0151] The nutritional composition according to the invention can be for example an infant formula, a starter infant formula, a follow-on or follow-up formula, a growing-up milk, a baby food, an infant cereal composition, a fortifier such as a human milk fortifier, a supplement such as a paediatric supplement, a pet food, or a pet food supplement. In some particular embodiments, the composition of the invention is an infant formula, a fortifier or a supplement that may be intended for the first 4 or 6 months of age. In a preferred embodiment the nutritional composition of the invention is an infant formula.

[0152] In some other embodiments, the nutritional composition of the present invention is a fortifier. The fortifier can be a breast milk fortifier (e.g. a human milk fortifier) or a formula fortifier such as an infant formula fortifier or a follow-on / follow-up formula fortifier.

[0153] When the nutritional composition is a supplement, it can be provided in the form of unit doses. In such cases it is particularly useful to define the amount of oligosaccharides and probiotics in terms of daily dose to be administered to the infant, young child or child. In some embodiments, the supplement is a paediatric supplement.

[0154] The composition according to the present invention may also comprise other types of oligosaccharide(s), polysaccharides and / or a fiber(s) and / or a precursor(s) thereof. The other oligosaccharide and / or fiber and / or precursor thereof may be selected from the list comprising human milk oligosaccharides (HMOs), galacto-oligosaccharides (GOS), fructooligosaccharides (FOS), xylooligosaccharides (XOS), cello-oligosaccharides (COS), arabinoxylans, arabinans, xylans, inulin, polydextrose, beta-glucans, pectins and any combination thereof and any derived preparations thereof (e.g. partial hydrolysis). They may be in an amount between 0 and 10% by weight of composition. In a particular embodiment, the nutritional composition can also contain at least one BMO (bovine milk derived oligosaccharide).

[0155] SCFAs may promote osteoblast differentiation, and thus bone mineralisation. The compositions of the invention for use in enhancing bone strength and / or growth may, for example, further comprise one or more SCFA, such as acetate, butyrate, or propionate.

[0156] SCFAs, such as acetate, butyrate, or propionate, may be produced, for example, by fermentation of oligosaccharides (e.g. HMOs of the invention) in the gut (e.g. by Bifodobacteria).

[0157] The composition of the invention may further comprise an SCFA precursor or an SCFA-originating ingredient.

[0158] Within the context of the present invention the expression “SCFA originating ingredient” identifies ingredients which by mammal / animal / microbial metabolism give rise to SCFA in the human / animal body of the subject assuming them. Non-limiting examples of SCFA originating ingredients are: glycerol derivatives such as mono-, di- and tri-acyl glycerol derivatives incorporating one or more SCFAs, such as for example tributirin, triacetin, tripropionin;

[0159] • phospholipid derivatives such as phophatidylcholine, phosphatidylethanolamine, phosphatidylserine, phospatidylethanol in which one or more acyl chains consist of SCFA;

[0160] • organic esters of SCFA, such as for example methyl, ethyl, propyl or iso-propyl esters (non-limiting examples of such SFCA organic esters are: ethyl butanoate, ethyl propionate, ethyl acetate etc); such esters may be naturally occurring or may be obtained via chemical reactions for example by condensation of a SCFA and an appropriate alcohol (for example ethylic alcohol);

[0161] • fibers or protein incorporating SCFAs, such as for example butyrated or acetylated fibers, for example butyrated or acetylated starch;

[0162] • fermentable fibers, such as for example fermentable oligosaccharides and starches. The fermentable fibers can include but are not restricted to pectins, mucilages, gums, galacto-oligosaccharides, oligofructan, inulin, polyfructoses, arabinoglactans, hemicelullose, oligosaccharides or mixtures of thereof.

[0163] • other types of oligosaccharide(s), polysaccharides and / or a fiber(s) and / or a precursor(s) thereof, as above described.

[0164] • HMO and HMO mixtures as above / below described.

[0165] The SCFA originating ingredient may generate SCFA according to different mechanisms occurring in the human or animal body.

[0166] For example, glycerol and triglycerides derivatives incorporating SCFAs may generate SCFA in human or animal intestine, by digestion occurring in the small intestine.

[0167] For example, fermentable fibers may generate SCFA in human or animal intestine, particularly in the colon, by fermentation achieved by microbiota naturally present therein.

[0168] For example, fibers or starch incorporating SCFAs may generate or liberate SCFA in human or animal intestine, by digestion occurring in the small or large intestine followed by fermentation of the non-digested part of the fiber achieved by microbiota naturally present in the colon. The SCFA originating ingredients may be incorporated in the compositions of the invention in the form of a free ingredient or they may be incorporated in the compositions in encapsulated form for example to mitigate the impact of flavors.

[0169] In some embodiments, the nutritional composition further comprises a short chain fatty acid (SCFA) precursor or SCFA-originating ingredient.

[0170] Preferably, the SCFA precursor or SCFA-originating ingredient comprises a fructooligosaccharide (FOS), galacto-oligosaccharide (GOS) or a HMO. In preferred embodiments, the SCFA precursor or SCFA-originating ingredient comprises a HMO. In particularly preferred embodiments, the SCFA precursor or SCFA-originating ingredient comprises a HMO and a probiotic (preferably B. infantis).

[0171] For example, the composition may further include HMOs, and mixtures thereof, such as described below.

[0172] Lacto-N-fucopentaose I (LNFP-I)

[0173] In some embodiments, the composition of the invention comprises or consists of human lactoferrin and the HMO lacto-N-fucopentaose I (LNFP-I).

[0174] In some embodiments, LNFP-I is present in a total amount of from 10 mg / L to 5000 mg / L of the composition according to the invention or of from 0.01 g / 100 g to 4 g / 100 g of the composition according to the invention.

[0175] In some embodiments, LNFP-I is present in a total amount of from 25 mg / L to 4000 mg / L of the composition according to the invention or of from 0.02 g / 100 g to 3.75 g / 100 g of the composition according to the invention. Suitably, LNFP-I is present in a total amount of from 50 mg / L to 2500 mg / L, for example from 60 mg / L to 2000 mg / L, for example from 80 mg / L to 1500 mg / L, for example from 100 mg / L to 1000 mg / L, for example from 200 mg / L to 800 mg / L of the composition according to the invention. Suitably, LNFP-I is present in a total amount of from 0.04 g / 100 g to 2 g / 100 g, for example from 0.05 g / 100 g to 1.6 g / 100 g, for example from 0.06 to 1.2 g / 100g, for example from 0.07 g / 100 g to 0.8 g / 100 g, for example from 0.1 g / 100g to 0.7 g / 100g of the composition (dry weight).

[0176] In one embodiment, the composition comprises from 0.015 wt.% to 3.8 wt.%, preferably from 0.08 wt.% to 1.2 wt.%, of lacto-N-fucopentaose I (LNFP-I) of the total wt.% of the composition.

[0177] LNFP-I may be isolated by chromatography or filtration technology from a natural source such as animal milks. Suitably, the animal milk as used herein may be cow, sheep, goat, camel or buffalo milk. Preferably, the animal milk is cow’s milk. Preferably, the LNFP-I may be produced by biotechnological means using specific fucosyltransferases and / or fucosidases either through the use of enzyme-based fermentation technology (recombinant or natural enzymes) or microbial fermentation technology. In the latter case, microbes may either express their natural enzymes and substrates or may be engineered to produce respective substrates and enzymes. Single microbial cultures and / or mixed cultures may be used. Fucosylated oligosaccharide formation can be initiated by acceptor substrates starting from any degree of polymerization (DP), from DP = 1 onwards. Suitable techniques for producing LNFP-I are known in the art (see, for example, Hu et al., Carbohydr Polym, 2022, 297: 120017 and Derya et al., J Biotechnol., 2020, 318: 31-38). Alternatively, LNFP-I may be produced by chemical synthesis from lactose as initial acceptor substrate building an LNT backbone and free fucose as final donor substrate or by starting from LNT for example, produced by biotechnology or chemical synthesis, and using fucose. Fucosylated oligosaccharides are also available for example from DSM of the Netherlands or from Elicityl of France.

[0178] HMO mixture

[0179] In some embodiments, the composition of the invention comprises a HMO mixture.

[0180] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

[0181] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyllactose (3FL).

[0182] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0183] In some embodiments, the HMO mixture does not consist of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0184] In some embodiments, the HMO mixture does not comprise 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT). In some embodiments, the HMO mixture does not consist of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and lacto-N-neotetraose (LNnT).

[0185] In some embodiments, the HMO mixture does not comprise 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and lacto-N-neotetraose (LNnT).

[0186] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0187] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and lacto-N-fucopentaose I (LNFP-I).

[0188] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-fucopentaose I (LNFP-I).

[0189] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0190] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0191] In one embodiment, the HMO mixture comprises 2FL in an amount of from 16 wt% to 85 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 31 wt% to 82 wt%, preferably from 41wt% to 70 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 16 wt% to 69 wt%, preferably from 22 wt% to 59 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 34 wt% to 85 wt%, preferably from 40 wt% to 71 wt%. Suitably, the HMO mixture may comprise 2FL in an amount of from 20 wt% to 60 wt%, preferably from 22 wt% to 55 wt%.

[0192] In one embodiment, the HMO mixture comprises LNT in an amount of from 4 wt% to 40 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 27 wt%, preferably from 14 wt% to 23 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 9 wt% to 24 wt%, preferably 12 wt% to 21 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 10 wt% to 40 wt%, preferably 12 wt% to 26 wt%. Suitably, the HMO mixture may comprise LNT in an amount of from 4 wt% to 30 wt%, preferably 6 wt% to 20 wt%.

[0193] In one embodiment, the HMO mixture comprises DFL in an amount of from 1 wt% to 14 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 11 wt%, preferably from 6 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 2 wt% to 10 wt%, preferably from 3 wt% to 8 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 4 wt% to 14 wt %, preferably from 5 wt% to 10 wt%. Suitably, the HMO mixture may comprise DFL in an amount of from 1 wt% to 12 wt %, preferably from 2 wt% to 8 wt%.

[0194] In one embodiment, the HMO mixture comprises 6SLand 3SL combined in an amount of from 7 wt% to 34 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 34 wt%, preferably from 11 wt% to 29 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 8 wt% to 26 wt%, preferably from 11 wt% to 22 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 9 wt% to 31 wt%, preferably from 10 wt% to 28 wt%. Suitably, the HMO mixture may comprise 6SL and 3SL combined in an amount of from 7 wt% to 23 wt%, preferably from 8 wt% to 22 wt%.

[0195] In one embodiment, the HMO mixture comprises 3FL in an amount of from 10 wt% to 50 wt%. Suitably, the HMO mixture may comprise 3FL in an amount of from 18 wt% to 50 wt%, preferably from 11 wt% to 43 wt%. Suitably, the HMO mixture may comprise 3FL in an amount of from 10 wt% to 50 wt%, preferably from 13 wt% to 46 wt%.

[0196] In one embodiment, the HMO mixture comprises LNnT in an amount of from 6 wt% to 30 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 6 wt% to 30 wt%, preferably from 7 wt% to 22 wt%. Suitably, the HMO mixture may comprise LNnT in an amount of from 3 wt% to 25 wt%, preferably from 5 wt% to 20 wt%.

[0197] In one embodiment, the HMO mixture comprises LNFP-I in an amount of from 2 wt% to 32 wt %. Suitably, the HMO mixture may comprise LNFP-I in an amount of from 5 wt% to 32 wt%, preferably from 10 wt% to 19 wt%. Suitably, the HMO mixture may comprise LNFP-I in an amount of from 2 wt% to 24 wt%, preferably from 5 wt% to 14 wt%.

[0198] In some embodiments, the HMO mixture consists or consists essentially of:

[0199] i. 60 wt% to 96 wt% of 2FL;

[0200] ii. 2 wt% to 25 wt% of 6SL; and

[0201] iii. 2 wt% to 35 wt% of 3SL. In some embodiments, the HMO mixture consists or consists essentially of:

[0202] i. 71 wt% to 80 wt% of 2FL;

[0203] ii. 4 wt% to 16 wt% of 6SL; and

[0204] iii. 4 wt% to 25 wt% of 3SL.

[0205] In some embodiments, the HMO mixture consists or consists essentially of:

[0206] i. 16 wt% to 69 wt% of 2FL;

[0207] ii. 9 wt% to 24 wt% of LNT ;

[0208] iii. 2 wt% to 10 wt% of DFL;

[0209] iv. 8 wt% to 26 wt% of 6SL and 3SL combined; and

[0210] v. 18 wt% to 50 wt% of 3FL.

[0211] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0212] i. 22 wt% to 59 wt% of 2FL;

[0213] ii. 12 wt% to 21 wt% of LNT ;

[0214] iii. 3 wt% to 8 wt% of DFL;

[0215] iv. 11 wt% to 22 wt% of 6SL and 3SL combined; and

[0216] v. 11 wt% to 43 wt% of 3FL.

[0217] In some embodiments, the HMO mixture consists or consists essentially of:

[0218] i. 34 wt% to 85 wt% of 2FL;

[0219] ii. 10 wt% to 40 wt% of LNT ;

[0220] iii. 4 wt% to 14 wt% of DFL;

[0221] iv. 9 wt% to 31 wt% of 6SL and 3SL combined; and

[0222] v. 6 wt% to 30 wt% of LNnT.

[0223] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0224] i. 40 wt% to 71 wt% of 2FL;

[0225] ii. 12 wt% to 26 wt% of LNT ;

[0226] iii. 5 wt% to 10 wt% of DFL; and

[0227] iv. 10 wt% to 28 wt% of 6SL and 3SL combined; and

[0228] v. 7 wt% to 23 wt% of LNnT.

[0229] In some embodiments, the HMO mixture consists or consists essentially of:

[0230] 20 wt% to 60 wt% of 2FL; ii. 4 wt% to 30 wt% of LNT ;

[0231] iii. 1 wt% to 12 wt % of DFL;

[0232] iv. 7 wt% to 23 wt% of 6SL and 3SL combined;

[0233] v. 10 wt% to 50 wt% of 3FL; and

[0234] vi. 3 wt% to 25 wt% of LNnT.

[0235] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0236] i. 22 wt% to 55 wt% of 2FL;

[0237] ii. 6 wt% to 20 wt% of LNT ;

[0238] iii. 2 wt% to 8 wt % of DFL;

[0239] iv. 8 wt% to 22 wt% of 6SL and 3SL combined;

[0240] v. 13 wt% to 46 wt% of 3FL and

[0241] vi. 5 wt% to 20 wt% of LNnT.

[0242] In some embodiments, the HMO mixture consists or consists essentially of:

[0243] i. 48 wt% to 77 wt% of 2FL;

[0244] ii. 3 wt% to 16 wt% of 6SL;

[0245] iii. 2 wt% to 23 wt% of 3SL; and

[0246] iv. 3 wt% to 39 wt% of LNFP-I.

[0247] In some embodiments, the HMO mixture consists or consists essentially of:

[0248] i. 58 wt% to 74 wt% of 2FL;

[0249] ii. 3 wt% to 15 wt% of 6SL;

[0250] iii. 3 wt% to 22 wt% of 3SL; and

[0251] iv. 7 wt% to 27 wt% of LNFP-I.

[0252] In some embodiments, the HMO mixture consists or consists essentially of:

[0253] i. 20 wt% to 46 wt% of 2FL;

[0254] ii. 11 wt% to 17 wt% of LNT ;

[0255] iii. 2 wt% to 7 wt% of DFL;

[0256] iv. 9 wt% to 21 wt% of 6SL and 3SL combined;

[0257] v. 9 wt% to 34 wt% of 3FL; and

[0258] vi. 5 wt% to 32 wt% of LNFP-I.

[0259] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0260] 22 wt% to 42 wt% of 2FL;

[0261] 1 ii. 12 wt% to 15 wt% of LNT ;

[0262] iii. 3 wt% to 6 wt% of DFL;

[0263] iv. 9 wt% to 19 wt% of 6SL and 3SL combined;

[0264] v. 11 wt% to 32 wt% of 3FL; and

[0265] vi. 10 wt% to 19 wt% of LNFP-I.

[0266] In some embodiments, the HMO mixture consists or consists essentially of:

[0267] i. 27 wt% to 41 wt% of 2FL;

[0268] ii. 8 wt% to 15 wt% of LNT ;

[0269] iii. 4 wt% to 6 wt% of DFL;

[0270] iv. 8 wt% to 18 wt% of 6SL and 3SL combined;

[0271] v. 13 wt% to 21 wt% of LNnT ; and

[0272] vi. 7 wt% to 33 wt% of LNPF-I.

[0273] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0274] i. 32 wt% to 39 wt% of 2FL;

[0275] ii. 10 wt% to 14 wt% of LNT ;

[0276] iii. 4 wt% to 6 wt% of DFL;

[0277] iv. 7 wt% to 15 wt% of 6SL and 3SL combined;

[0278] v. 16 wt% to 20 wt% of LNnT ; and

[0279] vi. 11 wt% to 23 wt% of LNPF-I .

[0280] In some embodiments, the HMO mixture consists or consists essentially of:

[0281] i. 29 wt% to 40 wt% of 2FL;

[0282] ii. 8 wt% to 13 wt% of LNT ;

[0283] iii. 3 wt% to 11 wt % of DFL;

[0284] iv. 3 wt% to 15 wt% of 6SL and 3SL combined;

[0285] v. 11 wt% to 35 wt% of 3FL;

[0286] vi. 1 wt% to 18 wt% of LNnT ; and

[0287] vii. 2 wt% to 24 wt% of LNFP-I.

[0288] In some preferred embodiments, the HMO mixture consists or consists essentially of:

[0289] i. 32 wt% to 39 wt% of 2FL;

[0290] ii. 9 wt% to 12 wt% of LNT ;

[0291] iii. 3 wt% to 11 wt % of DFL;

[0292] iv. 4 wt% to 15 wt% of 6SL and 3SL combined; v. 12 wt% to 35 wt% of 3FL;

[0293] vi. 1 wt% to 17 wt% of LNnT; and

[0294] vii. 4 wt% to 14 wt% of LNFP-I.

[0295] In some embodiments, the HMO mixture consists of 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT)

[0296] In some embodiments, the weight ratio of 2FL:LNnT is 10:1 to 1:2, preferably 2:1.

[0297] In some embodiments, the HMO mixture consists essentially of about 1 g / L 2FL and about 0.5 g / L LNnT.

[0298] In some embodiments, the nutritional composition further comprises 2'-fucosyllactose (2FL). When the composition is in liquid form, the total HMO concentration is typically in the range from 0.5 to 10 g / L, preferably in the range from 1 to 7.5 g / L. Specific examples of the concentration level of total HMO, when the composition is in liquid form, include 1 to 5 g / L, 1 to 4 g / L, 2 to 5 g / L, 1 to 3 g / L or 2 to 4 g / L.

[0299] When the composition is in solid form, the total HMO concentration is typically in the range from 0.35 to 7 wt% (g total HMO / 100 g dry composition), preferably in the range from 0.35 to 5 wt%. Specific examples of the concentration level of total HMO, when the composition is in dry form, include 0.5 to 3.5 wt% (g total HMO per 100 g dry composition), 0.5 to 2.5 wt%, 1 to 3.5 wt%, 0.5 to 2 wt% or 1 to 2.5 wt%.

[0300] When the nutritional composition is a supplement, it may comprise the HMO mixture as described herein and human lactoferrin, and no other additional nutrient on top of the excipients necessary to obtain a stable nutritional composition.

[0301] The nutritional composition of the present invention can be in solid (e.g. powder), liquid or gelatinous form. In a specific embodiment the nutritional composition is a supplement, wherein the supplement is in powder form and provided in a sachet, preferably a sachet with 0.1 to 20 g per sachet, for example 1 to 10 g per sachet, or in the form of a syrup, preferably a syrup with a total solid concentration of 5 to 75 g / 100 mL (5 to 75% (w / v)). When the supplement is in powder form, it may comprise a carrier. It is however preferred that the supplement is devoid of a carrier. When the supplement is in the form of a syrup, the components are preferably dissolved or suspended in water acidified with citrate.

[0302] In a particular embodiment the nutritional composition according to the invention is a hypoallergenic composition. In another particular embodiment the composition according to the invention is a hypoallergenic nutritional composition. Additional HMOs which may be included in the nutritional composition according to the present invention may be selected from the group consisting of lacto-N- fucopentaose (e.g. lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I, difucosyllacto-N-neohexaose II, para-lacto-N-neohexaose (para-LNnH), lacto-N-hexaose, lacto- N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose, lacto-N-octaose, lacto-N-neooctaose, iso-lacto-N-octaose, para- lacto-N-octaose, lacto-N-decaose, and any combination thereof.

[0303] In some embodiments, the composition according to the invention comprises at least one additional HMO.

[0304] In other embodiments, the composition according to the present invention is devoid of any further HMOs. Thus, the LNFP-I or HMO mixture as described herein may be the only HMOs in the composition of the invention.

[0305] Other ingredients

[0306] In some embodiments, the composition or combination of the present invention does not comprise at least one further probiotic (or probiotic strain), such as at least one further probiotic bacterial strain. In some embodiments, the composition or combination of the present invention does not comprise any Bifidobacterium, such as Bifidobacterium longum subsp. infantis, such as Bifidobacterium longum subsp. infantis LMG 11588 (also known as Bifidobacterium longum subsp. infantis NCC3039 or Bifidobacterium longum subsp. infantis ATCC 17930). Bifidobacterium longum subsp. infantis LMG 11588 is sold by the Belgian Coordinated Collections of Microorganisms (BCCM) under the LMG accession number LMG 11588.

[0307] The composition of the present invention can further comprise at least one further probiotic (or probiotic strain), such as at least one further probiotic bacterial strain.

[0308] The probiotic microorganisms most commonly used are principally bacteria and yeasts of the following genera: Lactobacillus spp., Lacticaseibacillus spp, Limosilactobacillus spp, Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp. In some particular embodiments, the probiotic is a probiotic bacterial strain. In some specific embodiments, it is particularly Bifidobacteria and / or Lactobacilli.

[0309] Suitable probiotic bacterial strains according to the present invention include Bifidobacterium animalis subsp. lactis CNCM I-3446 deposited according to the Budapest Treaty on 7th June 2005 at Collection Nationale Cultures De Microorganismes [French National Collection Of Microorganism Cultures] (CNCM), Institut Pasteur, 25 Rue Du Docteur Roux, F-75724 Paris Cedex 15 (France), or BL818 or Bifidobacterium animalis subsp. lactis sold inter alia by the Christian Hansen company of Denmark under the trademark Bb 12 (also known as DSM-15954), B. longum CNCM 1-2618 (B. longum NCC2705), Bifidobacterium breve sold by Danisco under the trademark Bb-03, Bifidobacterium breve sold by Morinaga under the trade mark M-16V, Bifidobacterium breve sold by Morinaga under the trade mark B-3, Bifidobacterium breve sold by sold by Yakult under the trade mark BBG-01, Bifidobacterium breve sold by Institut Rosell (Lallemand) under the trademark R0070, Lactobacillus rhamnosus ATCC 53103 available from Valio Oy of Finland under the trademark LGG, Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-2116, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivarius DSM 13084 sold by BLIS Technologies Limited of New Zealand under the designation KI2, Bifidobacterium longum subsp. infantis LMG 11588 (also known as DSM 20218; ATCC 17930; JCM1260) sold by the Belgian Coordinated Collections of Microorganisms (BCCM) under the LMG accession number LMG 11588, and Bifidobacterium longum subsp. infantis sold for example by Procter & Gamble Co. under the trademark Bifantis.

[0310] The composition according to the invention may contain from 10e3 to 10e12 cfu of the at least one (further) probiotic strain, more preferably between 10e7 and 10e12 cfu such as between 10e8 and 10e10 cfu of probiotic strain per g of composition or combination on a dry weight basis.

[0311] In one embodiment, the probiotics are viable. In another embodiment, the probiotics are nonreplicating or inactivated. There may be both viable probiotics and inactivated probiotics in some other embodiments. Probiotic components and metabolites can also be added.

[0312] In preferred embodiments, the nutritional composition comprises human lactoferrin, at least one HMO, and at least one probiotic particularly capable of metabolizing the HMO(s), such as Bifidobacterium longum subsp. infantis, particularly LMG 11588.

[0313] The nutritional composition according to the invention generally contains a protein source. The protein can be in an amount of from 1.6 to 3 g per 100 kcal. In some embodiments, especially when the composition is intended for premature infants, the protein amount can be between 2.4 and 4 g / 100kcal or more than 3.6 g / 100kcal (or 3.6 g / 100kcal or more). In some other embodiments the protein amount can be below 2.0 g per 100 kcal, e.g. between 1.8 to 2 g / 100 kcal, or in an amount below 1.8 g per 100 kcal.

[0314] Protein sources based on whey, casein and mixtures thereof may be used as well as protein sources based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof and may include alpha-lactalbumin and beta-lactoglobulin in any desired proportions.

[0315] In some advantageous embodiments the protein source is whey predominant (i.e. more than 50% of proteins are coming from whey proteins, such as 51%, 60% or 70%).

[0316] The proteins may be intact or hydrolysed or a mixture of intact and hydrolysed proteins. By the term “intact” is meant that the main part of the proteins are intact, i.e. the molecular structure is not altered, for example at least 80% of the proteins are not altered, such as at least 85% of the proteins are not altered, preferably at least 90% of the proteins are not altered, even more preferably at least 95% of the proteins are not altered, such as at least 98% of the proteins are not altered. In a particular embodiment, 100% of the proteins are not altered. The term “hydrolysed” means in the context of the present invention a protein which has been hydrolysed or broken down into its component amino acids. The proteins may be either fully or partially hydrolysed. It may be desirable to supply partially hydrolysed proteins (degree of hydrolysis between 2 and 20%), for example for infants or young children believed to be at risk of developing cow’s milk allergy. If hydrolysed proteins are required, the hydrolysis process may be carried out as desired and as is known in the art. For example, whey protein hydrolysates may be prepared by enzymatically hydrolysing the whey fraction in one or more steps. If the whey fraction used as the starting material is substantially lactose free, it is found that the protein suffers much less lysine blockage during the hydrolysis process. This enables the extent of lysine blockage to be reduced from about 15% by weight of total lysine to less than about 10% (or to about 10% or less) by weight of lysine; for example about 7% by weight of lysine which greatly improves the nutritional quality of the protein source.

[0317] In an embodiment of the invention at least 70% of the proteins are hydrolysed, preferably at least 80% of the proteins are hydrolysed, such as at least 85% of the proteins are hydrolysed, even more preferably at least 90% of the proteins are hydrolysed, such as at least 95% of the proteins are hydrolysed, particularly at least 98% of the proteins are hydrolysed. In a particular embodiment, 100% of the proteins are hydrolysed.

[0318] In one particular embodiment the proteins of the nutritional composition are hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree of hydrolysis (DH) of the protein can be between 8 and 40, or between 20 and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90 (or 10, 20, 40, 60, 80 or 90 or more).

[0319] The protein component can alternatively be replaced by a mixture or synthetic amino acid, for example for preterm or low birth weight infants. In a particular embodiment, the nutritional composition or the growing-up milk according to the invention is a hypoallergenic composition. In another particular embodiment, the composition according to the invention is a hypoallergenic nutritional composition or growing-up milk. The nutritional composition according to the present invention generally contains a carbohydrate source. This is particularly preferable in the case where the nutritional composition of the invention is an infant formula. In this case, any carbohydrate source conventionally found in infant formulae such as lactose, sucrose, saccharose, maltodextrin, starch and mixtures thereof may be used although one of the preferred sources of carbohydrates is lactose.

[0320] The nutritional composition according to the present invention generally contains a source of lipids. This is particularly relevant if the nutritional composition of the invention is an infant formula. In this case, the lipid source may be any lipid or fat which is suitable for use in infant formulae. Some suitable fat sources include palm oil, structured triglyceride oil, high oleic sunflower oil and high oleic safflower oil, medium-chain-triglyceride oil. The essential fatty acids linoleic and a-linolenic acid may also be added, as well small amounts of oils containing high quantities of preformed arachidonic acid and docosahexaenoic acid such as fish oils or microbial oils. The fat source may have a ratio of n-6 to n-3 fatty acids of about 5:1 to about 15: 1 ; for example about 8: 1 to about 10: 1.

[0321] The nutritional composition of the invention may also contain all vitamins and minerals understood to be essential in the daily diet and in nutritionally significant amounts. Minimum requirements have been established for certain vitamins and minerals. Examples of minerals, vitamins and other nutrients optionally present in the composition of the invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amounts of specific minerals and other vitamins will vary depending on the intended population.

[0322] If necessary, the nutritional composition of the invention may contain emulsifiers and stabilisers such as soy, lecithin, citric acid esters of mono- and di-glycerides, and the like. The nutritional composition of the invention may also contain other substances which may have a beneficial effect such as nucleotides, nucleosides, and the like. The nutritional composition of the invention may also contain carotenoid(s). In some particular embodiments of the invention, the nutritional composition of the invention does not comprise any carotenoid.

[0323] Manufacture of a nutritional composition

[0324] The nutritional composition according to the invention may be prepared in any suitable manner. A composition will now be described by way of example.

[0325] For example, a formula such as an infant formula may be prepared by blending together the protein source, the carbohydrate source and the fat source in appropriate proportions. If used, the emulsifiers may be included at this point. The vitamins and minerals may be added at this point but they are usually added later to avoid thermal degradation. Any lipophilic vitamins, emulsifiers and the like may be dissolved into the fat source prior to blending. Water, preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture. The temperature of the water is conveniently in the range between about 50°C and about 80°C to aid dispersal of the ingredients. Commercially available liquefiers may be used to form the liquid mixture.

[0326] The oligosaccharide(s) may be added at this stage, especially if the final product is to have a liquid form. If the final product is to be a powder, they may likewise be added at this stage if desired.

[0327] The liquid mixture is then homogenised, for example in two stages.

[0328] The liquid mixture may then be thermally treated to reduce bacterial loads, by rapidly heating the liquid mixture to a temperature in the range between about 80°C and about 150°C for a duration between about 5 seconds and about 5 minutes, for example. This may be carried out by means of steam injection, an autoclave or a heat exchanger, for example a plate heat exchanger.

[0329] Then, the liquid mixture may be cooled to between about 60°C and about 85°C for example by flash cooling. The liquid mixture may then be again homogenised, for example in two stages between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenised mixture may then be further cooled to add any heat sensitive components, such as vitamins and minerals. The pH and solids content of the homogenised mixture are conveniently adjusted at this point.

[0330] If the final product is to be a powder, the homogenised mixture is transferred to a suitable drying apparatus such as a spray dryer or freeze dryer and converted to powder. The powder should have a moisture content of less than about 5% (or about 5% or less) by weight. The oligosaccharide(s) may also or alternatively be added at this stage by dry-mixing or by blending them in a syrup form of crystals, along with the probiotic strain(s), and the mixture is spray-dried or freeze-dried.

[0331] If a liquid composition is preferred, the homogenised mixture may be sterilised then aseptically filled into suitable containers or may be first filled into the containers and then retorted.

[0332] Lactoferrin may be added at any stage during this procedure, but is preferably added after the heating step. Since lactoferrin is a protein it may be considered a part of the protein source. In another embodiment, the composition of the invention may be a supplement, such as a paediatric supplement. The supplement may be in the form of tablets, capsules, pastilles or a liquid for example. The supplement may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, cocompounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents and gel forming agents. The supplement may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.

[0333] Further, the supplement may contain an organic or inorganic carrier material suitable for oral or parenteral administration as well as vitamins, minerals trace elements and other micronutrients in accordance with the recommendations of Government bodies such as the USRDA.

[0334] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0335] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0336] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0337] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0338] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

[0339] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0340] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

[0341] Example 1

[0342] METHODS MC3T3-E1 subclone 4 culture and treatment conditions

[0343] The pre-osteoblastic cell line MC3T3-E1 subclone 4 (CRL-2593) was purchased from ATCC (Manassas; Virginia, USA). Cells were maintained in growth medium (GM) composed of ascorbic acid-free aMEM (ThermoFisher Scientific), supplemented with 10% fetal calf serum (FCS, ThermoFisher Scientific) and 1% penicillin / streptomycin. All culture media were refreshed every 2-3 days. Cells were passaged with trypsin / EDTA solution at less than 80% confluence.

[0344] To induce differentiation into osteoblasts, cells were seeded at 5 x 104cells / cm2and grown to confluency in GM for 24 h. Then, the medium was switched to differentiation medium (DM) composed of GM supplemented with p-glycerophosphate 10 mM, ascorbic acid 50|jg / ml and lactoferrin 100|jg / ml treatment solutions. The lactoferrins were purchased from Merck KGaA (Darmstadt, Germany) with the following catalog numbers: L1294 (high iron human lactoferrin, an iron saturated form; iron saturation of about 87.5% (determined using ICP-MS)), L0520 (low iron human lactoferrin, an iron depleted form; iron saturation of about 4.1% (determined using ICP-MS)) and L9507 (high iron bovine lactoferrin; iron saturation of about 80.5% (determined using ICP-MS)).

[0345] Osteoblast alkaline phosphatase activity

[0346] MC3T3-E1 subclone 4 cells were differentiated for 7 days and were then collected for alkaline phosphatase (ALP) activity measurement as described previously (Littlefield et al. (1990) A simple and sensitive microtiter plate estrogen bioassay based on stimulation of alkaline phosphatase in Ishikawa cells: Estrogenic action of delta 5 adrenal steroids. Endocrinology 127: 2757) with minor modifications. Briefly, cells were lysed by heat shock and collected in ALP buffer (1 M diethanolamine, 0.24 M MgCh, pH 9.8). Enzymatic reaction was monitored at 405 nm after 4-Nitrophenyl phosphate disodium salt hexahydrate addition. Michaelis-Menten kinetics were evaluated at 30 °C for 30 min. Vmax was used as a proxy for ALP activity. The activity value was normalized by protein content measured via the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

[0347] RESULTS The effect of different forms of lactoferrin on osteoblast alkaline phosphatase (ALP) activity was investigated.

[0348] The data obtained (Figure 1) show that human lactoferrin, in particular in low iron saturation form, shows a significant higher effect on ALP activity which is a marker of osteoblast differentiation, compared to the bovine lactoferrin.

[0349] Example 2

[0350] MC3T3-E1 subclone 4 culture and treatment conditions

[0351] MC3T3-E1 subclone 4 cells were cultured and treated as in example 1 , with the exception that a short-chain fatty acids (SCFA) cocktail and / or 3-hydroxybutyric acid (3HB) were added on top of p-glycerophosphate 10 mM, ascorbic acid 50pg / ml and lactoferrin 100pg / ml treatment solutions. The SCFA cocktail was made of a final overall SCFA concentration of 50pM, composed of 75% acetate, 20% propionate and 5% butyrate. These levels are based on the ones found after an in vitro digestion of a milk matrix containing 2’FL, DiFL (DFL), LNnT, LNT and 6’SLas described in WO2024 / 240924 A1 (Example 1). The 3HB (metabolite of HMO such as metabolized HMO mixture of 2’FL, DiFL (DFL), LNnT, LNT, 3’SL and 6S’L, as described in WO2021 / 255229 A1) was used at a final concentration of 10pM. DMSO was used as vehicle for 3HB at a final concentration of 0.1%. Thus 0.1% final concentration of DMSO was added in each treatment condition. As the conditions had to be spread over 2 cell culture plates, nontreated control wells were added and used to normalize the ALP activity. In these wells, cells were treated only with P-glycerophosphate 10 mM and 0.1% DMSO. SCFA and 3HB were purchased from Merck KGaA (Darmstadt, Germany) with the following catalog numbers: S2889 (sodium acetate), P1880 (sodium propionate), B5887 (sodium butyrate) and 166898 (3HB).

[0352] Osteoblast alkaline phosphatase activity

[0353] The ALP activity was assessed as in example 1, with the exception that the values obtained were further normalized with non-treated plate controls, to correct for a potential plate effect.

[0354] RESULTS

[0355] The effect of different forms of lactoferrin together with metabolites of HMOs on osteoblast alkaline phosphatase (ALP) activity was investigated.

[0356] The data obtained (Figure 2) show that human lactoferrin together with metabolites of HMOs, in particular in low iron saturation form, shows a significant higher effect on ALP activity which is a marker of osteoblast differentiation, compared to the bovine lactoferrin. Various preferred features and embodiments of the present invention will now be described with reference to the following numbered clauses:

[0357] 1. Human lactoferrin for use in enhancing bone strength and / or bone growth in a subject.

[0358] 2. Use of human lactoferrin for enhancing bone strength and / or bone growth in a subject.

[0359] 3. The lactoferrin for use or the use according to any one of clauses 1-2, wherein the lactoferrin is recombinant human lactoferrin, isolated human lactoferrin, or a combination thereof.

[0360] 4. The lactoferrin for use or the use according to any one of clauses 1-3, wherein the lactoferrin is recombinant human lactoferrin.

[0361] 5. The lactoferrin for use or the use according to any one of clauses 1-4, wherein the lactoferrin is iron-depleted lactoferrin or partially iron-saturated lactoferrin.

[0362] 6. The lactoferrin for use or the use according to any one of clauses 1-5, wherein the lactoferrin is partially iron-saturated lactoferrin.

[0363] 7. The lactoferrin for use or the use according to any one of clauses 1-6, wherein about 50% to 100% of the metal ion-binding sites present in the lactoferrin population do not have iron bound.

[0364] 8. The lactoferrin for use or the use according to any one of clauses 1-6, wherein about 40% to 60% of the metal ion-binding sites present in the lactoferrin population have iron bound.

[0365] 9. The lactoferrin for use or the use according to any one of clauses 1-8, wherein the lactoferrin is in the form of a nutritional composition.

[0366] 10. The lactoferrin for use or the use according to clause 9, wherein the nutritional composition further comprises a short chain fatty acid (SCFA) precursor or SCFA-originating ingredient.

[0367] 11. The lactoferrin for use or the use according to clause 10, wherein the SCFA precursor or SCFA-originating ingredient comprises a fructo-oligosaccharide (FOS), galactooligosaccharide (GOS) or a HMO, preferably wherein the SCFA precursor or SCFA-originating ingredient comprises a HMO, more preferably wherein the SCFA precursor or SCFA-originating ingredient comprises a HMO and a probiotic (preferably B. infantis). 12. The lactoferrin for use or the use according to any one of clauses 9-11, wherein the nutritional composition is selected from: an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.

[0368] 13. The lactoferrin for use or the use according to any one of clauses 9-12, wherein the nutritional composition is an infant formula.

[0369] 14. The lactoferrin for use or the use according to any one of clauses 9-13, wherein the nutritional composition comprises about 0.03 g to about 5 g lactoferrin per L or about 0.02 g to about 4 g lactoferrin per 100g, or wherein the composition or combination comprises about 0.1 g to about 5 g lactoferrin per L or about 0.1 g to about 4 g lactoferrin per 100g, preferably wherein the nutritional composition comprises about 0.05 g to about 2.5 g lactoferrin per L or about 0.04 g to about 2 g lactoferrin per 100g, or preferably wherein the composition or combination comprises about 0.1 g to about 2.5 g lactoferrin per L or about 0.1 g to about 2 g lactoferrin per 100g.

[0370] 15. The lactoferrin for use or the use according to any one of clauses 9-14, wherein the nutritional composition further comprises vitamin C.

[0371] 16. The lactoferrin for use or the use according to any one of clauses 9-15, wherein the nutritional composition further comprises LNFP-I.

[0372] 17. The lactoferrin for use or the use according to clause 16, wherein the nutritional composition comprises LNFP-I in atotal amount of from 25 mg / Lto4000 mg / Lof the nutritional composition or of from 0.02 g / 100 g to 3.75 g / 100 g of the nutritional composition (dry weight).

[0373] 18. The lactoferrin for use or the use according to clause 16, wherein the nutritional composition comprises LNFP-I in a total amount of from 50 mg / L to 2500 mg / L, preferably from 60 mg / L to 2000 mg / L, more preferably from 80 mg / L to 1500 mg / L of the nutritional composition or of from 0.04 g / 100 g to 2 g / 100 g, preferably from 0.05 g / 100 g to 1.6 g / 100 g, more preferably from 0.06 g / 100 g to 1.2 g / 100 g, most preferably from 0.07 g / 100 g to 0.8 g / 100 g of the nutritional composition (dry weight).

[0374] 19. The lactoferrin for use or the use according to any one of clauses 9-15, wherein the nutritional composition further comprises a HMO mixture comprising or consisting of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lacto-N-neotetraose (LNnT), and / or 3-fucosy I lactose (3FL), and optionally lacto-N-fucopentaose I (LNFP-I). 20. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), and 3'-sialyllactose (3SL).

[0375] 21. The lactoferrin for use or the use according to clause 20, wherein the HMO mixture consists essentially of:

[0376] i. 31 wt% to 82 wt% of 2FL;

[0377] ii. 10 wt% to 27 wt% of LNT ;

[0378] iii. 4 wt% to 11 wt% of DFL; and

[0379] iv. 9 wt% to 34 wt% of 6SL and 3SL combined.

[0380] 22. The lactoferrin for use or the use according to clause 20, wherein the HMO mixture consists essentially of:

[0381] i. 41 wt% to 70 wt% of 2FL;

[0382] ii. 14 wt% to 23 wt% of LNT ;

[0383] iii. 6 wt% to 10 wt% of DFL; and

[0384] iv. 11 wt% to 29 wt% of 6SL and 3SL combined.

[0385] 23. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and lacto-N-neotetraose (LNnT).

[0386] 24. The lactoferrin for use or the use according to clause 23, wherein the HMO mixture consists essentially of:

[0387] i. 34 wt% to 85 wt% of 2FL, preferably 40 wt% to 71 wt%;

[0388] ii. 10 wt% to 40 wt% of LNT, preferably 12 wt% to 26 wt%;

[0389] iii. 4 wt% to 14 wt% of DFL, preferably 5 wt% to 10 wt%;

[0390] iv. 9 wt% to 31 wt% of 6SL and 3SL combined, preferably 10 wt% to 28 wt%; and

[0391] v. 6 wt% to 30 wt% of LNnT, preferably 7 wt% to 22 wt%. 25. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), and 3-fucosyl lactose (3FL).

[0392] 26. The lactoferrin for use or the use according to clause 25, wherein the HMO mixture consists essentially of:

[0393] i. 16 wt% to 69 wt% of 2FL, preferably 22 wt% to 59 wt%;

[0394] ii. 9 wt% to 24 wt% of LNT, preferably 12 wt% to 21 wt%;

[0395] iii. 2 wt% to 10 wt% of DFL, preferably 3 wt% to 8 wt%;

[0396] iv. 8 wt% to 26 wt% of 6SL and 3SL combined, preferably 11 wt% to 22 wt%; and

[0397] v. 18 wt% to 50 wt% of 3FL, preferably 11 wt% to 43 wt%.

[0398] 27. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-neotetraose (LNnT).

[0399] 28. The lactoferrin for use or the use according to clause 27, wherein the HMO mixture consists essentially of:

[0400] i. 20 wt% to 60 wt% of 2FL, preferably 22 wt% to 55 wt%;

[0401] ii. 4 wt% to 30 wt% of LNT, preferably 6 wt% to 20 wt%;

[0402] iii. 1 wt% to 12 wt % of DFL, preferably 2 wt% to 8 wt%;

[0403] iv. 7 wt% to 23 wt% of 6SL and 3SL combined, preferably 8 wt% to 22 wt%; v. 10 wt% to 50 wt% of 3FL, preferably 13 wt% to 46 wt%; and

[0404] vi. 3 wt% to 25 wt% of LNnT, preferably 5 wt% to 20 wt%.

[0405] 29. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0406] 30. The lactoferrin for use or the use according to clause 29, wherein the HMO mixture consists essentially of: i. 27 wt% to 41 wt% of 2FL, preferably 32 wt% to 39 wt%;

[0407] ii. 8 wt% to 15 wt% of LNT, preferably 10 wt% to 14 wt%;

[0408] iii. 4 wt% to 6 wt% of DFL;

[0409] iv. 8 wt% to 18 wt% of 6SL and 3SL combined, preferably 7 wt% to 15 wt%;

[0410] v. 13 wt% to 21 wt% of LNnT, preferably 16 wt% to 20 wt%; and

[0411] vi. 7 wt% to 33 wt% of LNPF-I, preferably 11 wt% to 23 wt%.

[0412] 31. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), and lacto-N-fucopentaose I (LNFP-I).

[0413] 32. The lactoferrin for use or the use according to clause 31, wherein the HMO mixture consists essentially of:

[0414] i. 20 wt% to 46 wt% of 2FL, preferably 22 wt% to 42 wt% of 2FL;

[0415] ii. 11 wt% to 17 wt% of LNT, preferably 12 wt% to 15 wt% of LNT ;

[0416] iii. 2 wt% to 7 wt% of DFL, preferably 3 wt% to 6 wt% of DFL;

[0417] iv. 9 wt% to 21 wt% of 6SL and 3SL combined, preferably 9 wt% to 19 wt% of 6SL and 3SL combined;

[0418] v. 9 wt% to 34 wt% of 3FL, preferably 11 wt% to 32 wt% of 3FL; and

[0419] vi. 5 wt% to 32 wt% of LNFP-I, preferably 10 wt% to 19 wt% of LNFP-I.

[0420] 33. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL), 3-fucosyllactose (3FL), lacto-N-neotetraose (LNnT), and lacto-N-fucopentaose I (LNFP-I).

[0421] 34. The lactoferrin for use or the use according to clause 33, wherein the HMO mixture consists essentially of:

[0422] i. 29 wt% to 40 wt% of 2FL, preferably 32 wt% to 39 wt% of 2FL; ii. 8 wt% to 13 wt% of LNT, preferably 9 wt% to 12 wt% of LNT ;

[0423] iii. 3 wt% to 11 wt % of DFL;

[0424] iv. 3 wt% to 15 wt% of 6SL and 3SL combined, preferably 4 wt% to 15 wt% of 6SL and 3SL combined;

[0425] v. 11 wt% to 35 wt% of 3FL, preferably 12 wt% to 35 wt% of 3FL;

[0426] vi. 1 wt% to 18 wt% of LNnT, preferably 1 wt% to 17 wt% of LNnT; and

[0427] vii. 2 wt% to 24 wt% of LNFP-I, preferably 4 wt% to 14 wt% of LNFP-I.

[0428] 35. The lactoferrin for use or the use according to clause 19, wherein the HMO mixture consists of 2'-fucosyllactose (2FL) and lacto-N-neotetraose (LNnT)

[0429] 36. The lactoferrin for use or the use according to clause 35, wherein the weight ratio of 2FL:LNnT is 10:1 to 1:2, preferably 2:1.

[0430] 37. The lactoferrin for use or the use according to clause 35, wherein the HMO mixture consists essentially of about 1 g / L 2FL and about 0.5 g / L LNnT.

[0431] 38. The lactoferrin for use or the use according to any one of clauses 9-15, wherein the nutritional composition further comprises 2'-fucosyllactose (2FL).

[0432] 39. The lactoferrin for use or the use according to any one of clauses 9-15, wherein the nutritional composition further comprises a HMO mixture consisting of 2'-fucosyllactose (2FL), 6'-sialyllactose (6SL) and 3'-sialyllactose (3SL).

[0433] 40. The lactoferrin for use or the use according to clause 39, wherein the HMO mixture consists essentially of:

[0434] i. 60 wt% to 96 wt% of 2FL, preferably 71 wt% to 80 wt% of 2FL;

[0435] ii. 2 wt% to 25 wt% of 6SL, preferably 4 wt% to 16 wt% of 6SL; and

[0436] iii. 2 wt% to 35 wt% of 3SL, preferably 4 wt% to 25 wt% of 3SL.

[0437] 40. The lactoferrin for use or the use according to any one of clauses 9-15, wherein the nutritional composition further comprises a HMO mixture consisting of 2'-fucosyllactose (2FL), 6'-sialyllactose (6SL), 3'-sialyllactose (3SL) and lacto-N-fucopentaose I (LNFP-I). 41. The lactoferrin for use or the use according to clause 40, wherein the HMO mixture consists essentially of:

[0438] i. 48 wt% to 77 wt% of 2FL, preferably 58 wt% to 74 wt% of 2FL;

[0439] ii. 3 wt% to 16 wt% of 6SL, preferably 3 wt% to 15 wt% of 6SL;

[0440] iii. 2 wt% to 23 wt% of 3SL, preferably 3 wt% to 22 wt% of 3SL; and

[0441] iv. 3 wt% to 39 wt% of LNFP-I, preferably 7 wt% to 27 wt% of LNFP-I.

[0442] 39. The lactoferrin for use or the use according to any one of clauses 1-38, wherein the subject is human.

[0443] 40. The lactoferrin for use or the use according to any one of clauses 1-39, wherein the subject is an infant, a young child, a child, or an adult.

[0444] 41. The lactoferrin for use or the use according to any one of clauses 1-40, wherein the subject is an infant.

[0445] 42. The lactoferrin for use or the use according to any one of clauses 1-41, wherein the subject suffered from and / or is suffering from stunted growth and / or faltering growth.

[0446] 43. The lactoferrin for use or the use according to any one of clauses 1-42, wherein the subject was born preterm or with low-birth weight or experienced intra-uterine growth retardation.

[0447] 44. The lactoferrin for use or the use according to any one of clauses 1-43, wherein the lactoferrin enhances bone mineralisation.

[0448] 45. The lactoferrin for use or the use according to any one of clauses 1-44, wherein the lactoferrin promotes osteoblast mineralisation and / or osteoblast differentiation.

[0449] 46. The lactoferrin for use or the use according to any one of clauses 1-45, wherein the lactoferrin improves one or more bone parameter selected from: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax).

[0450] 47. The lactoferrin for use or the use according to any one of clauses 1-46, wherein the lactoferrin promotes catch-up growth, preferably wherein catch-up growth is determined using height velocity. 48. A method of enhancing bone strength and / or bone growth in a subject comprising administering human lactoferrin to the subject, wherein the lactoferrin is optionally in the form of a nutritional composition, such as selected from: an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement.

[0451] 49. The lactoferrin for use or the use according to any one of clauses 1-47, wherein the subject is an infant, a young child or a child, preferably an infant.

[0452] 50. The lactoferrin for use or the use according to clause 49, wherein the subject is an infant or a young child that was born by C-section and / or preterm, and / or small for gestational age (SGA), and / or with a low, very low, or extremely low weight at birth (LWB, VLBW, or ELBW) and / or that experienced intra-uterine growth retardation (IIIGR).

[0453] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. (Original in Electronic Form)

[0454] (This sheet is not part of and does not count as a sheet of the international application)

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[0460] 3 / 4

[0461] PCT

[0462] (Original in Electronic Form)

[0463] (This sheet is not part of and does not count as a sheet of the international application)

[0464]

[0465] FOR RECEIVING OFFICE USE ONLY

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[0467] (Original in Electronic Form)

[0468] (This sheet is not part of and does not count as a sheet of the international application)

[0469] FOR INTERNATIONAL BUREAU USE ONLY

[0470]

Claims

CLAIMS1. Human lactoferrin for use in enhancing bone strength and / or bone growth in a subject.

2. The lactoferrin for use according to claim 1, wherein the lactoferrin is recombinant human lactoferrin, isolated human lactoferrin, or a combination thereof, preferably recombinant human lactoferrin.

3. The lactoferrin for use according to any one of claims 1-2, wherein the lactoferrin is iron-depleted lactoferrin or partially iron-saturated lactoferrin, preferably partially iron-saturated lactoferrin.

4. The lactoferrin for use according to any one of claims 1-4, wherein about 40% to 60% of the metal ion-binding sites present in the lactoferrin population have iron bound.

5. The lactoferrin for use according to any one of claims 1-4, wherein the lactoferrin is in the form of a nutritional composition.

6. The lactoferrin for use according to claim 5, wherein the nutritional composition further comprises a short chain fatty acid (SCFA) precursor or SCFA-originating ingredient, preferably wherein the SCFA precursor or SCFA-originating ingredient comprises a fructooligosaccharide (FOS), galacto-oligosaccharide (GOS) or a HMO, more preferably wherein the SCFA precursor or SCFA-originating ingredient comprises a HMO, even more preferably wherein the SCFA precursor or SCFA-originating ingredient comprises a HMO and a probiotic (preferably B. infantis).

7. The lactoferrin for use according to claim 5 or 6, wherein the nutritional composition is selected from: an infant formula, a starter infant formula, a follow-on or follow-up formula, a baby food, an infant cereal composition, a growing-up-milk, a fortifier such as a human milk fortifier, or a supplement such as a paediatric supplement.

8. The lactoferrin for use according to any one of claims 5-7, wherein the nutritional composition comprises about 0.03 g to about 5 g lactoferrin per L or about 0.02 g to about 4 g lactoferrin per 100g, preferably wherein the nutritional composition comprises about 0.05 g to about 2.5 g lactoferrin per L or about 0.04 g to about 2 g lactoferrin per 100g.

9. The lactoferrin for use according to any one of claims 5-8, wherein the nutritional composition further comprises vitamin C.

10. The lactoferrin for use according to any one of claims 1-9, wherein the subject is human, such as an infant, a young child, a child, or an adult.4711. The lactoferrin for use according to any one of claims 1-10, wherein the subject is an infant, a young child, or a child.

12. The lactoferrin for use according to any one of claims 1-11, wherein the lactoferrin enhances bone mineralisation.

13. The lactoferrin for use according to any one of claims 1-12, wherein the lactoferrin promotes osteoblast mineralisation and / or osteoblast differentiation.

14. The lactoferrin for use according to any one of claims 1-13, wherein the lactoferrin improves one or more bone parameter selected from: bone mineral density (BMD), trabecular bone volume fraction (BV / TV), cortical bone volume (Ct.BV), and bone ultimate force (FMax).

15. A method of enhancing bone strength and / or bone growth in a subject comprising administering human lactoferrin to the subject.

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