Complex coacervates of lactoferrin and osteopontin

Abstract

The present invention relates to a complex coacervate comprising lactoferrin and osteopontin, a method for preparing the same, and a composition comprising the same.Furthermore, the present invention relates to a complex comprising lactoferrin and osteopontin in the treatment and / or prevention of metabolic and / or inflammatory diseases.Furthermore, the present invention relates to a complex comprising lactoferrin and osteopontin for promoting bone development, growth, strength and / or healing, or for preventing and / or treating bone diseases.

Description

[Technical field]

[0001] The present invention relates to a complex coacervate comprising lactoferrin and osteopontin, a method for preparing the same, and a composition comprising the same.Furthermore, the present invention relates to a complex comprising lactoferrin and osteopontin in the treatment and / or prevention of metabolic and / or inflammatory diseases.Furthermore, the present invention relates to a complex comprising lactoferrin and osteopontin for promoting bone development, growth, strength and / or healing, or for preventing and / or treating bone diseases.

[0002] [Background technology] Lactoferrin (LF) and osteopontin (OPN) have been identified as having beneficial health benefits and therefore attempts have been made to use these proteins in nutritional or pharmaceutical products.

[0003] Lactoferrin (LF) is an iron-binding glycoprotein found in the milk of most mammalian species. The protein is typically present in human early milk and mature milk at concentrations of about 4.91 g / L and 2.10 g / L, respectively, but in bovine milk at approximately 10-fold lower concentrations. LF is a protein with associated biological functions, including antibacterial, anti-inflammatory, and immunomodulatory effects. In several clinical studies in infant populations, LF has been associated with a reduced incidence of late-onset sepsis and necrotizing enterocolitis.

[0004] LF is known to bind to anionic proteins in the form of soluble complexes. When lactoferrin is added during wet mixing of an infant formula blend, the protein undergoes denaturation by unfolding and refolding under the influence of temperature and pH, resulting in protein destabilization and increased viscosity. This phenomenon makes it very difficult to add lactoferrin in a wet state (i.e., before drying) to products such as infant formula.

[0005] Therefore, it has been reported to add lactoferrin to products such as solid form infant formula by dry mixing after the base powder of the product is produced.However, such a process is problematic because lactoferrin added in solid form needs to be sterilized.To achieve the required sterilization level, lactoferrin needs to be subjected to sterilization techniques.Most sterilization techniques include the use of high heat, which can result in the denaturation of lactoferrin.Other sterilization techniques, such as membrane filtration, are available, but can be expensive and require specific equipment.Addition of sterilized lactoferrin to the final product also requires specific and precise aseptic administration equipment.

[0006] Osteopontin (OPN) is a low-abundance, acidic, highly phosphorylated glycoprotein that is also present in higher concentrations in human milk compared to cow's milk. The mean OPN concentrations in human milk, cow's milk, and infant formula have been reported to be 138 mg / L, 18 mg / L, and 9 mg / L, respectively. Several biological functions have been attributed to OPN, including the ability to stimulate immune system development, brain development, and intestinal development. OPN supplementation with infant formula has also been reported to reduce the incidence of fever, lower levels of proinflammatory TNF-α, and higher levels of interleukin-2, altering plasma cytokine patterns.

[0007] To overcome the challenges associated with sterilization of lactoferrin, it would be highly desirable to develop a combined form of lactoferrin and osteopontin that would allow them to be incorporated into a wet mix along with other ingredients of infant formula and aseptically processed or spray dried in the infant formula composition.

[0008] Additionally, it would also be highly desirable to enhance the bioactivity and / or bioavailability of lactoferrin, especially in combination with osteopontin, for administration to a subject, especially in the case of metabolic and / or inflammatory diseases.

[0009] In particular, it is highly desirable to improve the bioactivity and / or bioavailability of lactoferrin, particularly in combination with osteopontin, in a subject after ingestion, especially for the treatment and / or prevention of metabolic and / or inflammatory diseases.

[0010] Furthermore, lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin strongly stimulates the proliferation and differentiation of primary osteoblasts and also acts as a survival factor that inhibits apoptosis induced by serum deprivation. Lactoferrin also affects and can strongly inhibit osteoclast formation (Naot, D., et al., 2005. Clinical Medicine & Research, 3(2), pp.93-101).

[0011] Studies have shown that osteopontin also plays a role in bone metabolism and bone homeostasis. Osteopontin is a key factor in neuron-mediated and endocrine-regulated bone mass and is involved in the biological activities of several bone-related cells, such as proliferation, migration, and adhesion. Osteopontin has been demonstrated to be closely related to the development and progression of many bone-related diseases, including osteoporosis (Si, J., et al., 2020. Medical science monitor: international medical journal of experimental and clinical research, 26, pp. e919159-1).

[0012] It is highly desirable to enhance the bioactivity and / or bioavailability of lactoferrin and / or osteopontin in order to promote bone metabolism and / or bone homeostasis.

[0013] To date, attempts to form coacervates between lactoferrin and osteopontin have not proven successful.

[0014] [Summary of the Invention] The present invention relates to a complex coacervate comprising lactoferrin and osteopontin.

[0015] The present invention also relates to a method for the preparation of a complex coacervate according to any of the preceding claims, the method comprising the steps of: a. providing separate aqueous solutions containing lactoferrin and osteopontin; b. mixing individual aqueous solutions containing lactoferrin and osteopontin at a pH of 4 to 6, preferably pH 4.5 to 5.5, more preferably pH 4.8 to 5.2, and even more preferably pH 5, wherein the individual aqueous solutions containing lactoferrin and osteopontin are adjusted so that the protein mass ratio of lactoferrin to osteopontin is in the range of 2 to 8, preferably in the range of 3 to 6, more preferably in the range of 3.2 to 5.5, more preferably in the range of 3.5 to 5, even more preferably in the range of 3.8 to 4.2, and even more preferably in the range of 4; Includes.

[0016] The present invention also relates to a composition comprising a complex coacervate according to the invention.

[0017] The present invention also relates to a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin, or a composition comprising a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin, for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0018] The present invention also relates to a method for treating or preventing metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a composition comprising a complex coacervate.

[0019] The present invention also relates to a complex coacervate comprising lactoferrin and osteopontin for use in promoting bone development, growth, strength and / or healing, or in the prevention and / or treatment of bone diseases.

[0020] The present invention also relates to a method for promoting bone development, growth, strength and / or healing, or for preventing and / or treating a bone disease, comprising administering to a subject in need thereof a therapeutically effective amount of a complex coacervate comprising lactoferrin and osteopontin.

[0021] Additional features and advantages of the present invention are described in, or will be apparent from, the following description of the presently preferred embodiments which take place in conjunction with the drawings. [Brief description of the drawings]

[0022] [Figure 1] Optical microscope images (40x magnification) of lactoferrin (LF)-osteopontin (OPN) complex coacervates (pH 5, 5% wt / vol protein, prepared at 4:1 LF:OPN mass protein ratio) isolated and dispersed in ultrapure water to confirm the presence of spherical liquid coacervates and the absence of irregular solid coprecipitates. Panel A, Panel B: Phase contrast microscopy; Panel C: Darkfield microscopy. All images were taken using a LabCam® smartphone microscope adapter. [Diagram 2] Differential scanning calorimetry thermograms of 5% wt / vol solutions of lactoferrin (dashed line) and osteopontin (dotted line) at pH 5.0 (FIG. 1, above), and of a LF / OPN complex coacervate with a protein concentration of 27.4% (wt / wt) at pH 5.0 (protein mass ratio 4:1, prepared at 5% wt / vol protein). [Diagram 3]Effects of adding gastrointestinal digestates of lactoferrin (LF), osteopontin (OPN), LF-OPN soluble complex (SC), or LF-OPN complex coacervate (CC) on an intestinal cell inflammation model. All samples are added at 0.35 mg protein equivalent / mL. Escherichia coli O111:B4 lipopolysaccharide (LPS)-induced NF-κB activation in HT-29 clone 34 cells was used as an inflammation model. LPS (20 ng / mL) and human whey (5% v / v) are added to all wells. All data are normalized to give 100 relative luminescence units (RLU) for LPS treatment and represent the mean ± standard error of triplicate measurements from three independent experiments. "*" represents a statistically significant difference (P<0.05) from 100% RLU treatment. [Figure 4-1] Effects of lactoferrin-osteopontin soluble complex (soluble), lactoferrin-osteopontin coacervate complex (coacervate), and lactoferrin-osteopontin blend (blend) on bone development, growth, and strength. C57 / bl6 wild-type mice were orally supplemented with three different osteopontin-lactoferrin mixtures (soluble, blend, or coacervate complex, n=10 per group) between postnatal days 2 and 28. From days 28 to 170, all mice were fed the same amount of standard chow. At the end of the study, femurs were harvested to assess the following bone microarchitecture parameters: (A) trabecular bone volume and tissue volume ratio (BV / TV, %); (B) trabecular bone mineral density (Tb.BMD, mg HA / ccm); (C) cortical bone volume (Ct.BV, mm3); (D) medio-lateral diameter (ML diameter, mm); (E) antero-posterior diameter (AP diameter, mm); (F) force yield (N); and (G) stiffness (N / mm). (H) and (I) show examples of trabecular and cortical bone structures obtained by micro-CT, respectively. (J) shows the orientation in the medio-lateral and antero-posterior diameters. [Figure 4-2] Effects of lactoferrin-osteopontin soluble complex (soluble), lactoferrin-osteopontin coacervate complex (coacervate), and lactoferrin-osteopontin blend (blend) on bone development, growth, and strength. C57 / bl6 wild-type mice were orally supplemented with three different osteopontin-lactoferrin mixtures (soluble, blend, or coacervate complex, n=10 per group) between postnatal days 2 and 28. From days 28 to 170, all mice were fed the same amount of standard chow. At the end of the study, femurs were harvested to assess the following bone microarchitecture parameters: (A) cancellous bone volume and tissue volume ratio (BV / TV, %); (B) cancellous bone mineral density (Tb.BMD, mg HA / ccm); (C) cortical bone volume (Ct.BV, mm3); (D) medio-lateral diameter (ML diameter, mm); (E) anterior-posterior diameter (AP diameter, mm); (F) force yield (N); and (G) stiffness (N / mm). (H) and (I) show examples of cancellous and cortical bone structures obtained by micro-CT, respectively. (J) shows the orientation in the medio-lateral and antero-posterior diameters. [Figure 4-3]Effects of lactoferrin-osteopontin soluble complex (soluble), lactoferrin-osteopontin coacervate complex (coacervate), and lactoferrin-osteopontin blend (blend) on bone development, growth, and strength. C57 / bl6 wild-type mice were orally supplemented with three different osteopontin-lactoferrin mixtures (soluble, blend, or coacervate complex, n=10 per group) between postnatal days 2 and 28. From days 28 to 170, all mice were fed the same amount of standard chow. At the end of the study, femurs were harvested to assess the following bone microarchitecture parameters: (A) cancellous bone volume and tissue volume ratio (BV / TV, %); (B) cancellous bone mineral density (Tb.BMD, mg HA / ccm); (C) cortical bone volume (Ct.BV, mm3); (D) medio-lateral diameter (ML diameter, mm); (E) anterior-posterior diameter (AP diameter, mm); (F) force yield (N); and (G) stiffness (N / mm). (H) and (I) show examples of cancellous and cortical bone structures obtained by micro-CT, respectively. (J) shows the orientation in the medio-lateral and antero-posterior diameters.

[0023] [Mode for carrying out the invention] definition As used herein, the following terms have the following meanings.

[0024] The term "complex coacervate" is well known in the art. Complex coacervates are understood as spherical droplets composed of at least two different classes of proteins that are held together primarily by electrostatic forces from the surrounding aqueous liquid. Cooper et al. [Current Opinion in Colloid and Interface Science, (2005), 10, 52-78] define complex coacervates by the separation of a polymer solution composed of at least two types of macromolecules (typically oppositely charged polyelectrolytes) into two immiscible liquid phases. In this case, complex coacervates are defined as either a macroscopic phase concentrated with macromolecules obtained after phase separation by association, or concentrated droplets of macromolecules obtained after mixing two dispersions containing oppositely charged macromolecules, i.e., proteins in this particular case. Complex coacervates are formed by the aggregation of macromolecular complexes formed between two oppositely charged macromolecules (proteins or polysaccharides) to lower the free energy of the mixture, as thermodynamically pointed out by Schmitt et al. [Handbook of Hydrocolloids, Second Edition. Woodhead Publishing, 2009, pp. 420-476].

[0025] In general, the formation of macromolecular complexes and subsequent coacervates is due to electrostatic interactions, and coacervate formation occurs when aggregates of macromolecular complexes reach the electrostatic limit of colloidal stability, i.e., when the surface ζ potential is between -15 and +15 mV. Complex coacervates are distinct from soluble complexes.

[0026] The term "infant" means a child under the age of 12 months.

[0027] The term "young children" refers to children between 1 and 7 years of age. The expression "nutritional composition" refers to a composition that provides nutrition to a subject. This nutritional composition is usually administered orally or intravenously and usually comprises a lipid or fat source and a protein source.

[0028] In a particular embodiment, the composition of the present invention is a "synthetic nutritional composition". The expression "synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological means, which mixture can be chemically identical to a mixture naturally occurring in mammalian milk (i.e., the synthetic composition is not human milk).

[0029] As used herein, the expression "infant formula" or IF refers to a food intended for specific nutritional use in infants during the first month of life, which meets the nutritional requirements of infants falling within this category by the milk itself (Article 2(c) of Commission Directive 91 / 321 / EEC 2006 / 141 / EC of 22 December 2006 on infant and follow-on formulas). The expression also refers to nutritional compositions intended for infants and as defined in Codex Alimentarius (Codex STAN72-1981), and special infant foods, including foods for specific medical purposes. The expression "infant formula" encompasses both "infant starter formulas" and "follow-up formulas" or "follow-on formulas".

[0030] A "follow-up formula" or "follow-on formula" is given from 6 months onwards. Such formulas constitute the main liquid component of the increasingly diverse diet of infants in this category. The expression "infant food" means a food intended for a specific nutritional use by infants or young children during the first few years of life. The expression "infant cereal composition" means a food intended for a specific nutritional use by infants or young children during the first few years of life.

[0031] The term "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula.

[0032] The term "probiotic" means live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (FAO / WHO, 2002). Microbial cells are broadly defined as bacteria or yeasts. All percentages (%) are by weight unless otherwise stated.

[0033] Method for producing the composite Complex coacervates The present invention relates to a complex coacervate comprising lactoferrin and osteopontin.

[0034] In certain embodiments, complex coacervates may contain protein in an amount of 50% w / w or less. In certain embodiments, complex coacervates may preferably contain protein in an amount of 40% w / w or less. In certain embodiments, complex coacervates may more preferably contain protein in an amount of 35% w / w or less.

[0035] In certain embodiments, the complex coacervates may contain protein in an amount of at least 5% w / w. In certain embodiments, the complex coacervates may preferably contain protein in an amount of at least 15% w / w. In certain embodiments, the complex coacervates may more preferably contain protein in an amount of at least 25% w / w.

[0036] In certain embodiments, the complex coacervate may contain protein in an amount of 5 to 50% w / w. In certain embodiments, the complex coacervate may preferably contain protein in an amount of 15 to 40% w / w. In certain embodiments, the complex coacervate may more preferably contain protein in an amount of 25 to 35% w / w.

[0037] The amount of protein in a complex coacervate can be determined using the method according to AOAC 991.20-1994.

[0038] In certain embodiments, complex coacervates may contain water in an amount of 95% w / w or less. In certain embodiments, complex coacervates may contain water in an amount of 85% w / w or less. In certain embodiments, complex coacervates may even more preferably contain water in an amount of 75% w / w or less.

[0039] In certain embodiments, the complex coacervates may contain water in an amount of at least 50% w / w. In certain embodiments, the complex coacervates may contain water in an amount of at least 60% w / w. In certain embodiments, the complex coacervates may even more preferably contain water in an amount of at least 65% w / w.

[0040] In certain embodiments, the complex coacervates may contain water in an amount of 50-95% w / w. In certain embodiments, the complex coacervates may contain water in an amount of 60-85% w / w. In certain embodiments, the complex coacervates may contain water in an amount of 65-75% w / w, even more preferably.

[0041] The amount of water and protein in complex coacervates can be measured using methods according to ISO 5537:2004.

[0042] In certain embodiments, the complex coacervates have a particle size of at least 500 nm in the shortest dimension. In certain embodiments, the complex coacervates preferably have a diameter of at least 600 nm in the shortest dimension. In certain embodiments, the complex coacervates more preferably have a diameter of at least 700 nm in the shortest dimension. In certain embodiments, the complex coacervates more preferably have a diameter of at least 900 nm in the shortest dimension.

[0043] The diameter of the complex coacervates can be measured using dynamic light scattering or by microscopy.

[0044] In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is 8 or less. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably 6 or less. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably 5.5 or less. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably 5 or less. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably 4.2 or less.

[0045] In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is at least 2. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably at least 3. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably at least 3.2. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably at least 3.5. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably at least 3.8.

[0046] In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is in the range of 2 to 8. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably in the range of 3 to 6. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably in the range of 3.2 to 5.5. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably in the range of 3.5 to 5. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably in the range of 3.8 to 4.2. In certain embodiments, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably 4.

[0047] In certain embodiments, the complex coacervates have a zeta potential in the range of -15 to +15 mV. In certain embodiments, the complex coacervates may preferably have a zeta potential in the range of -10 to +10 mV. In certain embodiments, the complex coacervates may preferably have a zeta potential in the range of -8 to +5 mV.

[0048] Zeta potential can be measured using a Malvern Zetasizer Nano-ZS (Malvern Instruments Inc., Worchestershire, UK) equipped with Malvern Zetasizer software 7.02.

[0049] Manufacturing method The present invention also relates to a method for producing a complex coacervate according to the invention, the method comprising the steps of: a. providing separate aqueous solutions containing lactoferrin and osteopontin; b. mixing individual aqueous solutions containing lactoferrin and osteopontin at a pH of 4 to 6, preferably pH 4.5 to 5.5, more preferably pH 4.8 to 5.2, and even more preferably pH 5, wherein the individual aqueous solutions containing lactoferrin and osteopontin are adjusted so that the protein mass ratio of lactoferrin to osteopontin is in the range of 2 to 8, preferably in the range of 3 to 6, more preferably in the range of 3.2 to 5.5, more preferably in the range of 3.5 to 5, even more preferably in the range of 3.8 to 4.2, and even more preferably in the range of 4; Includes.

[0050] According to the present invention, in step a., separate aqueous solutions containing lactoferrin and osteopontin are provided, whereby it is understood that a separate aqueous solution containing lactoferrin and a separate solution of osteopontin are prepared and provided.

[0051] In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is adjusted to a specific pH before mixing. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is adjusted to a pH of 4 to 6, preferably a pH of 4.5 to 5.5, more preferably a pH of 4.8 to 5.2, and even more preferably a pH of 5.

[0052] In an alternative specific embodiment, the individual aqueous solutions containing lactoferrin and osteopontin are not adjusted to a specific pH prior to mixing.

[0053] According to the present invention, in step b., the individual aqueous solutions containing lactoferrin and osteopontin are mixed at a pH of 4 to 6. It is thereby understood that the individual aqueous solutions containing lactoferrin and osteopontin are mixed and adjusted to a pH of 4 to 6 by addition of an acid, preferably an aqueous HCl solution, or a base, preferably an aqueous NaOH solution.

[0054] In certain embodiments, the individual aqueous solutions containing lactoferrin and osteopontin are mixed at a pH of 4.5 to 5.5. In certain embodiments, the individual aqueous solutions containing lactoferrin and osteopontin are mixed preferably at a pH of 4.8 to 5.2. In certain embodiments, the individual aqueous solutions containing lactoferrin and osteopontin are mixed even more preferably at a pH of 5.

[0055] According to the present invention, in step b., the individual aqueous solutions containing lactoferrin and osteopontin are prepared so that the protein mass ratio of lactoferrin to osteopontin in the mixed solution is in the range of 2-8.

[0056] In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the protein mass ratio in the mixed solution is in the range of 3 to 6. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is more preferably adjusted so that the protein mass ratio in the mixed solution is in the range of 3.2 to 5.5. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is more preferably adjusted so that the protein mass ratio in the mixed solution is in the range of 3.5 to 5. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is even more preferably adjusted so that the protein mass ratio in the mixed solution is in the range of 3.8 to 4.2. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is even more preferably adjusted so that the protein mass ratio in the mixed solution is in the range of 4.

[0057] In a specific embodiment, in step b., the ionic strength in the mixed aqueous solution corresponds to 30 mM or less of added salt. By this, it is understood that the mixed aqueous solution does not contain more than 30 mM of added salt, which may be added when preparing individual solutions of lactoferrin and osteopontin and / or added during step b. of mixing individual solutions of lactoferrin and osteopontin, i.e., the total amount of added salt contained in the mixed solution. By this, it is understood that the salt is not positively and / or negatively charged lactoferrin and / or osteopontin. In a specific embodiment, the salt is understood as an inorganic salt. In a specific embodiment, the salt is understood as NaCl.

[0058] In certain embodiments, the ionic strength in the mixed aqueous solution preferably corresponds to 20 mM or less of salt. In certain embodiments, the ionic strength in the mixed aqueous solution more preferably corresponds to 10 mM or less of salt. In certain embodiments, the ionic strength in the mixed aqueous solution is more preferably 5 mM or less. In certain embodiments, the ionic strength in the mixed aqueous solution even more preferably corresponds to 0.2 mM or less of salt.

[0059] In a particular embodiment, in step b., the individual aqueous solutions containing lactoferrin and osteopontin are adjusted so that the total protein concentration in the mixed solution is below the point of self-suppression.

[0060] In certain embodiments, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is less than 8% by weight / volume.In certain embodiments, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is preferably less than 6% by weight / volume.

[0061] In certain embodiments, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is more than 2% by weight / volume.In certain embodiments, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is more than 4% by weight / volume.

[0062] In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is in the range of 2 to 8 wt / vol %. In a specific embodiment, each aqueous solution containing lactoferrin and osteopontin is preferably adjusted so that the total protein concentration in the mixed solution is in the range of 4 to 6 wt / vol %.

[0063] In certain embodiments, the complex coacervates formed in the methods according to the invention can be isolated by any method known to one of skill in the art.

[0064] In certain embodiments, the complex coacervates may be subjected to a drying process such as spray drying, belt drying, tumble drying, and / or freeze drying.

[0065] Compositions Comprising Complex Coacervates The present invention also relates to a composition comprising a complex coacervate according to the invention.

[0066] In certain embodiments, the composition may comprise a complex coacervate according to the invention and a soluble complex of lactoferrin and osteopontin, hi certain embodiments, the composition comprises a complex coacervate according to the invention and a soluble complex of lactoferrin and osteopontin obtained from a method for producing a complex coacervate according to the invention.

[0067] The composition may be any type of composition in which the complex may be incorporated, such as a composition in the form of a food or beverage product, an animal feed product, a dietary supplement for humans or animals, a pharmaceutical composition, or a cosmetic composition. The product may be in solid, liquid, or semi-liquid form.

[0068] Food and beverage products include all products intended for oral consumption by humans for the purpose of providing nutrition and / or pleasure. The product may be, for example, a nutritional composition for infants and / or small children, for pregnant or lactating women or women who wish to become pregnant, for individuals who require special nutrition due to poor health, or for the elderly, etc. More preferably, the nutritional composition is selected from infant formulas, infant cereals, follow-up formulas, growing-up milk, and milk products for pregnant and lactating women or women who wish to become pregnant. Other examples of food and beverage products include milk products or dairy products such as yogurt, soups, sauces, sweet and savory snacks, powdered drinks, and cereal products.

[0069] The product may also be in the form of an animal feed product or a dietary supplement for animals. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc.

[0070] Dietary supplements typically exist in the form of liquids, gels, powders, or tablets or capsules. Powdered supplements typically include supplements that are to be dissolved in water or sprinkled on food or beverages. Such supplements are intended to provide additional nutrients and / or health benefits to the subjects who consume such foods, as well as other beneficial ingredients, such as lactoferrin, lactadherin, and / or lysozyme. Dietary supplements according to the present invention can be used to provide nutrients and / or health benefits to humans and animals, as defined above. Dietary supplements include, for example, powdered supplements that are added to breast milk, for example, for premature or low birth weight infants. Dietary supplements also include supplements for pregnant or lactating women, or for women who wish to become pregnant.

[0071] Pharmaceutical products include, for example, drop, syrup, powder, tablet, or capsule products intended for the treatment or prevention of an adverse medical condition in a subject in need thereof.

[0072] Cosmetic compositions are typically intended for a cosmetic effect on the body and may be for topical application or may be administered by the oral route.

[0073] The compositions of the present invention preferably contain a therapeutically effective amount of the complex coacervate of the present invention.

[0074] In a preferred embodiment, the composition of the invention is an infant formula, a starter infant formula, a follow-on formula, a baby food, an infant cereal composition, a growing up milk, a fortifier such as a human milk fortifier, or a food supplement. In such compositions, the complex coacervates of the invention are preferably present in an amount to provide from 0.001 to 3 g, preferably from 0.01 to 2 g, more preferably from 0.1 to 1 g, of lactoferrin per litre of composition.

[0075] All types of compositions according to the invention can be formulated and manufactured according to the knowledge of the person skilled in the art. The complex coacervates of the invention are advantageously robust enough to be processed together with the other raw materials of the composition.

[0076] For example, in the manufacture of spray-dried products such as powdered infant formula, growing-up milk, or follow-up formula, the complex coacervates of the present invention are robust enough to be added into a wet mix and spray-dried together with other raw materials of the product. This is advantageous in terms of process economics, since aseptic dosing and dry mixing of sensitive proteins such as lactoferrin is not required. In addition, the incorporation of the complex into the wet mix is ​​advantageous in terms of product structure. In particular, the complex coacervates of the present invention will be mixed homogeneously with other raw materials. In contrast, dry-mixed powders may result in inhomogeneous products due to differences in the properties of the powders to be mixed, such as differences in density or particle size. Inhomogeneity may lead to inaccurate dosing of proteins.

[0077] Therefore, an object of the present invention is also a method for the preparation of a composition selected from an infant formula, a follow-up formula or a growing-up milk in powder form, comprising the steps of preparing a product concentrate comprising the complex coacervate of the present invention and spray-drying said product concentrate. The preparation of the wet mixture and the spray-drying are carried out according to the general knowledge of the person skilled in the art.

[0078] The present invention also provides a method for the preparation of a composition selected from liquids, infant formulas, starter infant formulas, follow-on formulas, baby foods, infant cereal compositions, growing-up milks, fortifiers such as human milk fortifiers, or food supplements, comprising the steps of mixing a complex of the present invention into a liquid product base and aseptically processing the product base containing the complex. The preparation and aseptic processing of the liquid product base are carried out according to the general knowledge of the person skilled in the art.

[0079] The compositions of the present invention may further comprise at least one probiotic, such as a probiotic bacterial strain (ie, a probiotic strain).

[0080] The most commonly used probiotic microorganisms are bacteria and yeasts, mainly from the following genera: Lactobacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp. In some particular embodiments, the probiotic is a probiotic strain. In some particular embodiments, this is in particular Bifidobacteria and / or Lactobacilli.

[0081] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 available under the trade name LGG from Valio Oy (Finland), Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM 1-21 16, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivarius DSM 13084 sold under the designation KI2 by BLIS Technologies Limited (New Zealand), Bifidobacterium lactis CNCM 1-3446, among others, Bb from the Christian Hansen company (Denmark). Examples of such bacteria include Bifidobacterium lactis CNCM 1-3446 sold under the trademark BB536 by Morinaga Milk Industry Co., Ltd. (Japan), Bifidobacterium longum ATCC BAA-999 sold under the trademark BB536 by Morinaga Milk Industry Co., Ltd. (Japan), Bifidobacterium breve sold under the trademark Bb-03 by Danisco, Bifidobacterium breve sold under the trademark M-16V by Morinaga, Bifidobacterium infantis sold under the trademark Bifantis by Procter & Gamble Co., and Bifidobacterium breve sold under the trademark R0070 by Institut Rosell (Lallemand). The compositions according to the invention typically contain 10e3-10e12 cfu of probiotic strain per gram of composition on a dry weight basis, more preferably 10e7-10e12 cfu of probiotic strain per gram of composition. In one embodiment, the probiotic is live. In another embodiment, the probiotic is non-replicating or inactivated.In some other embodiments, both viable and inactivated probiotics may be present.

[0082] The compositions of the present invention may further comprise at least one type of non-digestible oligosaccharide other than the aforementioned human milk oligosaccharides (e.g., prebiotics), typically in an amount of 0.3-10% by weight of the composition.

[0083] Prebiotics are usually indigestible in the sense that they are not broken down or absorbed in the stomach or small intestine. Thus, they remain intact when they enter the large intestine, where they are selectively fermented by beneficial bacteria. Examples of prebiotics include certain oligosaccharides, such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS). Combinations of prebiotics, such as 90% GOS and 10% short-chain fructooligosaccharides, such as the product by BENEO-Orafti sold under the trademark Orafti® Oligofructose (formerly Raftilose®), or 10% inulin, such as the product by BENEO-Orafti sold under the trademark Orafti® Inulin (formerly Raftiline®), may be used. A particularly preferred combination of prebiotics is 70% short-chain fructooligosaccharides and 30% inulin, which is the product sold by BENEO-Orafti under the trademark "Prebio 1".

[0084] The composition of the invention may further comprise at least one phage (bacteriophage) or a mixture of phages, preferably against pathogenic Streptococci, Haemophilus, Moraxella, and Staphylococci. The composition according to the invention may be a nutritional composition, a preparation, or a food product.

[0085] The composition according to the invention may be a nutritional composition, such as a synthetic nutritional composition, the composition according to the invention may be an infant formula, a starter infant formula, a follow-on formula, a baby food, an infant cereal composition, a growing up milk, a fortifier such as a human milk fortifier, or a food supplement.

[0086] When the composition is a nutritional supplement, it can be provided in the form of a unit dose. In some embodiments, the composition of the present invention is typically an infant formula.

[0087] The compositions of the present invention are typically used for infants or young children born by Caesarean section.

[0088] These infants and young children represent a particular group of subjects requiring particular needs and care, and the inventors have surprisingly found that compositions comprising at least one human milk oligosaccharide and / or precursor thereof are particularly effective for use in reducing the incidence of necrotizing enterocolitis in these infants born by Caesarean section.

[0089] The compositions according to the invention may be used in term or preterm infants born by Caesarean section.

[0090] Advantageously, the composition of the invention is intended for use in full-term or premature infants, especially those born by Caesarean section.

[0091] In some embodiments, the compositions of the present invention are for use in infants who are small for gestational age and born by Caesarean section, hi some embodiments, the compositions according to the present invention may be for use before and / or during the weaning period.

[0092] The compositions of the present invention may be in solid (eg, powder), liquid, or gelatinous form.

[0093] As it is particularly intended for infants born by Caesarean section, the composition may advantageously be a nutritional composition to be taken in liquid form.The composition of the present invention may be a nutritionally complete formula such as an infant formula, a starter formula, a follow-on formula, or a fortifier such as a human milk fortifier.

[0094] The composition according to the invention also generally contains a protein source, preferably in an amount of less than 2.0 g per 100 kcal, and even more preferably in an amount of less than 1.8 g per 100 kcal. The type of protein is not considered to be critical in the present invention, provided that the minimum requirements for essential amino acid content are met and satisfactory growth is guaranteed. Thus, not only soy-based protein sources may be used, but also protein sources based on whey, casein, and mixtures thereof. As for whey protein, the protein source may be based on acid whey or sweet whey or mixtures thereof, and may contain α-lactalbumin and β-lactoglobulin in any desired ratio.

[0095] The protein may be a protein in its native state or a hydrolyzed protein, or a mixture of proteins in its native state and hydrolyzed protein. The term "intact" means that the majority of the protein is in its native state, i.e., its molecular structure is unchanged, for example, at least 80% of the protein is unchanged, for example, at least 85% of the protein is unchanged, preferably at least 90% of the protein is unchanged, even more preferably at least 95% of the protein is unchanged, for example, at least 98% of the protein is unchanged. In a particular embodiment, the protein is completely unchanged.

[0096] The term "hydrolyzed" in the context of the present invention means that a protein has been hydrolyzed or broken down into its constituent peptides or amino acids.

[0097] The protein may be either fully hydrolyzed or partially hydrolyzed. For example, it may be desirable to provide partially hydrolyzed protein (degree of hydrolysis 2-20%) to infants or young children who are considered to be at risk of developing cow's milk allergy. If hydrolyzed protein is required, the hydrolysis process may be carried out as desired and as known in the art. For example, whey protein hydrolysates may be prepared by enzymatic hydrolysis of whey protein fractions in one or more steps. It has been found that if the whey fraction used as raw material is substantially lactose-free, the protein undergoes significantly less lysine blackage during the hydrolysis process. This allows the degree of lysine blackage to be reduced from about 15% total lysine by weight to less than about 10% lysine by weight, e.g., about 7% lysine by weight, significantly improving the nutritional value of the protein source. In one embodiment of the invention, at least 70% of the protein is hydrolyzed, preferably at least 80% of the protein is hydrolyzed, such as at least 85% of the protein is hydrolyzed, even more preferably at least 90% of the protein is hydrolyzed, such as at least 95% of the protein is hydrolyzed, especially at least 98% of the protein is hydrolyzed. In a particular embodiment, 100% of the protein is hydrolyzed.

[0098] In a particular embodiment, the composition according to the invention is a hypoallergenic composition. In another particular embodiment, the composition according to the invention is a hypoallergenic nutritional composition.

[0099] The composition according to the present invention usually contains a carbohydrate source.Containing a carbohydrate source is particularly preferred when the nutritional composition of the present invention is an infant formula.In this case, any carbohydrate source such as lactose, saccharose, maltodextrin, starch and their mixtures that are conventionally found in infant formulas may be used, but the preferred carbohydrate source is lactose.

[0100] The composition according to the invention generally contains a lipid source. This is particularly appropriate when the nutritional composition of the invention is an infant formula. In this case, the lipid source may be any lipid or fat suitable for use in infant formula. Some suitable fat sources include palm olein, high oleic sunflower oil, and high oleic safflower oil. The essential fatty acids linoleic acid and olinolenic acid may also be added, as well as small amounts of oils, such as fish oils or microbial oils rich in preformed arachidonic acid and docosahexaenoic acid. The fat source preferably has a ratio of n-6 fatty acids to n-3 fatty acids of about 5:1 to about 15:1, for example about 8:1 to about 10:1.

[0101] The composition of the present invention also preferably contains all vitamins and minerals that are understood to be essential in the daily diet and in nutritionally meaningful amounts. For certain vitamins and minerals, minimum requirements have been established. Examples of minerals, vitamins, and other nutrients that may be optionally present in the composition of the present invention include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D, folic acid, inositol, niacin, biotin, pantothenic acid, choline, calcium, phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine, and L-carnitine. Minerals are usually added in salt form. The presence and amount of certain minerals and other vitamins will vary depending on the target population. If necessary, the composition of the present invention may contain emulsifiers and stabilizers, such as soybean, lecithin, citrate esters of mono- and diglycerides, and the like.

[0102] The compositions of the invention may also contain other substances, such as nucleotides, nucleosides, etc., which may have beneficial effects.

[0103] The compositions according to the invention can be prepared in any suitable manner. The compositions will now be described by way of example.

[0104] For example, a formula, such as an infant formula, can be prepared by blending together a protein source, a carbohydrate source, and a fat source in the appropriate proportions. If used, an emulsifier can be included at this point. Vitamins and minerals may be added at this point, but are typically added later to avoid thermal degradation. Any lipophilic vitamins and emulsifiers, etc., can be dissolved in the fat source prior to blending. Water, preferably reverse osmosis treated water, can then be mixed to form a liquid mixture. The temperature of the water is suitably in the range of about 50°C to about 80°C to aid in the dispersion of the ingredients. A commercially available liquefying device can be used to form the liquid mixture.

[0105] If the final product is a powder, this may be added at this stage as well, if desired.The liquid mixture is then homogenized, for example, in two stages.

[0106] The liquid mixture can then be subjected to a heat treatment to reduce the bacterial load, for example, by rapidly heating the liquid mixture for about 5 seconds to about 5 minutes to a temperature in the range of about 80° C. to about 150° C. This heating may be performed by steam injection, by autoclave, or by a heat exchanger, for example a plate heat exchanger.

[0107] The liquid mixture may then be cooled, for example by flash cooling, to about 60°C to about 85°C. The liquid mixture may then be homogenized again, for example in two stages, at about 10 MPa to about 30 MPa in the first stage and about 2 MPa to about 10 MPa in the second stage. The homogenized mixture may then be further cooled and any heat-sensitive ingredients, such as vitamins and minerals, may be added. The pH and solids content of the homogenized mixture are typically adjusted at this point.

[0108] If the final product is a powder, the homogenized mixture is transferred to a suitable drying device, such as a spray dryer or freeze dryer, and converted to a powder. The powder should have a moisture content of less than about 5% by weight. Human milk oligosaccharide(s) and / or precursors thereof may be added at this stage, along with the probiotic strain(s) (if used), by dry mixing or by blending them in crystalline syrup form, and the mixture is spray-dried or freeze-dried.

[0109] If a liquid composition is preferred, the homogenized mixture may be sterilized and then aseptically filled into suitable containers, or may be filled into the containers first and then retorted.

[0110] Therapeutic and prophylactic uses The complex coacervates of the invention or the compositions of the invention can be advantageously used in therapy. The invention therefore also provides such complex coacervates and such compositions for use in therapy or prophylaxis. In a particularly preferred embodiment, the invention provides complex coacervates of the invention and compositions comprising such complex coacervates for use in therapy or prophylaxis.

[0111] Treatment is intended herein as the cure or prevention of a disease or dysfunction of the body, and also encompasses prophylactic treatment, i.e., the prevention of adverse medical conditions. Treatment is also intended herein to include human and animal treatment. In other words, the present invention relates to a method of treating a subject, comprising administering to the subject a therapeutically effective amount of a conjugate or product according to the present invention.

[0112] The present invention therefore relates to a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin, or a composition comprising a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin, for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0113] Thus, the present invention also relates to a method for treating or preventing metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a complex coacervate.

[0114] The definitions and embodiments for the complex coacervates of the invention and compositions comprising complex coacervates described herein above apply mutatis mutandis to their uses in therapy and prophylaxis.

[0115] The complexes, particularly complex coacervates, and compositions comprising the complexes, particularly complex coacervates, are preferably administered to a subject in need thereof.

[0116] In certain embodiments, the subject is an infant, a young child, or a toddler. In certain embodiments, the subject is an infant. In certain embodiments, the subject is an infant born at term or prematurely, particularly an infant born by Caesarean section.

[0117] The complex coacervates of the present invention are particularly advantageous because they provide lactoferrin to a subject that is highly bioactive even after gastrointestinal digestion, thereby providing lactoferrin to a subject that is bioactive after digestion, thereby providing therapeutic and / or prophylactic benefits of lactoferrin and / or osteopontin even after gastrointestinal digestion.

[0118] The complex coacervates (e.g., in the form of nutritional compositions, food supplements, etc.) may be administered by any suitable route, for example, orally, enterally, or parenterally. In a preferred embodiment, the complex coacervates are administered orally.

[0119] The complex coacervates (e.g., in the form of nutritional compositions, food supplements, etc.) may be administered in any suitable dosage, for example to provide a therapeutically effective amount of the complex coacervates. The complex coacervates may be administered in a dosage of at least 100 mg / kg / day, at least 200 mg / kg / day, at least 300 mg / kg / day, at least 400 mg / kg / day, at least 500 mg / kg / day, or at least 1000 mg / kg / day. The complex coacervates may be administered in a dosage of up to 10000 mg / kg / day, up to 5000 mg / kg / day, up to 4000 mg / kg / day, up to 3000 mg / kg / day, up to 2000 mg / kg / day. In some embodiments, the complex coacervates are administered in a dosage of 100-10000 mg / kg / day, preferably in a dosage of 500-5000 mg / kg / day, more preferably in a dosage of 1000-2000 mg / kg / day.

[0120] Use in promoting bone metabolism and / or bone homeostasis When administered to a subject, the complex coacervates can promote normal bone metabolism and bone homeostasis. Several studies have shown that lactoferrin and osteopontin play a role in bone metabolism and bone homeostasis (see, for example, Naot, D., et al., 2005. Clinical Medicine & Research, 3(2), pp. 93-101; and Si, J., et al., 2020. Medical science monitor: international medical journal of experimental and clinical research, 26, pp. e919159-1).

[0121] Thus, the present invention also provides a complex coacervate comprising lactoferrin and osteopontin for use in promoting bone development, growth, strength and / or healing, or in the prevention and / or treatment of bone disease.

[0122] The present invention also provides a method for promoting bone development, growth, strength and / or healing, or for preventing and / or treating a bone disease, comprising administering to a subject in need thereof a therapeutically effective amount of a complex coacervate comprising lactoferrin and osteopontin.

[0123] The present invention also provides the use of a complex coacervate comprising lactoferrin and osteopontin for the manufacture of a medicament for promoting bone development, growth, strength and / or healing, or for the prevention and / or treatment of a bone disease.

[0124] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in promoting bone development.As used herein, "promoting bone development" can refer to supporting normal bone metabolism and / or bone homeostasis, for example, during childhood and adolescence.During childhood and adolescence, bone is shaped by a process called modeling, which forms new bone at some sites in the bone and removes old bone from other sites of the same bone.Bone remodeling is a lifelong process in which mature bone tissue is removed from the skeleton and new bone tissue is formed.Supporting normal bone metabolism and / or bone homeostasis can refer to supporting normal modeling and / or remodeling of bone (see, for example, Allen, MR and Burr, DB, 2014.Bone modeling and remodeling.Basic and applied bone biology (pp.75-90).Academic Press). Supporting normal bone metabolism and / or bone homeostasis may result in normal bone anatomy and physiology (Clarke, B., 2008. Clinical journal of the American Society of Nephrology, 3 (Supplement 3), pp. S131-S139).

[0125] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in promoting bone growth and / or strength. As used herein, "promoting bone growth and / or strength" may refer to supporting normal bone growth and / or bone strength, for example during childhood and adolescence. Supporting normal bone growth and / or bone strength may result in normal bone anatomy and physiology. Suitable methods and parameters for measuring bone growth and bone strength are known to those skilled in the art (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469(8), pp.2128-2138). Suitably, normal bone growth and / or bone strength may be measured using one or more bone parameters selected from cancellous bone volume and tissue volume ratio (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medial-lateral diameter, anterior-posterior diameter, bone yield strength, and bone stiffness. Suitable methods for measuring these parameters are available to those skilled in the art.

[0126] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in promoting bone healing. As used herein, "promoting bone healing" may refer to supporting normal bone healing, for example after fracture. Fracture is one of the most frequent injuries to the musculoskeletal system. Although fracture treatment has improved greatly in recent decades, the majority of all fractures still exhibit complications, including delayed healing and nonunion (Claes, L., et al., 2012. Nature Reviews Rheumatology, 8(3), pp.133-143). Thus, supporting normal bone healing may prevent, for example, delayed union and / or nonunion. Increasing age may increase the risk of delayed union or nonunion. The complex coacervate may prevent and / or reduce the frequency and / or occurrence and / or severity and / or duration of fracture.

[0127] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of bone disease. As used herein, the term "bone disease" may refer to a medical condition that affects bone, particularly a medical condition associated with a decrease in bone organic matrix. Suitably, the bone disease may be a metabolic bone disease. As used herein, the term "metabolic bone disease" may refer to a bone disorder caused by a deficiency of minerals such as calcium, phosphorus, magnesium, or vitamin D (see, for example, Mays, S., 2007. Advances in human palaeopathology, pp. 215-251). Such disorders may include low bone density, osteoporosis, osteopenia, rickets, osteomalacia, Paget's disease of bone, hypophosphatasia, scurvy, and osteitis fibrosa cystica. In some embodiments, the bone disease is selected from low bone density, osteopenia, osteoporosis, osteomalacia, and rickets.

[0128] Metabolic bone disease is common in premature and / or low birth weight infants and / or infants who have experienced suboptimal intrauterine nutrition, resulting in an increased risk of fractures in these populations (Arch Dis Child Fetal Neonatal Ed 2002 86:F82-F85). Infants, children, and adolescents with growth retardation due to malnutrition and / or disease are also frequently affected by these conditions.

[0129] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of low bone density. Bone mineral density or bone mineral density (BMD) is the amount of bone mineral in bone tissue and is used in clinical medicine as an indirect indicator of osteoporosis and fracture risk. The indicator is measured by a procedure called densitometry. BMD test provides an individual with a measurement called T-score, which is a number obtained from comparing the individual's bone mineral density with the optimal bone mineral density. A subject with low bone mineral density may have a bone mineral density that is more than 1.0 standard deviation lower than the average peak bone mass (average of young healthy adults) measured by DXA (dual energy x-ray absorptiometry).

[0130] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of osteopenia. Osteopenia is a condition characterized by a deficiency of organic bone matrix resulting in a lower than normal amount of bone tissue. Osteopenia can be defined as a bone mineral density 1.0-2.5 standard deviations below the mean peak bone mass as measured by DXA.

[0131] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of osteoporosis. Osteoporosis (from the Greek word "porous bones") is a bone disease that results in an increased risk of fracture. The disease is characterized by under-formation of bone, excessive bone loss, or a combination of both. In osteoporosis, bone mineral density (BMD) is reduced, bone microarchitecture deteriorates, and the amount and type of protein in bone is altered. Osteoporosis may be defined as a bone mineral density that is 2.5 standard deviations or more below the mean peak bone mass measured by DXA.

[0132] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of osteomalacia or rickets. Osteomalacia is a condition in which bone mineral density (BMD) and bone mineral content (BMC) are lower than normal. Osteomalacia in children is usually associated with rickets. Osteomalacia or rickets may show symptoms as diffuse body pain, muscle weakness, and bone fragility. The most common cause of this disease is a lack of vitamin D, which is normally obtained from diet and sun exposure.

[0133] The complex coacervate may enhance bone growth and / or bone strength when administered to a subject. For example, the complex coacervate may enhance bone growth and / or bone strength when administered to a subject, compared to a subject not administered the complex coacervate or a subject administered a different form of lactoferrin and osteopontin (e.g., as a blend or soluble complex). Suitably, the complex coacervate enhances bone growth and / or bone strength compared to the same dose of lactoferrin and osteopontin provided as a soluble complex. Suitably, the complex coacervate enhances bone growth and / or bone strength compared to the same dose of lactoferrin and osteopontin provided as a blend. Suitably, the complex coacervate increases one or more bone parameters selected from cancellous bone volume and tissue volume ratio (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), medial-lateral diameter, anterior-posterior diameter, bone yield strength, and bone stiffness. Suitable methods for determining these parameters are available to those skilled in the art.

[0134] The subject may be any suitable subject. Suitably, the subject may be a mammal. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an animal, preferably the animal is a pet. The pet may be an animal selected from dogs, cats, birds, fish, rodents such as mice, rats and guinea pigs, rabbits, etc.

[0135] The present invention is particularly suitable for infants and young children who are at risk of bone disease, have a family history of bone disease, or have already experienced at least one, preferably several, episodes of bone fracture.The present invention is also particularly suitable for infants and young children who were born prematurely or with low birth weight, or experienced intrauterine growth retardation, or experienced growth retardation due to malnutrition, or experienced diseases such as Crohn's disease and / or celiac disease and / or cancer, or were treated with drugs that cause malabsorption, anorexia, and / or metabolic bone disease, such as chemotherapeutic agents and / or corticosteroids.The present invention is particularly preferred for use with infants and children who were born prematurely or with low birth weight, or experienced intrauterine growth retardation, or were intrauterine malnutrition, or experienced growth retardation.

[0136] In some embodiments, the subject is a juvenile, adolescent, child, or infant. The term "juvenile" may refer to an individual who has not yet reached adulthood. The term "adolescent" may refer to an individual during the period from the onset of adolescence to adulthood. The term "child" may refer to an individual at the stage between birth and adolescence.

[0137] In some embodiments, the subject was born prematurely or with low birth weight, or experienced intrauterine growth retardation. The term "premature infant" may refer to an infant born at a gestational age of at least 37 weeks. The term "low birth weight infant" may refer to an infant whose birth weight is less than 2500 g.

[0138] In some embodiments, the subject has experienced stunting. The definition of stunting may refer to a "height for age" value less than two standard deviations from the WHO Child Growth Standards median (see, for example, De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pp. 12-26). In some embodiments, the subject has experienced stunting due to malnutrition or has experienced a disease such as anorexia, Crohn's disease, and / or celiac disease. In some embodiments, the subject has experienced stunting due to treatment with drugs that cause malabsorption, anorexia, and / or metabolic bone disease, such as chemotherapeutic agents and / or corticosteroids.

[0139] The invention may also be applied to adolescents or adults who are at risk of bone disease or who have experienced at least one, preferably several, episodes of bone fracture, or who were born prematurely or with low birth weight, or who have experienced intrauterine growth retardation, or who have experienced stunting due to malnutrition, or who have experienced diseases such as Crohn's disease and / or celiac disease and / or cancer, or who have been treated with drugs such as chemotherapeutic agents and / or corticosteroids that cause malabsorption, anorexia, and / or metabolic bone disease, or who have experienced growth retardation due to disease or malnutrition or use of drugs during infancy and / or childhood (including adolescence).

[0140] In some embodiments, the subject is an adolescent or adult. In some embodiments, the subject is an adult, preferably the subject is an elderly person. In some embodiments, the subject is at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. The subject may have one or more bone fractures. The subject may have or be at risk of having one or more delayed unions and / or one or more nonunions.

[0141] [Example] f 1.Material Lactoferrin (LF) powder has a total protein, ash, and moisture content of 98.7% (wt / wt, Dumas nitrogen x 6.25), 0.37% (wt / wt), and 0.42% (wt / wt), respectively, as measured according to the official AOAC method (AOAC, 2005). The purity of LF is 95% of total protein (wt / wt, by HPLC at 214 nm). The spray-dried bovine osteopontin (OPN) powder used has a total protein, moisture, and ash content of 89.6% (wt / wt, Dumas nitrogen x 7.17), 2.22% (wt / wt), and 9.2% (wt / wt), respectively. The purity of OPN is 99% of total protein according to the supplier's specifications.

[0142] 2. Preparation of Complex Coacervates Individual protein solutions of LF and OPN were prepared by magnetically stirring the respective powders in ultrapure water (18.2 MΩ.cm) at 25°C for approximately 2 h, followed by magnetic stirring overnight at 4°C (approximately 18 h) to ensure complete rehydration.

[0143] Individual protein solutions of LF and OPN were mixed in 50 mL polypropylene centrifuge tubes. After pH adjustment, these solutions were centrifuged at 3007 relative centrifugal force (RCF) for 20 minutes at 20°C, accelerating the palm separation using a Sorvall RC 5C Plus centrifuge fitted with a Sorvall GSA rotor. For the remainder of the experiment, the mixed solutions were allowed to settle under static conditions at 4°C for approximately 18 hours.

[0144] For samples in which phase separation occurred, mass balance was established by isolating the phases with a micropipette prior to mass measurement using a four-point analytical balance.

[0145] Complex coacervates of lactoferrin and osteopontin are formed at a LF:OPN ratio (protein mass basis) of 2 at pH 4, at LF:OPN ratios (protein mass basis) of 4 and 6 at pH 5, and at a LF:OPN ratio (protein mass basis) of 8 at pH 6.

[0146] Complex coacervates of lactoferrin and osteopontin were formed with the highest coacervation yield at pH 5 and a LF:OPN mixing ratio (on a mass protein basis) of 4. The highest coacervate yield refers to the mixed solution where the greatest % of the total nitrogen is present in the coacervate phase or the least % of the total nitrogen is present in the supernatant phase, e.g., as soluble complexes.

[0147] 3. Measurement of the structure of complex coacervates The microscopic appearance of the LF and OPN complex coacervates was imaged using a Leica DM1000 optical microscope (Leica Microsystems GmbH, DE) equipped with a LabCam® smartphone adapter (iDu Optics, USA) to facilitate digital image capture. The microscope was operated at 40x magnification in both phase contrast and dark field operation modes. To prepare samples for imaging, the LF and OPN complex coacervates (prepared at a 4:1 LF:OPN mass mix ratio, pH 5, and 5% w / v protein) were first isolated and dispersed in water, after which approximately 20 μL of sample was placed between a microscope slide and a cover slip. To ensure representative image capture, samples were imaged at least three times.

[0148] FIG. 1 shows that micrometer-scale LF-OPN coprecipitates and / or self-aggregates of either protein did not coexist with the complex coacervates, as irregularly shaped (solid) flocs were absent and all visible objects were essentially spherical (liquid) in appearance.

[0149] 4. Measurement of thermal stability of complex coacervates In light of the fact that the thermal stability of lactoferrin in complex coacervates is greater than that of lactoferrin alone, the effect of complex coacervation of lactoferrin with osteopontin on lactoferrin and osteopontin is shown by differential scanning calorimetry.

[0150] Differential scanning calorimetry was performed on a T2500 Discovery Differential Scanning Calorimeter (TA Instruments, Crawley, UK). Approximately 75 μL of sample (6%, weight / volume, protein) was transferred to a high-volume stainless steel pan and then zero The pan was sealed using a press (TA Instruments, Crawley, UK). An equal volume of ultrapure water was used as a reference. Samples were equilibrated at 20°C and then heated from 20°C to 100°C at 1°C / min, followed by cooling to 20°C at 10°C / min. Measurements were performed in triplicate and heat flow curves were processed using Trios 8.32 software.

[0151] FIG. 2 shows differential scanning calorimetry thermograms of 5% wt / vol solutions of lactoferrin (dashed line) and osteopontin (dotted line) at pH 5.0 ( FIG. 1 , above), and of a LF / OPN complex coacervate at pH 5.0 (protein mass ratio 4:1, prepared at 5% wt / vol protein) with a protein concentration of 27.4% (wt / wt).

[0152] FIG. 2 thus shows that the LF / OPN complex coacervate has a heat capacity that is different from that of the individual components, and the T of the individual uncomplexed protein peaks is m This shows that the shift is

[0153] Thus, the thermograms in FIG. 2 confirm the formation of complex coacervates between LF and OPN, which resulted in improved thermal stability of LF.

[0154] 5. Measurement of biological activity after gastrointestinal digestion 5.1. Preparation of comparative diets and diets according to the invention A diet containing a combination of infant formula (IF) and protein powders for simulated infant gastrointestinal digestion was dry mixed and then hydrated.

[0155] Combinations were prepared by adding IF (having a protein content of 10.46 wt / wt%, fat of 28.7 wt / wt%, carbohydrate of 53.8 wt / wt%, moisture of 2.2 wt / wt%, and ash of 2.16 wt / wt%) with (1) LF achieving 600 mg / L upon hydration, (2) OPN achieving 150 mg / L, and (3) a soluble complex of LF and OPN or (4) a complex coacervate of LF and OPN achieving 600 mg LF / L.

[0156] Meals were prepared by hydrating the relevant powders in ultrapure water (18.2 MΩ.cm) to a total protein concentration of 1.34% (weight / volume). All meals had the same protein concentration to ensure consistent enzyme:substrate ratios between samples for simulated digestion. Protein concentrations were selected based on the recommended reconstitution ratios for the model formula used.

[0157] 5.2. Simulation of infant gastrointestinal digestion Simulation of infant gastrointestinal digestion was performed using the static in vitro method proposed by Menard et al. (O. Menard et al., Food, Chemistry, 2018, 240, 338-345). The model is based on physiological findings in term infants and the justification for all parameters is available in Menard et al.

[0158] Digestions were performed in 40 mL conical screw-cap amber glass vials that were soaked in 6% (vol / vol) nitric acid to remove residuals before each digestion. The vials were placed in a 37°C water bath and agitated at 50 rpm for the duration of the digestion process (1 h in the stomach and 1 h in the intestine).

[0159] Three independent tests were performed on gastric (pH 5.3) and gastrointestinal (intestinal stage pH 6.6) digestion of each sample. Gastric lipase (19 U / mL) and pepsin (268 U / mL) activities were achieved using rabbit stomach extract (RGE70, Lipolytech, France) and porcine pepsin (Sigma P6887).

[0160] Samples were taken at sequential time points and instantly inactivated by one of three methods depending on the endpoint analysis (described in each section).

[0161] 5.3.Cell culture HT-29 clone 34 cells were used. This cell line is capable of expressing a reporter gene for secreted alkaline phosphatase (SEAP) after activation of the NF-κB signaling pathway, as described in Goulding et al., Food Chemistry, 362, 130142. HT-29 clone 34 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol / vol) heat-inactivated fetal bovine serum (FBS) and 1% (vol / vol) non-essential amino acids. Cells were maintained in T75 culture flasks in an incubator at 37°C with 5% CO2.

[0162] 5.4. Cell culture experiments HT-29 clone 34 cells were cultured as described above. For the experiments, 2 × 10 cells were cultured in 24-well format cell culture plates (Greiner Bio-One, Austria). 5Cells were seeded at 100 / mL and cultured for 3 days before treatment to ensure monolayer confluence. For cell treatment, the medium was removed and treatments were prepared in fresh medium and added to the wells to a final well volume of 1 mL. Cells were treated with either undigested or digested forms of the relevant diet. All treatments were filtered using a 0.45 μm sterile filter before cell administration. Gastric digests harvested for cell culture analysis were preserved by raising the pH to above 7 by using 0.1 M phosphate buffer (to inhibit pepsin activity) before freezing, while gastrointestinal digests were preserved by adding Pefabloc® (to inhibit serine protease activity) to a final concentration of 1 mM before freezing. Human whey (HMS) was added at a concentration of 5% (vol / vol) of the well volume to provide a source of effector molecules, including soluble CD14 (sCD14). The HMS used was prepared as previously reported in Goulding et al., Food Chemistry, 362, 130142. The sCD14 concentration in the HMS was 48 μg / mL. Ethical approval and prior informed consent for the use of human milk donor samples for research purposes was obtained for the samples in this study. Ethical approval was obtained from the Clinical Research Ethics Committee of Cork Teaching Hospitals, Cork, Ireland. Lipopolysaccharide (LPS) from Gram-negative Escherichia coli O111:B4 serotype was used in the experiments (Sigma, L2630). LPS was added at a concentration of 20 ng / mL to all wells except the negative control. After administration of the treatments, the cells were incubated at 37°C and 5% CO2 for 16 hours, after which the cell supernatants were harvested and subsequently analyzed. Phosphatase activity of SEAP was quantified by bioluminescence using the Phosphalight kit (Applied Biosystems, MA, USA) as a marker of NF-κB activation. Bioluminescence was quantified as relative luminescence units (RLU) using a Varioskan Flash multiwell plate reader (Thermo Scientific, MA, USA) with an integration time of 1000 ms.For graphical representation of the data, the LPS treatment was set as 100 relative luminescence units and all other data were normalized to this.

[0163] 5.5. Behavior of Undigested and Digested Lactoferrin, Osteopontin, and Their Complexes or Complex Coacervates in an Intestinal Cell Inflammation Model The effect of adding gastrointestinal digesta of LF, OPN, SC, and CC to the intestinal cell inflammation model is shown in Figure 3. NF-κB transcription factor was activated when cells were stimulated with lipopolysaccharide (LPS, 20 ng / mL) of E. coli O111:B4 serotype. LF, OPN, and all their combinations, when undigested and native, at 0.35 mg protein / mL, produced a statistically significant inhibition of LPS-induced NF-κB activation.

[0164] When intestinal cells were treated with gastrointestinal digests, the LF and OPN digests did not inhibit LPS-induced NF-κB activation, whereas the SC and CC digests both produced statistically significant inhibition. The inhibition of LPS-induced NF-κB activation by the SC and CC gastrointestinal digests was not statistically significant with respect to each other, but was significant when compared with LF alone.

[0165] FIG. 3 shows the effect of adding gastrointestinal digesta of lactoferrin (LF), osteopontin (OPN), LF-OPN soluble complex (SC), or LF-OPN complex coacervate (CC) on an intestinal cell inflammation model. All samples are added at 0.35 mg protein equivalent / mL. Escherichia coli O111:B4 lipopolysaccharide (LPS)-induced NF-κB activation in HT-29 clone 34 cells was used as the inflammation model. LPS (20 ng / mL) and human whey (5% v / v) are added to all wells. All data are normalized to give 100 relative luminescence units (RLU) for LPS treatment and represent the mean ± standard error of triplicate measurements from three independent experiments. "*" represents a statistically significant difference (P<0.05) from 100% RLU treatment.

[0166] Figure 3 shows that the ability of LF to inhibit LPS-induced NF-κB activation requires that LF not be fully proteolyzed. Figure 3 also shows that digestion of pre-complexed or pre-coacervated LF-OPN powders resulted in altered gastrointestinal digesta bioactivity in this model of enterocyte inflammation.

[0167] 6. Lactoferrin-Osteopontin Coacervate Complexes and Bone Development, Growth, and Strength C57 / bl6 wild type mice were orally supplemented between postnatal days 2 and 28 with three different osteopontin-lactoferrin mixtures (soluble, blended, or coacervate complex, n=10 per group): · Bovine lactoferrin-osteopontin soluble complex (soluble) solution, 20% w / v in distilled water, 1250 mg / kg / day, N=8. Bovine lactoferrin-osteopontin coacervate complex (coacervate) solution, 20% weight / volume in distilled water, 1250 mg / kg / day, N=9. Bovine lactoferrin-osteopontin blend (blend) solution, 1000 mg / kg / day of bovine lactoferrin powder 20% w / v in distilled water + 250 mg / kg / day of bovine osteopontin powder, N=9.

[0168] All mice were fed the same amount of standard diet from days 28 to 170. At the end of the study, femurs were harvested to evaluate bone microarchitectural parameters.

[0169] Micro-computed tomography (μCT UCT35, Scanco Medical AG, Basserdorf Switzerland) was used to evaluate the microarchitecture of trabecular and cortical bone investigated in the distal metaphysis and mid-shaft of the femur, respectively, as previously reported (Bonnet N, et al. J Bone Miner Res. 2017; doi / 10 / 1002). Briefly, trabecular and cortical bone areas were evaluated using 10um isotropic voxels. For the femoral trabecular area, 100 slices of primary trabecular bone were analyzed, taken from 100 slices of secondary trabecular bone below the distal growth plate, to remove the primary trabecular bone. Femoral cortical structure was evaluated by 50 continuous CT slides in the mid-shaft of the femur. Morphometric variables were calculated from binarized images using a direct 3D method that does not rely on a priori assumptions about the underlying structure (Bonnet N, et al. Med Phys 2009;36(4):1286-97).

[0170] Bone is composed of cortical (or compact) bone and cancellous (or spongy) bone. Cortical bone accounts for approximately 80% of the bone mass in the human body and has a smaller surface area than cancellous bone due to its lower porosity. Cancellous bone is located at the ends of long bones and accounts for approximately 20% of the total skeletal mass. Examples of cancellous and cortical structures are shown in Figures 4H and 4I, respectively.

[0171] For the cancellous bone region, the bone volume to tissue volume ratio (BV / TV) and cancellous bone mineral density (Tb.BMD, mg HA / ccm) were evaluated. The results are shown in Figure 4A and Figure 4B, respectively. For the cortical bone in the midshaft of the femur, the cortical bone volume (Ct.BV, mm 3 ), anterior-posterior diameter, and medial-lateral diameter were measured (see FIG. 4J). The results are shown in FIG. 4C, FIG. 4D, and FIG. 4E, respectively.

[0172] To test the biomechanical properties of the bone, we performed three-point bending tests as previously reported (Turner CH, Burr DB. Bone 1993;14:595-608). Load was applied to failure in compression mode at a nominal deformation rate of 2 mm / min. Load-displacement curves were recorded during the test. Yield force and stiffness results are shown in Figure 4F and Figure 4G, respectively.

[0173] Overall, the results show that mice supplemented with lactoferrin-osteopontin coacervate complexes exhibited improved bone development, growth, and strength compared to mice supplemented with different forms of lactoferrin-osteopontin, e.g., mice supplemented with lactoferrin-osteopontin coacervate complexes had significantly higher cancellous BV / TV, cancellous bone mineral density, and cortical bone volume.

[0174] Embodiment Various preferred features and embodiments of the present invention are described with reference to the following numbered paragraphs.

[0175] 1. A complex coacervate containing lactoferrin and osteopontin.

[0176] 2. A complex coacervate according to paragraph 1 comprising protein in an amount of from 2.5 to 50% w / w, preferably in an amount of from 15 to 40% w / w, more preferably in an amount of from 25 to 35% w / w.

[0177] 3. A complex coacervate according to any one of paragraphs 1 and 2, comprising water in an amount of 50 to 95% w / w, preferably in an amount of 60 to 85% w / w, even more preferably in an amount of 65 to 75% w / w.

[0178] 4. A complex coacervate according to any one of paragraphs 1 to 3 having a diameter in its shortest dimension of at least 500 nm, preferably at least 600 nm in its shortest dimension, more preferably at least 700 nm in its shortest dimension, more preferably at least 900 nm in its shortest dimension.

[0179] 5. A complex coacervate according to any one of paragraphs 1 to 4, having a zeta potential in the range of -15 to +15 mV, preferably in the range of -10 to +10 mV, more preferably in the range of -8 to +5 mV.

[0180] 6. A method for producing a complex coacervate according to any one of paragraphs 1 to 5, comprising the steps of: a. providing separate aqueous solutions containing lactoferrin and osteopontin; b. mixing the individual aqueous solutions containing lactoferrin and osteopontin at a pH of 4 to 6, preferably 4.5 to 5.5, more preferably 4.8 to 5.2, and even more preferably 5, wherein the individual aqueous solutions containing lactoferrin and osteopontin are adjusted so that the protein mass ratio of lactoferrin to osteopontin is in the range of 2 to 8, preferably in the range of 3 to 6, more preferably in the range of 3.2 to 5.5, more preferably in the range of 3.5 to 5, even more preferably in the range of 3.8 to 4.2, and even more preferably in the range of 4; A method comprising:

[0181] 7. The method according to paragraph 6, wherein in step b., the ionic strength in the mixed aqueous solution is equal to or less than 30 mM, preferably equal to or less than 20 mM, more preferably equal to or less than 10 mM, more preferably equal to or less than 5 mM, even more preferably equal to or less than 0.2 mM of added salt, preferably added inorganic salt, more preferably equal to or less than added NaCl.

[0182] 8. A method according to any one of paragraphs 6 and 7, wherein in step b., the individual aqueous solutions containing lactoferrin and osteopontin are adjusted to have a total protein concentration in the range of 2 to 8% weight / volume, preferably in the range of 4 to 6% weight / volume.

[0183] 9. A composition comprising a complex coacervate according to any one of paragraphs 1 to 5.

[0184] 10. The composition according to paragraph 9, selected from the group consisting of a food composition, a pet food composition, a beverage, a nutritional formula, or a nutraceutical.

[0185] 11. The composition according to any one of paragraphs 9 and 10, which is an infant formula, a starter infant formula, a follow-on formula, a baby food, an infant cereal composition, growing up milk, a fortifier such as a human milk fortifier, or a food supplement.

[0186] 12. A complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0187] 13. A method for treating or preventing metabolic disorders, in particular overweight, obesity, pre-diabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a composition comprising a complex coacervate.

[0188] 14. A complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, for use in promoting bone development, growth, strength and / or healing, or in the prevention and / or treatment of a bone disease.

[0189] 15. A method for promoting bone development, growth, strength and / or healing, or preventing and / or treating a bone disease, by administering to a subject a complex comprising lactoferrin and osteopontin, preferably a complex coacervate, or a composition comprising a complex comprising lactoferrin and osteopontin, preferably a complex coacervate.

Claims

1. A complex coacervate comprising lactoferrin and osteopontin for use in promoting bone development, growth, strength and / or healing in a subject, or in preventing and / or treating a bone disease in a subject.

2. 2. A complex coacervate for use according to claim 1, comprising protein in an amount of 5 to 50% w / w, preferably in an amount of 15 to 40% w / w, more preferably in an amount of 25 to 35% w / w.

3. 3. A complex coacervate for use according to claim 1 or 2, comprising water in an amount of 50 to 95% w / w, preferably in an amount of 60 to 85% w / w, more preferably in an amount of 65 to 75% w / w.

4. 4. A complex coacervate for use according to any one of claims 1 to 3, having a diameter in its shortest dimension of at least 500 nm, preferably at least 600 nm in its shortest dimension, more preferably at least 700 nm in its shortest dimension, and even more preferably at least 900 nm in its shortest dimension.

5. 5. A complex coacervate for use according to any one of claims 1 to 4, having a zeta potential in the range of -15 to +15 mV, preferably in the range of -10 to +10 mV, more preferably in the range of -8 to +5 mV.

6. The complex coacervate for use according to any one of claims 1 to 5, which is administered in the form of a composition.

7. 7. The complex coacervate for use according to claim 6, wherein the composition is selected from the group consisting of a food composition, a pet food composition, a beverage, a nutritional formula, or a nutraceutical.

8. 8. The complex coacervate for use according to claim 6 or 7, wherein the composition is an infant formula, a starter infant formula, a follow-on formula, a baby food, an infant cereal composition, a growing up milk, a fortifier, or a food supplement.

9. 9. The complex coacervate for use according to any one of claims 1 to 8, which is administered orally.

10. 10. The complex coacervate for use according to any one of claims 1 to 9, administered at a dose of 100 to 10000 mg / kg / day, preferably at a dose of 500 to 5000 mg / kg / day, more preferably at a dose of 1000 to 2000 mg / kg / day.

11. 11. The complex coacervate for use according to any one of claims 1 to 10, wherein said complex coacervate enhances bone growth and / or bone strength, preferably wherein said complex coacervate increases one or more bone parameters selected from cancellous bone volume to tissue volume ratio (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct. BV), medial-lateral diameter, anterior-posterior diameter, bone yield strength, and bone stiffness.

12. 12. The complex coacervate for use according to any one of claims 1 to 11, wherein the bone disease is a metabolic bone disorder, preferably wherein the bone disease is low bone density, osteopenia, osteoporosis, osteomalacia, or rickets.

13. The subject is a young person, an adolescent, a child, or an infant, preferably (i) the subject was born preterm or of low birth weight, or experienced intrauterine growth retardation; (ii) the subject suffers from stunting due to malnutrition or suffers from a disease such as anorexia, Crohn's disease, and / or celiac disease; and / or (iii) the subject experiences stunting due to treatment with drugs that cause malabsorption, anorexia, and / or metabolic bone disease; A complex coacervate for use according to any one of claims 1 to 12.

14. The complex coacervate for use according to any one of claims 1 to 12, wherein the subject is an adult, preferably the subject has one or more bone fractures.

15. 15. The complex coacervate for use according to any one of claims 1 to 14, wherein the subject is a human being or the subject is an animal, preferably the subject is a human being.

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