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 for use 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 use in promoting bone development, growth, strength and / or healing or in the prevention and / or treatment of 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. In addition, the present invention relates to a complex comprising lactoferrin and osteopontin for treating and / or preventing metabolic diseases and / or inflammatory diseases. In addition, the present invention relates to a complex comprising lactoferrin and osteopontin for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases. Background Art

[0002] Lactoferrin (LF) and osteopontin (OPN) have been identified to have 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 mammals. This protein is normally found in concentrations of approximately 4.91 g / L and 2.10 g / L in early and mature human milk, respectively, while the concentration in bovine milk is approximately 1 / 10 of that. LF is a protein with relevant biological functions, including antimicrobial, anti-inflammatory, and immunomodulatory effects. Several clinical studies in infant populations have linked LF to 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 infant formula blends, the protein undergoes stretch denaturation and refolding under the influence of temperature and pH, resulting in protein instability and increased viscosity. This phenomenon makes the addition of lactoferrin in a wet state (i.e., before drying) in products such as infant formula very complicated.

[0005] Therefore, it has been described to add lactoferrin in solid form to products such as infant formula by dry mixing after the base powder of the product has been prepared. However, this method is challenging because the lactoferrin added in solid form needs to be sterile. In order to achieve the required sterility level, the lactoferrin needs to be subjected to sterilization techniques. Most sterilization techniques involve the use of high heat, which can lead to denaturation of the lactoferrin. Other sterilization techniques, such as membrane filtration, can be used, but these sterilization techniques can be costly and require specific equipment. In addition, the addition of sterilized lactoferrin in the final product requires specific and accurate sterile dosing equipment.

[0006] Osteopontin (OPN) is a minor, acidic, highly phosphorylated glycoprotein that is also present in higher concentrations in human milk than in bovine milk. The average OPN concentrations in human milk, bovine milk, and infant formula have been reported to be 138 mg / L, 18 mg / L, and 9 mg / L, respectively. OPN has several biological functions, including the ability to stimulate immune, brain, and intestinal development. It has also been reported that supplementation of infant formula with OPN reduces the incidence of fever and alters plasma cytokine patterns, resulting in decreased levels of proinflammatory TNF-α and increased levels of interleukin-2.

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

[0008] Furthermore, it would be highly desirable to enhance the bioactivity and / or bioavailability of lactoferrin, particularly when administered in combination with osteopontin to a subject, particularly a subject suffering from a metabolic disease and / or an inflammatory disease.

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

[0010] In addition, lactoferrin can also promote bone growth. At physiological concentrations, lactoferrin effectively stimulates the proliferation and differentiation of primary osteoblasts, and also acts as a survival factor for inhibiting apoptosis induced by serum deprivation. Lactoferrin also affects osteoclast formation and can effectively inhibit osteoclastogenesis (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 homeostasis. Osteopontin is an important factor in neuron-mediated and endocrine regulation of bone mass, and is involved in biological activities such as proliferation, migration and adhesion of several bone-related cells. It has been shown that osteopontin is closely related to the occurrence and development of many bone-related diseases (including osteoporosis) (Si, J. et al., 2020. Medical science monitor: international medical journal of experimental and clinical research, 26, p. e919159-1).

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

[0013] Attempts to form aggregates of lactoferrin and osteopontin have so far appeared unsuccessful. Summary of the invention

[0014] The present invention relates to complex coacervates comprising lactoferrin and osteopontin.

[0015] The present invention also relates to a method for preparing a complex coacervate according to any of the preceding claims, wherein the method comprises the following steps:

[0016] a. providing a separate aqueous solution comprising lactoferrin and osteopontin,

[0017] b. mixing the separate aqueous solutions comprising lactoferrin and osteopontin at a pH of 4 to 6, preferably at a pH of 4.5 to 5.5, more preferably at a pH of 4.8 to 5.2, even more preferably at a pH of 5, and wherein the separate aqueous solutions comprising lactoferrin and osteopontin are adjusted such 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.

[0018] The invention also relates to a composition comprising the complex coacervate according to the invention.

[0019] The present invention also relates to a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, prediabetes or diabetes and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0020] The present invention also relates to a method for treating or preventing metabolic disorders, in particular overweight and obesity, prediabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate comprising lactoferrin and osteopontin.

[0021] 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 preventing and / or treating bone diseases.

[0022] The present invention also relates to a method for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of a complex coacervate comprising lactoferrin and osteopontin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Additional features and advantages of the present invention are described in, and will be apparent from, the following description of presently preferred embodiments given with reference to the accompanying drawings, in which:

[0024] Figure 1 : Optical microscopic images (40x magnification) of lactoferrin (LF)-osteopontin (OPN) complex coacervates (prepared at pH 5, 5% w / v protein, LF:OPN mass protein ratio 4:1) that were separated and dispersed in ultrapure water to confirm the presence of spherical liquid coacervates and the absence of irregular solid coprecipitates. Panels A, B: phase contrast microscopy; Panel C: dark field microscopy. All images were acquired using Smartphone microscope adapter capture.

[0025] Figure 2 : 5% w / v solutions of lactoferrin (dashed line) and osteopontin (dotted line) at pH 5.0 (upper Figure 1 ) and a LF / OPN complex coacervate (prepared at a protein mass ratio of 4:1, 5% w / v protein) at pH 5.0, which has a protein concentration of 27.4% (w / w).

[0026] Figure 3 : Effect of adding gastrointestinal digests of lactoferrin (LF), osteopontin (OPN), LF-OPN soluble complex (SC) or LF-OPN complex coacervate (CC) to an enterocyte inflammation model. All samples were added at 0.35 mg protein equivalent / mL. The inflammation model used was NF-κB activation in HT-29 clone 34 cells induced by Escherichia coli O111:B4 lipopolysaccharide (LPS). LPS (20 ng / mL) and human milk serum (5% v / v) were added to all wells. All data were normalized to 100 relative luminescence units (RLU) for LPS treatment and represent the mean ± standard error of three replicate measurements from three independent experiments. "*" represents a statistically significant difference (P<0.05) from 100% RLU treatment.

[0027] Figure 4: Effects of lactoferrin-osteopontin soluble complex (soluble), lactoferrin-osteopontin coacervate complex (coacervate) and lactoferrin-osteopontin blend (coacervate) on bone development, growth and strength. C57 / bl6 wild-type mice were orally supplemented with three different osteopontin-lactoferrin mixtures (soluble, blended or coacervate complexes, n=10 per group) between days 2 and 28 after birth. From day 28 to day 170, all mice received the same amount of standard diet. At the end of the study, femurs were collected to evaluate the following bone microstructural parameters: (A) trabecular bone volume and tissue volume fraction (BV / TV, %); (B) trabecular bone mineral density (Tb.BMD, mg HA / ccm); (C) cortical bone volume (Ct.BV, mm 3 ); (D) medial-lateral diameter (ML diameter, mm); (E) anterior-posterior diameter (AP diameter, mm); (F) yield force (N); and (G) stiffness (N / mm). (H) and (I) show exemplary trabecular and cortical structures obtained by micro-CT, respectively. (J) shows the direction of the medial-lateral diameter and the anterior-posterior diameter; DETAILED DESCRIPTION

[0028] definition

[0029] As used herein, the following terms have the following meanings.

[0030] The term "complex coacervate" is well defined in the art. Complex coacervates are understood to be spherical droplets composed of at least two different types of proteins, which 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 coacervate by separating a macromolecular solution composed of at least two macromolecules (usually oppositely charged polyelectrolytes) into two immiscible liquid phases. In this case, complex coacervates are defined as macroscopic phases concentrated in macromolecules obtained after associating phase separation or droplets concentrated in macromolecules obtained after mixing two dispersions containing oppositely charged macromolecules (i.e., proteins in this particular case). Thermodynamically, complex coacervates are formed by the aggregation of macromolecular complexes formed between two oppositely charged macromolecules (proteins or polysaccharides) to reduce the free energy of the mixture, as pointed out by Schmitt et al. [Handbook of Hydrocolloids, Second Edition, Woodhead Publishing, 2009, Pages 420-476]. Typically, macromolecular complexes and subsequent condensate formation are mediated by electrostatic interactions and condensates form when aggregates of macromolecular complexes reach the electrostatic limit of colloidal stability, i.e., surface zeta potentials between -15 mV and +15 mV. Complex condensates are distinct from soluble complexes.

[0031] The term "infant" refers to a child under the age of 12 months.

[0032] The term "young child" refers to a child between the ages of one and seven years. The expression "nutritional composition" refers to a composition for nourishing an individual. Such nutritional compositions are usually administered orally or intravenously and they usually include a lipid or fat source and a protein source.

[0033] In a specific embodiment, the composition of the invention is a "synthetic nutritional composition". The expression "synthetic nutritional composition" refers to a mixture obtained by chemical and / or biological methods, the chemical properties of which may be identical to the mixture naturally present in mammalian milk (ie, the synthetic composition is not breast milk).

[0034] As used herein, the expression "infant formula" or IF refers to a food intended for specific nutritional purposes in the first few months after the birth of an infant, and which itself meets the nutritional needs of such people (in accordance with Article 2 (c) of Directive 91 / 321 / EEC 2006 / 141 / EC of the European Commission of 22 December 2006 on infant formula and follow-up infant formula). It also refers to a nutritional composition intended for infants, and as defined in the Codex Alimentarius Commission (Codex STAN 72-1981) and infant special products (including foods for special medical purposes). The expression "infant formula" covers both "starter infant formula" and "follow-up formula" or "follow-on formula".

[0035] "Second infant formula" or "follow-on formula" is given from the 6th month onwards. Infant formula constitutes the main liquid element in the gradually diversified diet of such persons. The expression "baby food" means a foodstuff intended for specific nutritional use by infants or young children during the first years of life. The expression "infant cereal composition" means a foodstuff intended for specific nutritional use by infants or young children during the first years of life.

[0036] The term "fortifier" refers to a liquid or solid nutritional composition suitable for mixing with breast milk or infant formula.

[0037] The term "probiotic" refers to live microorganisms that confer a health benefit to the host when administered in adequate amounts (FAO / WHO, 2002). The microbial cells are generally bacteria or yeast. Unless otherwise indicated, all percentages are by weight. Method for preparing the composite

[0038] Complex coacervate

[0039] The present invention relates to complex coacervates comprising lactoferrin and osteopontin.

[0040] In a particular embodiment, the complex coacervate may comprise protein in an amount of no more than 50% w / w. In a particular embodiment, the complex coacervate may preferably comprise protein in an amount of no more than 40% w / w. In a particular embodiment, the complex coacervate may more preferably comprise protein in an amount of no more than 35% w / w.

[0041] In a particular embodiment, the complex coacervate may comprise protein in an amount of at least 5% w / w. In a particular embodiment, the complex coacervate may preferably comprise protein in an amount of at least 15% w / w. In a particular embodiment, the complex coacervate may more preferably comprise protein in an amount of at least 25% w / w.

[0042] In a specific embodiment, the complex coacervate may comprise protein in an amount of 5% w / w to 50% w / w. In a specific embodiment, the complex coacervate may preferably comprise protein in an amount of 15% w / w to 40% w / w. In a specific embodiment, the complex coacervate may more preferably comprise protein in an amount of 25% w / w to 35% w / w.

[0043] The amount of protein in the complex coacervate can be determined using a method according to AOAC 991.20-1994.

[0044] In a specific embodiment, the complex coacervate may comprise water in an amount of no more than 95% w / w. In a specific embodiment, the complex coacervate may comprise water in an amount of no more than 85% w / w. In a specific embodiment, the complex coacervate may even more preferably comprise water in an amount of no more than 75% w / w.

[0045] In a particular embodiment, the complex coacervate may comprise water in an amount of at least 50% w / w. In a particular embodiment, the complex coacervate may comprise water in an amount of at least 60% w / w. In a particular embodiment, the complex coacervate may even more preferably comprise water in an amount of at least 65% w / w.

[0046] In a specific embodiment, the complex coacervate may comprise water in an amount of 50% w / w to 95% w / w. In a specific embodiment, the complex coacervate may comprise water in an amount of 60% w / w to 85% w / w. In a specific embodiment, the complex coacervate may even more preferably comprise water in an amount of 65% w / w to 75% w / w.

[0047] The amount of water and protein in the complex coacervate can be determined using the method according to ISO 5537:2004.

[0048] In a specific embodiment, the complex coacervate has a particle size of at least 500 nm in the shortest dimension. In a specific embodiment, the complex coacervate preferably has a diameter of at least 600 nm in the shortest dimension. In a specific embodiment, the complex coacervate more preferably has a diameter of at least 700 nm in the shortest dimension. In a specific embodiment, the complex coacervate more preferably has a diameter of at least 900 nm in the shortest dimension.

[0049] The diameter of the complex coacervates can be determined using dynamic light scattering or by microscopy.

[0050] In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is not greater than 8. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably not more than 6. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably not more than 5.5. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably not more than 5. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably not more than 4.2.

[0051] In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is at least 2. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably at least 3. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably at least 3.2. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably at least 3.5. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably at least 3.8.

[0052] In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is in the range of 2 to 8. In a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is preferably in the range of 3 to 6. In a specific embodiment, 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 a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is more preferably in the range of 3.5 to 5. In a specific embodiment, 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 a specific embodiment, the protein mass ratio of lactoferrin to osteopontin in the complex coacervate is even more preferably 4.

[0053] In a specific embodiment, the complex coacervate has a zeta potential in the range of -15 mV and +15 mV. In a specific embodiment, the complex coacervate may preferably have a zeta potential in the range of -10 mV and +10 mV. In a specific embodiment, the complex coacervate may preferably have a zeta potential in the range of -8 mV to +5 mV.

[0054] Zeta potential can be measured using a Malvern Zetasizer Nano-ZS (Malvern Instruments Inc., Worcchshire, UK) equipped with Malvern Zetasizer software 7.02.

[0055] Preparation method

[0056] The present invention also relates to a method for preparing the complex coacervate according to the present invention, wherein said method comprises the following steps:

[0057] a. providing a separate aqueous solution comprising lactoferrin and osteopontin,

[0058] b. mixing the separate aqueous solutions comprising lactoferrin and osteopontin at a pH of 4 to 6, preferably at a pH of 4.5 to 5.5, more preferably at a pH of 4.8 to 5.2, even more preferably at a pH of 5, and wherein the separate aqueous solutions comprising lactoferrin and osteopontin are adjusted such 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.

[0059] According to the present invention, in step a., a separate aqueous solution comprising lactoferrin and osteopontin is provided. Thus, it is to be understood that a separate aqueous solution comprising lactoferrin and a separate solution of osteopontin are prepared and provided.

[0060] In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are adjusted to a specific pH value before mixing. In a specific embodiment, the separate solutions comprising lactoferrin and osteopontin are adjusted to a pH value of 4 to 6, preferably to a pH value of 4.5 to 5.5, more preferably to a pH value of 4.8 to 5.2, even more preferably to a pH value of 5.

[0061] In an alternative embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are not adjusted to a specific pH value prior to mixing.

[0062] According to the present invention, in step b., separate aqueous solutions comprising lactoferrin and osteopontin are mixed at a pH value of 4 to 6. Thus, it is understood that separate aqueous solutions comprising lactoferrin and osteopontin are mixed and the pH value is adjusted to 4 to 6 by adding an acid (preferably an aqueous HCl solution) or a base (preferably an aqueous NaOH solution).

[0063] In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are mixed at a pH value of 4.5 to 5.5. In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably mixed at a pH value of 4.8 to 5.2. In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are even more preferably mixed at a pH value of 5.

[0064] According to the present invention, in step b., the separate aqueous solution containing lactoferrin and osteopontin is adjusted so that the protein mass ratio of lactoferrin to osteopontin in the mixed solution is 2 to 8.

[0065] In a specific embodiment, it is preferred to adjust the separate aqueous solution containing lactoferrin and osteopontin so that the protein mass ratio in the mixed solution is in the range of 3 to 6. In a specific embodiment, it is more preferred to adjust the separate aqueous solution containing lactoferrin and osteopontin so that the protein mass ratio in the mixed solution is in the range of 3.2 to 5.5. In a specific embodiment, it is more preferred to adjust the separate aqueous solution containing lactoferrin and osteopontin so that the protein mass ratio in the mixed solution is in the range of 3.5 to 5. In a specific embodiment, it is even more preferred to adjust the separate aqueous solution containing lactoferrin and osteopontin so that the protein mass ratio in the mixed solution is in the range of 3.8 to 4.2. In a specific embodiment, it is even more preferred to adjust the separate aqueous solution containing lactoferrin and osteopontin so that the protein mass ratio in the mixed solution is in the range of 4.

[0066] In a specific embodiment, in step b., the ionic strength in the mixed aqueous solution is not higher than 30 mM added salt. It is therefore understood that the mixed aqueous solution does not contain more than 30 mM of added salt, i.e. the total amount of added salt contained in the mixed solution, and that they may be added in particular when providing separate solutions of lactoferrin and osteopontin and / or when mixing separate solutions of lactoferrin and osteopontin during step b. Salt is therefore understood to be lactoferrin and / or osteopontin that is not positively and / or negatively charged. In a specific embodiment, salt is understood to be an inorganic salt. In a specific embodiment, salt is understood to be NaCl.

[0067] In a specific embodiment, the ionic strength in the mixed aqueous solution is preferably not higher than 20 mM salt. In a specific embodiment, the ionic strength in the mixed aqueous solution is more preferably not higher than 10 mM salt. In a specific embodiment, the ionic strength in the mixed aqueous solution is more preferably not higher than 5 mM. In a specific embodiment, the ionic strength in the mixed aqueous solution is more preferably not higher than 0.2 mM.

[0068] In a specific embodiment, in step b., the separate aqueous solutions comprising lactoferrin and osteopontin are adjusted such that the total protein concentration in the mixed solution is below the autoinhibition point.

[0069] In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted such that the total protein concentration in the mixed solution is less than 8% w / v. In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted such that the total protein concentration in the mixed solution is preferably less than 6% w / v.

[0070] In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted such that the total protein concentration in the mixed solution is greater than 2% w / v. In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted such that the total protein concentration in the mixed solution is preferably greater than 4% w / v.

[0071] In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted so that the total protein concentration in the mixed solution is in the range of 2% w / v to 8% w / v. In a specific embodiment, the separate aqueous solutions comprising lactoferrin and osteopontin are preferably adjusted so that the total protein concentration in the mixed solution is preferably in the range of 4% w / v to 6% w / v.

[0072] In a particular embodiment, the complex coacervates formed in the process according to the invention can be separated by any method known to the skilled person.

[0073] In a particular embodiment, the complex coacervate may be subjected to a drying step, such as spray drying, belt drying, drum drying and / or freeze drying.

[0074] Compositions comprising complex coacervates

[0075] The invention also relates to a composition comprising the complex coacervate according to the invention.

[0076] In a specific embodiment, the composition may comprise a complex coacervate according to the invention and a soluble complex of lactoferrin and osteopontin. In a specific embodiment, the composition comprises a complex coacervate according to the invention and a soluble complex of lactoferrin and osteopontin obtained from the method for preparing a complex coacervate according to the invention.

[0077] The composition may be any type of composition into which the complex may be incorporated, such as a composition in the form of a food or beverage product, an animal feed product, a nutritional supplement for humans or animals, a pharmaceutical composition or a cosmetic composition. The product may be in solid, liquid or semi-liquid form.

[0078] For the purpose of providing nutrition and / or pleasure, food and beverage products include all products intended to be taken orally by humans. It can be, for example, a nutritional composition, such as for infants and / or young children, for pregnant women or lactating women or women who wish to be pregnant, for individuals who need special nutrition due to poor health, or for the elderly. More preferably, the nutritional composition is selected from infant formula, infant cereals, two-stage infant formula, growing milk and the dairy products for pregnant women and lactating women or women who wish to be pregnant. Other examples of food and beverage products include dairy products such as dairy products or yogurt, soups, sauces, sweet snacks and salty snacks, powdered beverages and cereal products.

[0079] The product can also be in the form of an animal food product or a nutritional supplement for an animal. Preferably, the animal is a mammal. Examples of animals include primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc.

[0080] Nutritional supplements are usually in the form of liquids, gels, powders or tablets or capsules. Powder supplements usually cover supplements to be dissolved in water or sprayed on food or beverages. Such supplements are intended to provide additional nutrients and / or health benefits to the individual consuming it, as well as other beneficial ingredients, such as lactoferrin, lactadhesin and / or lysozyme. Supplements according to the present invention can be used to provide nutrients and / or health benefits to humans and animals, as defined above. Nutritional supplements include, for example, powder supplements added to breast milk, for example for premature or low birth weight infants. It also includes supplements for pregnant or lactating women or women who wish to become pregnant.

[0081] Pharmaceutical products include, for example, drops, syrups, powders, tablets or capsules intended to treat or prevent an adverse medical condition in an individual in need thereof.

[0082] Cosmetic compositions are generally intended to have an aesthetic effect on the body and may be used topically or may be administered by the oral route.

[0083] The compositions of the invention preferably comprise a therapeutically effective amount of the complex coacervates of the invention.

[0084] In a preferred embodiment, the composition of the invention is an infant formula, a first infant formula, an infant formula, a baby food, an infant cereal composition, a growing-up milk, a fortifier (such as a human milk fortifier) ​​or a supplement. In such a composition, the complex coacervate of the invention is preferably present in an amount providing 0.001 g to 3 g, preferably 0.01 g to 2 g, more preferably 0.1 g to 1 g lactoferrin per liter of the composition.

[0085] All types of compositions according to the invention can be formulated and manufactured according to the knowledge of a person skilled in the art.The complex coacervates of the invention are advantageously sufficiently robust to be processed together with the other ingredients of the composition.

[0086] For example, in the manufacture of spray-dried products, such as infant formulas, growing-up milks or second-stage infant formulas in powder form, the complex coacervates of the present invention are sufficiently stable to be added to a wet mix and spray-dried together with the other ingredients of the product. This is advantageous from the perspective of process economy, since aseptic dosing and dry mixing of sensitive proteins such as lactoferrin are not required. In addition, from the perspective of product structure, incorporating the complex into a wet mix is ​​advantageous. Specifically, the complex coacervates of the present invention will be mixed with the other ingredients in a uniform manner. In contrast, dry-mixed powders can result in an inhomogeneous product due to different properties of the mixed powders, such as, for example, differences in density or particle size. Inhomogeneity can result in inaccurate dosing of the protein.

[0087] Therefore, a method for preparing a composition selected from an infant formula, a second infant formula or a growing-up milk in powder form is also an object of the present invention, which method comprises preparing a product concentrate comprising the complex coacervate of the present invention and spray drying the product concentrate. The preparation of the wet mix and the spray drying are carried out according to the general knowledge of a person skilled in the art.

[0088] The present invention also provides a method for preparing a composition selected from a liquid, an infant formula, a first infant formula, an infant formula, a baby food, an infant cereal composition, a growing-up milk, a fortifier (e.g., a human milk fortifier) ​​or a supplement, the method comprising mixing the complex of the present invention into a liquid product base and aseptically processing the product base containing the complex. The preparation of the liquid product base and aseptic processing are performed according to the general knowledge of those skilled in the art.

[0089] The composition of the present invention may further comprise at least one probiotic (or probiotic strain), such as a probiotic bacterial strain.

[0090] The most commonly used probiotic microorganisms are mainly most bacteria and yeasts of the following genera: Lactobacillus spp., Streptococcus spp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp. In some embodiments, the probiotics are probiotic strains. In some embodiments, they are specifically bifidobacteria and / or lactobacilli.

[0091] Suitable probiotic bacterial strains include Lactobacillus rhamnosus ATCC 53103 available from Valio Oy, Finland under the trade name LGG, Lactobacillus rhamnosus CGMCC 1.3724 sold under the trade name KI2 by BLIS Technologies Limited, New Zealand, Lactobacillus paracasei CNCM 1-21 16, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivarius DSM 13084, Bifidobacterium lactis CNCM 1-21 16 sold under the trade name Bb12 by Christian Hansen company, Denmark. 1-3446, 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 in Canada. The composition according to the present invention generally comprises 10e3 to 10e12 cfu of the probiotic strain, more preferably between 10e7 and 10e12 cfu of the probiotic strain per gram of the composition based on dry weight. In one embodiment, the probiotic is alive. In another embodiment, the probiotic is non-replicating or inactivated. In some other embodiments, live probiotics and inactivated probiotics may be present at the same time.

[0092] In addition to the human milk oligosaccharides mentioned above, the composition of the present invention may also comprise at least one non-digestible oligosaccharide (eg prebiotic), typically in an amount of 0.3% to 10% by weight of the composition.

[0093] Prebiotics are generally non-digestible in the sense that they are not broken down and absorbed in the stomach or small intestine and thus remain intact as they pass through the stomach and small intestine to the colon where they are selectively fermented by beneficial bacteria. Examples of prebiotics include certain oligosaccharides, such as fructooligosaccharides (FOS) and galacto-oligosaccharides (GOS). Combinations of prebiotics may be used, such as 90% GOS with 10% short chain oligofructose (e.g., marketed by BENEO-Orafti under the trademark FROS). Fructo-oligosaccharides (formerly ) or a combination of 90% GOS and 10% inulin (for example sold under the trademark Inulin by the company BENEO-Orafti Inulin (formerly A particularly preferred prebiotic combination is a combination of 70% short-chain fructo-oligosaccharides and 30% inulin, which is the product sold under the trademark "Prebio 1" by the company BENEO-Orafti.

[0094] The composition of the invention may also comprise at least one bacteriophage (bacteriophage) or a mixture of bacteriophages, preferably against pathogenic Streptococci, Haemophilus, Moraxella and Staphylococci. The composition according to the invention may be a nutritional composition, preparation or food product.

[0095] The composition according to the invention may be, for example, a nutritional composition, such as a synthetic nutritional composition. It may be an infant formula, a first 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 supplement.

[0096] When the composition is a supplement, it may be provided in unit dosage form.In some embodiments, the composition of the invention is typically an infant formula.

[0097] The composition of the present invention is generally used for babies or young children born by caesarean section.

[0098] These infants or young children represent a special subject group with special needs requiring special care and the inventors have surprisingly found that a composition comprising at least one human milk oligosaccharide and / or a precursor thereof is particularly effective in reducing the incidence of necrotizing enterocolitis in these infants born by caesarean section.

[0099] The composition according to the present invention can be used for babies born at term or prematurely by caesarean section.

[0100] Advantageously, the composition of the invention is used in full-term infants or premature infants, in particular infants born by caesarean section.

[0101] In some embodiments, the composition of the present invention is for use in infants born by cesarean section who are small for gestational age.In some embodiments, the composition according to the present invention may be used prior to and / or during the weaning period.

[0102] The composition of the present invention may be in the form of a solid (eg, powder), liquid or gel.

[0103] As it is particularly intended for babies born by caesarean section, the composition may advantageously be a nutritional composition for consumption in liquid form.The composition may be a nutritionally complete formula, such as an infant formula, a first infant formula, a follow-on formula or a fortifier, such as a human milk fortifier.

[0104] The composition according to the invention generally also contains a protein source, preferably in an amount of less than 2.0 g / 100 kcal, even more preferably less than 1.8 g / 100 kcal. The type of protein is considered to be immaterial for the present invention, as long as the minimum requirements for the content of essential amino acids are met and satisfactory growth is ensured. Thus, protein sources based on whey, casein and mixtures thereof may be used, as well as protein sources based on soy. As far as whey proteins are concerned, the protein source may be based on acid whey or sweet whey or mixtures thereof, and may contain alpha-lactalbumin and beta-lactoglobulin in any desired ratio.

[0105] The protein may be intact or hydrolyzed, or a mixture of intact and hydrolyzed proteins. The term "intact" means that the major part of the protein is intact, i.e. the molecular structure is not changed, for example at least 80% of the protein is not changed, such as at least 85% of the protein is not changed, preferably at least 90% of the protein is not changed, even more preferably at least 95% of the protein is not changed, such as at least 98% of the protein is not changed. In a specific embodiment, 100% of the protein is not changed.

[0106] In the context of the present invention, the term "hydrolyzed" refers to a protein that has been hydrolyzed or broken down into its constituent peptides or amino acids.

[0107] The protein can be fully hydrolyzed or partially hydrolyzed. For example, for infants and young children who are considered to be at risk of cow's milk allergy, it may be desirable to provide partially hydrolyzed protein (degree of hydrolysis between 2% and 20%). If hydrolyzed protein is required, a hydrolysis process can be performed as required and as known in the art. For example, a whey protein hydrolyzate can be prepared by enzymatic hydrolysis of the whey fraction in one or more steps. If the whey fraction used as a raw material is substantially lactose-free, it is found that the protein undergoes much less lysine blockage during the hydrolysis process. This makes it possible to reduce the degree of lysine blocking from about 15% by weight of total lysine to less than about 10% by weight of lysine; for example, about 7% by weight of lysine, which greatly improves the nutritional quality of the protein source. 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, in particular at least 98% of the protein is hydrolyzed. In a specific embodiment, 100% of the protein is hydrolyzed.

[0108] 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.

[0109] The composition according to the invention typically comprises a carbohydrate source. This is particularly preferred in the case where the nutritional composition of the invention is an infant formula. In this case, any carbohydrate source typically present in infant formulas may be used, such as lactose, sucrose, maltodextrin, starch and mixtures thereof, but a preferred carbohydrate source is lactose.

[0110] The composition according to the invention generally comprises a lipid source. This is particularly relevant in the case where the nutritional composition of the invention is an infant formula. In this case, the lipid source may be any lipid or fat suitable for infant formula. Preferred fat sources include palmitoleic acid, high oleic sunflower oil and high oleic safflower oil. The essential fatty acids linoleic acid and alpha-linolenic acid may also be added, as well as small amounts of oils containing large amounts of preformed arachidonic acid and docosahexaenoic acid, such as fish oil or microbial oils. The ratio of n-6 fatty acids to n-3 fatty acids of the fat source is preferably from about 5:1 to about 15:1; for example from about 8:1 to about 10:1.

[0111] The composition of the present invention also preferably comprises all vitamins and minerals that are considered to be necessary for daily diet, and these vitamins and minerals are nutritionally significant amounts. The minimum requirements of certain vitamins and minerals have been determined. The examples of minerals, vitamins and other nutrients 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 the form of salts. The presence and amount of specific minerals and other vitamins will vary according to the target group. If necessary, the composition of the present invention may include emulsifiers and stabilizers, such as soybeans, lecithin, citric acid monoglyceride and citric acid diglyceride etc.

[0112] The compositions of the present invention may also contain other substances that may have beneficial effects, such as nucleotides, nucleosides, and the like.

[0113] The composition according to the invention may be prepared by any suitable means. The composition will now be described by way of example.

[0114] For example, formula foods such as infant formula foods can be prepared by blending a protein source, a carbohydrate source, and a fat source in an appropriate ratio. If used, an emulsifier can be added at this time. Vitamins and minerals can be added at this time, but they are usually added later to avoid thermal degradation. Before blending, any lipophilic vitamins, emulsifiers, etc. can be dissolved in a fat source. Water (preferably water subjected to reverse osmosis) can then be mixed to form a liquid mixture. The water temperature is suitably in the range of about 50°C to about 80°C to help disperse the ingredients. A commercially available liquefier can be used to form a liquid mixture.

[0115] If the final product is a powder, these ingredients can also be added at this stage if desired.The liquid mixture is then homogenized, for example in two stages.

[0116] The liquid mixture may then be heat treated to reduce the bacterial load, for example by rapidly heating the liquid mixture to a temperature in the range of about 80°C to about 150°C for a duration between about 5 seconds and about 5 minutes. This may be performed by steam injection, an autoclave, or a heat exchanger (e.g., a plate heat exchanger).

[0117] The liquid mixture is then cooled, for example by flash cooling, to between about 60°C and about 85°C. The liquid mixture is then homogenized again, for example in two stages, between about 10 MPa and about 30 MPa in the first stage and between about 2 MPa and about 10 MPa in the second stage. The homogenized mixture can then be further cooled to allow for the addition of any heat-sensitive components, such as vitamins and minerals. The pH and solids content of the homogenized mixture are conveniently adjusted at this point.

[0118] If the final product is to be 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 moisture content of the powder should be less than about 5% by weight. Human milk oligosaccharides and / or their precursors may be added at this stage by dry mixing them with the probiotic strain (if used) or by mixing them with the probiotic strain in the form of a crystalline syrup and then spray drying or freeze drying the mixture.

[0119] If a liquid composition is preferred, the homogenised mixture may be sterilised and then filled into suitable containers under aseptic conditions or filled into containers first and then retorted.

[0120] Use in treatment and prevention

[0121] The complex coacervates of the invention or the compositions of the invention can advantageously be used in therapy. Therefore, the present invention also provides such complex coacervates and such compositions for use in therapy or prevention. In a particularly preferred aspect, the present invention provides complex coacervates of the invention and compositions comprising such complex coacervates for use in therapy or prevention.

[0122] Treatment herein is intended to cure or prevent a disease or disorder in the body, and also encompasses prophylactic treatment, i.e., prevention of an adverse medical condition. Treatment herein is also intended to include human and animal treatment. In other words, the present invention relates to a method for treating an individual, the method comprising administering to the individual a therapeutically effective amount of a compound or product according to the present invention.

[0123] Therefore, the present invention relates to a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, prediabetes or diabetes and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0124] Therefore, the present invention also relates to a method for treating or preventing metabolic disorders, in particular overweight and obesity, prediabetes or diabetes and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate comprising lactoferrin and osteopontin.

[0125] The definitions and embodiments of the complex coacervates and compositions comprising the complex coacervates of the present invention as described above apply mutatis mutandis to their use in therapy and prevention.

[0126] The complexes, in particular the complex coacervates, and compositions comprising the complexes, in particular the complex coacervates, are preferably administered to a subject in need thereof.

[0127] In a specific embodiment, the subject is an infant, a toddler or a toddler. In a specific embodiment, the subject is an infant. In a specific embodiment, the subject is a full-term infant or a premature infant, particularly an infant born by caesarean section.

[0128] The complex coacervates according to the invention are particularly beneficial because they provide the subject with highly biologically active lactoferrin even after gastrointestinal digestion. Thus, the complex coacervates according to the invention provide the subject with biologically active lactoferrin after digestion, so that the therapeutic and / or preventive effect of lactoferrin and / or osteopontin can be provided even after gastrointestinal digestion.

[0129] The complex coacervate (eg, in the form of a nutritional composition, a supplement, etc.) may be administered by any suitable route, such as by oral, enteral or parenteral administration. In a preferred embodiment, the complex coacervate is administered orally.

[0130] The complex coacervate (e.g., in the form of a nutritional composition, a supplement, etc.) can be administered in any suitable dose, for example, to provide a therapeutically effective amount of the complex coacervate. The complex coacervate can be administered at a dose 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 coacervate can be administered at a dose of 10000 mg / kg / day or less, 5000 mg / kg / day or less, 4000 mg / kg / day or less, 3000 mg / kg / day or less, 2000 mg / kg / day or less. In some embodiments, the complex coacervate is administered at a dose of 100 mg / kg / day to 10000 mg / kg / day, preferably at a dose of 500 mg / kg / day to 5000 mg / kg / day, and more preferably at a dose of 1000 mg / kg / day to 2000 mg / kg / day.

[0131] In promoting Bone metabolism and / or homeostasis Uses in

[0132] When administered to a subject, the complex coacervate can promote normal bone metabolism and homeostasis. Studies have shown that lactoferrin and osteopontin play a role in bone metabolism and 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).

[0133] Therefore, the present invention also provides a complex coacervate comprising lactoferrin and osteopontin for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases.

[0134] The present invention also provides a method for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases, which comprises administering a therapeutically effective amount of a complex coacervate comprising lactoferrin and osteopontin to a subject in need thereof.

[0135] The present invention also provides use of the complex coacervate containing lactoferrin and osteopontin for preparing a medicament for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases.

[0136] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for promoting bone development. As used herein, "promoting bone development" may refer to supporting normal bone metabolism, for example, in childhood and adolescence, and / or homeostasis. In childhood and adolescence, bones are shaped by a process known as modeling, which allows new bone to be formed in one part and old bone to be removed from another part 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 homeostasis may refer to supporting normal bone modeling and / or reconstruction (see, for example, Allen, MR and Burr, DB, 2014. Bone modeling and remodeling, in Basic and applied bone biology (pages 75-90), Academic Press). Supporting normal bone metabolism and / or homeostasis can lead to normal bone anatomy and physiology (Clarke, B., 2008. Clinical journal of the American Society of Nephrology, 3 (Suppl 3), pp. S131-S139).

[0137] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for promoting bone growth and / or strength. As used herein, "promoting bone growth and / or strength" may refer to supporting normal bone growth and / or strength, for example, in childhood and adolescence. Supporting normal bone growth and / or strength may result in normal bone anatomy and physiology. Suitable methods and parameters for determining bone growth and bone strength will be known to the skilled person (see, for example, Donnelly, E., 2011. Clinical Orthopaedics and Related Research, 469 (8), pp. 2128-2138). Suitably, one or more bone parameters selected from the following may be used to determine normal bone growth and / or strength: trabecular bone volume and tissue volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), inner and outer diameters, anterior and posterior diameters, bone yield force, and bone stiffness. Suitable methods for determining these parameters are available to those skilled in the art.

[0138] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for promoting bone healing. As used herein, "promoting bone healing" may refer to supporting normal bone healing, such as healing after a fracture. Fractures are one of the most common injuries to the musculoskeletal system. Although fracture treatment has improved significantly in recent decades, most of all fractures still show delayed healing and complications including non-union (Claes, L. et al., 2012. Nature Reviews Rheumatology, 8 (3), pp. 133-143). Therefore, supporting normal bone healing can, for example, prevent delayed healing and / or non-union. Increasing age may increase the risk of delayed healing or non-union. The complex coacervate can prevent and / or reduce the frequency and / or occurrence and / or severity and / or duration of fractures.

[0139] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for preventing and / or treating bone diseases. As used herein, the term "bone disease" may refer to a medical condition that affects bone, particularly a medical condition that relates to a reduction in bone organic matrix. Suitably, 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, e.g., 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, hypophosphatase, scurvy and osteitis fibrosa cystica. In some embodiments, bone disease is selected from low bone density, osteopenia, osteoporosis, osteomalacia and rickets.

[0140] Metabolic bone diseases are common in premature infants and / or infants with low birth weight and / or infants who have suffered from inadequate nutrition in utero, and result in an increased risk of fractures in these groups (Arch Dis Child Fetal Neonatal Ed 2002 86:F82-F85). Infants, children, and adolescents whose growth is retarded due to malnutrition and / or illness are also frequently affected by these conditions.

[0141] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for preventing and / or treating low bone density. Bone density or bone mineral density (BMD) is the amount of bone mineral in bone tissue and is used as an indirect indicator of osteoporosis and fracture risk in clinical medicine. It is measured by a procedure called densitometry. The BMD test provides a measurement result called a T score for an individual, which is a numerical value obtained by comparing the individual's bone density with the optimal bone density. Subjects with low bone density may have a bone mineral density that is more than 1.0 standard deviation lower than the average peak bone mass (average value for young healthy adults) measured by DXA (dual-energy x-ray absorptiometry).

[0142] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in preventing and / or treating osteopenia. Osteopenia is a condition characterized by a lack of organic bone matrix resulting in less than normal bone tissue mass. Osteopenia can be defined as a bone mineral density between 1.0 and 2.5 standard deviations below the mean peak bone mass as measured by DXA.

[0143] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for use in the prevention and / or treatment of osteoporosis. Osteoporosis ("porous bone" from Greek) is a bone disease that leads to an increased risk of fractures. The disease is characterized by too little bone formation, too much bone loss, or a combination of both. In osteoporosis, bone mineral density (BMD) decreases, bone microarchitecture degenerates, and the amount and type of protein in the bone changes. Osteoporosis can be defined as a bone mineral density that is 2.5 standard deviations or more below the mean peak bone mass measured by DXA.

[0144] The present invention provides a complex coacervate comprising lactoferrin and osteopontin for preventing and / or treating osteomalacia or rickets. Osteomalacia is a condition in which bone mineral density (BMD) and bone mineral content (BMC) are below normal levels. Osteomalacia in children is often associated with rickets. Osteomalacia or rickets may manifest as symptoms such as diffuse pain throughout the body, muscle weakness and bone brittleness. The most common cause of the disease is vitamin D deficiency, which is usually obtained from diet and sunlight exposure.

[0145] When administered to a subject, the complex coacervate can increase bone growth and / or bone strength. For example, when the complex coacervate is administered to a subject, it can increase bone growth and / or bone strength compared to a subject to which the complex coacervate is not administered or to which different forms of lactoferrin and osteopontin (e.g., as a blend or soluble complex) are administered. Suitably, the complex coacervate increases bone growth and / or bone strength when compared to the same dose of lactoferrin and osteopontin provided as a soluble complex. Suitably, the complex coacervate increases bone growth and / or bone strength when 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 the following: trabecular bone volume and tissue volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), inner and outer diameters, anterior and posterior diameters, bone yield force, and bone stiffness. Suitable methods for determining these parameters are available to those skilled in the art.

[0146] The subject can be any suitable subject. Suitably, the subject can be a mammal. In preferred embodiments, the subject is a human. In other embodiments, the subject is an animal, preferably wherein the animal is a pet. The pet can be an animal selected from dogs, cats, birds, fish, rodents (such as mice, rats and guinea pigs, rabbits) and the like.

[0147] The present invention is particularly suitable for infants and young children who are at risk of bone disease, have a family history of bone disease, or have experienced at least one, preferably several, fracture episodes. The present invention is also particularly suitable for infants and young children who are born prematurely or have a low birth weight or experience intrauterine growth retardation, or infants and young children who suffer growth retardation due to malnutrition or experience diseases (such as Crohn's disease and / or celiac disease and / or cancer), or infants and young children treated with drugs (such as chemotherapeutic drugs and / or corticosteroids) that cause malabsorption, anorexia, and / or metabolic bone diseases. The present invention is particularly preferably used for infants and children who are born prematurely or have a low birth weight or experience intrauterine growth retardation or suffer from intrauterine malnutrition or suffer from growth delay.

[0148] In some embodiments, the subject is a teenager, a teenager, a child, or an infant. The term "juvenile" may refer to an individual who has not yet reached adulthood. The term "teenager" may refer to an individual from the beginning of puberty to adulthood. The term "child" may refer to an individual between the birth stage and puberty.

[0149] In some embodiments, the subject is premature or has a low birth weight or experiences intrauterine growth retardation. The term "preterm infant" may refer to an infant whose gestational age is less than 37 weeks at birth. The term "low birth weight infant" may refer to an infant whose live birth weight is less than 2,500 g.

[0150] In some embodiments, the subject suffers from growth retardation. The definition of growth retardation may refer to a "height for age" value that is less than two standard deviations of the median value of the WHO child growth standard (see, e.g., De Onis, M. and Branca, F., 2016. Maternal & child nutrition, 12, pages 12-26). In some embodiments, the subject suffers from growth retardation due to malnutrition or experiencing diseases such as anorexia, Crohn's disease and / or celiac disease. In some embodiments, the subject suffers from growth retardation due to treatment with drugs (such as chemotherapeutic drugs and / or corticosteroids) that cause malabsorption, anorexia and / or metabolic bone disease.

[0151] The present invention may also be applied to adolescents or adults who are at risk of bone disease or who have experienced at least one, preferably several, bone fracture episodes, or who were born prematurely or with low birth weight or who experienced intrauterine growth retardation, or who suffer growth retardation due to malnutrition or who experience disease (such as Crohn's disease and / or celiac disease and / or cancer), or who are treated with drugs that cause malabsorption, anorexia and / or metabolic bone disease (such as chemotherapeutic drugs and / or corticosteroids), or who suffer growth delay during infancy and / or childhood (including adolescence) due to disease or malnutrition or drug use.

[0152] In some embodiments, the subject is a teenager or an adult. In some embodiments, the subject is an adult, preferably wherein the subject is an elderly person. In some embodiments, the subject is at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, or at least 80 years old. The subject may have one or more fractures. The subject may have one or more delayed unions and / or one or more nonunions, or may be at risk of these occurring.

[0153] Example

[0154] f

[0155] 1. Materials

[0156] According to the AOAC official method (AOAC, 2005), the total protein, ash and moisture contents of lactoferrin (LF) powder were 98.7% (w / w, Dumas nitrogen x6.25), 0.37% (w / w) and 0.42% (w / w), respectively. The purity of LF was 95% of the total protein (w / w, measured by HPLC at 214 nm). The total protein, moisture and ash contents of the spray-dried bovine osteopontin (OPN) powder used were 89.6% (w / w, Dumas nitrogen x7.17), 2.22% (w / w), 9.2% (w / w), respectively. According to the supplier's instructions, the purity of OPN was 99% of the total protein.

[0157] 2. Preparation of Complex Coacervates

[0158] Separate protein solutions of LF and OPN were prepared by hydrating their respective powders in ultrapure water (18.2 MΩ.cm) with magnetic stirring for about 2 h at 25°C, followed by magnetic stirring at 4°C overnight (~18 h) to ensure complete rehydration.

[0159] The separate protein solutions of LF and OPN were mixed in 50 mL polypropylene centrifuge tubes. After pH adjustment, these solutions were centrifuged at 20°C for 20 minutes using a Sorvall RC 5C Plus centrifuge with a Sorvall GSA rotor at a relative centrifugal force (RCF) of 3007 to accelerate phase separation. For the remainder of the experiment, the mixed solutions were given time to settle naturally under static conditions at 4°C for ~18 hours.

[0160] In samples where phase separation occurred, mass balance determinations were performed by separating the phases using a micropipette prior to mass measurement using a 4-point analytical balance.

[0161] Complex coacervates of lactoferrin and osteopontin were formed at pH 4 with a LF:OPN mixing ratio (based on protein mass) of 2; at pH 5 with a LF:OPN mixing ratio (based on protein mass) of 4 and 6; and at pH 6 with a LF:OPN mixing ratio (based on protein mass) of 8.

[0162] Complex coacervates of lactoferrin and osteopontin were formed with the highest coacervation yield at pH 5 and a LF:OPN mixing ratio (based on protein mass) of 4. The maximum coacervation yield refers to the maximum % of total nitrogen present in the coacervate phase or the lowest % of total nitrogen present in the supernatant phase in the mixed solution, such as, for example, as soluble complexes.

[0163] 3. Measurement of complex condensate structure

[0164] The microscopic appearance of the complex coacervates of LF and OPN was imaged using a Leica DM1000 optical microscope (Leica Microsystems GmbH, DE) equipped with A smartphone adapter (iDu Optics, USA) was used to facilitate digital image capture. The microscope was operated at 40× magnification in phase contrast and dark field operation modes. To prepare samples for imaging, complex coacervates of LF and OPN (prepared at a 4:1 LF:OPN mass mixing ratio, pH 5, 5% w / v protein) were first isolated and dispersed in water, after which ~20 μL of the sample was placed between a microscope slide and a cover glass. The samples were imaged at least 3 times to ensure that representative images were captured.

[0165] Figure 1 It was shown that micron-sized LF-OPN coprecipitates and / or self-aggregates of either protein do not coexist with complex coacervates, as all visible entities appeared spherical (liquid) in nature, with no irregularly shaped (solid) floccules present.

[0166] 4. Measurement of thermal stability of complex coacervates

[0167] The effect of complex coacervation of lactoferrin and osteopontin on lactoferrin and osteopontin was demonstrated by differential scanning calorimetry analysis, with the thermal stability of lactoferrin in the complex coacervate being better than that of lactoferrin alone.

[0168] Differential scanning calorimetry analysis was performed using a T2500 Discovery Differential Scanning Calorimeter (TA Instruments, Crawley, UK). About 75 μL of sample (6%, w / v, protein) was transferred to a high volume stainless steel pan and then sealed using a T0 press (TA Instruments, Crawley, UK). Ultrapure water of similar volume was used as a reference. The sample was balanced at 20°C, then heated from 20°C to 100°C at 1°C / min, and then cooled to 20°C at 10°C / min. Measured in triplicate, and heat flow curves were processed using Trios 8.32 software.

[0169] Figure 2 5% w / v solutions of lactoferrin (dashed line) and osteopontin (dotted line) at pH 5.0 are shown (top Figure 1 ) and a LF / OPN complex coacervate (prepared at a protein mass ratio of 4:1, 5% w / v protein) at pH 5.0, which has a protein concentration of 27.4% (w / w).

[0170] therefore, Figure 2 It is shown that the heat capacity of the LF / OPN complex coacervate is different from that of the individual components and results in a T of the individual uncomplexed protein peak. m An offset occurs.

[0171] therefore, Figure 2 The thermograms in confirm the formation of complex coacervates between LF and OPN, which leads to the improved thermal stability of LF.

[0172] 5. Measurement of bioactivity after gastrointestinal digestion

[0173] 5.1. Preparation of the comparative diet and the diet according to the invention

[0174] The meal that was subjected to simulated infant gastrointestinal digestion, which involved a combination of infant formula (IF) and protein powder, was dry mixed before hydration.

[0175] The combinations prepared were: IF (having a protein content of 10.46% w / w, 28.7% w / w fat, 53.8% w / w carbohydrates, 2.2% w / w moisture and 2.16% w / w ash), with the addition of (1) LF to reach 600 mg / L when hydrated, (2) OPN to reach 150 mg / L, and (3) a soluble complex of LF and OPN, or (4) a complex coacervate of LF and OPN to reach 600 mg LF / L.

[0176] Diets were prepared by hydrating the relevant powders to a total protein concentration of 1.34% (w / v) in ultrapure water (18.2 MΩ.cm). All diets had the same protein concentration to ensure consistent enzyme: substrate ratios between simulated digestion samples. The protein concentration was selected based on the recommended reconstitution rate of the model formulation used.

[0177] 5.2. Simulating infant gastrointestinal digestion

[0178] The static in vitro method proposed by Ménard et al. (O. Ménard et al., Food, Chemistry, 2018, 240, 338-345) was used to simulate infant gastrointestinal digestion. The model is based on physiological findings in full-term infants, and the settings of all parameters can be obtained from the method of Ménard et al.

[0179] Digestion was performed in 40 mL conical screw-cap amber glass bottles, which were soaked in 6% (v / v) nitric acid before each digestion to remove residues. Bottles were placed in a 37°C water bath and stirred at 50 rpm during digestion (1 hour gastric and 1 hour intestinal).

[0180] Three independent experiments were performed for each sample for gastric (pH 5.3) and gastrointestinal (intestinal stage pH 6.6) digestion. Gastric lipase (19 U / mL) and pepsin activity (268 U / mL) were obtained using rabbit gastric extract (RGE70, Lipolytech, France) and porcine pepsin (Sigma P6887).

[0181] Serial time point samples were collected and transiently inactivated by one of three techniques according to endpoint analysis (described in each section).

[0182] Cell culture

[0183] HT-29 clone 34 cells were used. This cell line allows expression of a reporter gene for secretory 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 medium (DMEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) non-essential amino acids. Cells were stored in T75 culture flasks in an incubator at 37°C with 5% CO2.

[0184] Cell culture experiments

[0185] HT-29 clone 34 cells were cultured as described above. In the experiment, cells were cultured at 2 × 10 5 Cells were seeded at 100 cells / mL in 24-well cell culture plates (Greiner Bio-One, Austria) and cultured for 3 days before treatment to ensure monolayer confluence. For cell treatments, the culture medium was removed and treatments were prepared in fresh culture medium and added to the wells to a final well volume of 1 mL. Cells were treated with the relevant diet in either undigested or digested form. All treatments were filtered using a 0.45 μm sterile filter prior to cell administration. Gastric digests collected for cell culture analysis were preserved by raising the pH to above 7 using 0.1 M phosphate buffer prior to freezing (to inhibit pepsin activity), while gastric digests collected for cell culture analysis were preserved by adding 0.1 M phosphate buffer prior to freezing. (to inhibit serine protease activity) to a final concentration of 1 mM to preserve gastrointestinal digests. Human milk serum (HMS) was added at a concentration of 5% (v / v) of the pore volume to provide a source of effector molecules, including soluble CD14 (sCD14). The HMS used was prepared as previously described in Goulding et al., Food Chemistry, 362, 130142. The sCD14 concentration of HMS was 48 μg / mL. For the samples in this study, ethical approval and prior informed consent for the use of donor human milk samples for research purposes were obtained. The Clinical Research Ethics Committee of the Cork Teaching Hospital in Cork, Ireland granted ethical approval. The lipopolysaccharide (LPS) used in the experiment was Gram-negative Escherichia coli O111: B4 serotype (Sigma, L2630). LPS was added to all wells except the negative control at a concentration of 20 ng / mL. After application, the cells were incubated at 37°C and 5% CO2 for 16 hours, and then the cell supernatant was recovered and analyzed subsequently. Phosphalight kit (Applied Biosystems, MA, USA) was used as a marker for NF-κB activation, and the phosphatase activity of SEAP was quantified by bioluminescence. Bioluminescence was quantified as relative luminescence units (RLU) using a Varioskan Flash multi-well plate reader (Thermo Scientific, MA, USA) with an integration time of 1000 ms. For graphical representation of data, LPS treatment was set to 100 relative luminescence units, and all other data were normalized to this.

[0186] 5.5. Undigested and digested lactoferrin, osteopontin, and their complexes or complex aggregates in enterocyte inflammation Behavior in the model

[0187] Figure 3 The effect of adding gastrointestinal digests of LF, OPN, SC and CC to an enterocyte inflammation model is shown. When cells are stimulated with lipopolysaccharide (LPS, 20 ng / mL) of E. coli O111:B4 serotype, the NF-κB transcription factor is activated. When in undigested intact form, 0.35 mg protein / mL of LF, OPN and all combinations thereof resulted in statistically significant inhibition of LPS-induced NF-κB activation.

[0188] When enterocytes were treated with gastric digest, LF and OPN digest did not inhibit LPS-induced NF-κB activation, whereas both SC and CC digests caused statistically significant inhibition. The inhibition of LPS-induced NF-κB activation by gastric digests of SC and CC was not statistically significantly different, but was significantly different compared to LF alone.

[0189] Figure 3The effect of adding gastrointestinal digests of lactoferrin (LF), osteopontin (OPN), LF-OPN soluble complex (SC) or LF-OPN complex coacervate (CC) to an enterocyte inflammation model is shown. All samples were added at 0.35 mg protein equivalent / mL. The inflammation model used was NF-κB activation in HT-29 clone 34 cells induced by Escherichia coli O111:B4 lipopolysaccharide (LPS). LPS (20 ng / mL) and human milk serum (5% v / v) were added to all wells. All data were normalized to 100 relative luminescence units (RLU) for LPS treatment and represent the mean ± standard error of three replicates from three independent experiments. "*" represents a statistically significant difference (P<0.05) from 100% RLU treatment.

[0190] Figure 3 It was shown that the ability of LF to inhibit LPS-induced NF-κB activation requires that LF is not completely proteolyzed.It was also shown that in this enterocyte inflammation model, digestion of pre-complexed or pre-agglomerated LF-OPN powders resulted in altered bioactivity of the gastrointestinal digesta.

[0191] 6. Lactoferrin-osteopontin coacervate complex and bone development, growth and strength

[0192] C57 / bl6 wild-type mice were orally supplemented with three different osteopontin-lactoferrin mixtures (soluble, blended or coacervated complexes, n=10 per group) between postnatal days 2 and 28:

[0193] ●Bovine lactoferrin-osteopontin soluble complex (soluble) solution, 1250 mg / kg /

[0194] d, 20% w / v in distilled water, N=8.

[0195] Bovine lactoferrin-osteopontin coacervate complex (coacervate) solution, 1250 mg / kg / day,

[0196] 20% w / v in distilled water, N=9.

[0197] - Bovine lactoferrin-osteopontin blend (blend) solution, 1000 mg / kg / day of bovine lactoferrin powder + 250 mg / kg / day of bovine osteopontin powder, 20% w / v in distilled water, N=9.

[0198] All mice received the same amount of standard diet from day 28 to day 170. At the end of the study, femurs were collected to evaluate bone microarchitectural parameters.

[0199] The trabecular and cortical bone microstructures studied at the distal metaphysis and femoral midshaft were evaluated using micro-computed tomography (μCT UCT35, Scanco Medical AG, Basserdorf Switzerland), as previously described in the literature (Bonnet N et al., J Bone Miner Res. 2017; doi: 10: 1002). In short, trabecular and cortical bone areas were evaluated using isotropic 10um voxels. For the femoral trabecular area, in order to eliminate the main cancellous tissue, 100 main cancellous tomographic images obtained from 100 tomographic images of the secondary cancellous tissue under the distal growth plate were analyzed. The femoral cortical structure was evaluated using 50 consecutive CT tomographic images located at the femoral midshaft. Using a direct three-dimensional technique that does not rely on previous assumptions about the underlying structure, morphological variables were calculated from binarized images (Bonnet N et al., Med Phys 2009; 36 (4): 1286-97).

[0200] Bone is composed of cortical bone (or compact bone) and trabecular bone (or cancellous bone). Cortical bone accounts for approximately 80% of the body's bone mass and has a lower surface area than trabecular bone due to its lower porosity. Trabecular bone is located at the ends of long bones and accounts for approximately 20% of the total mass of the skeleton. Exemplary trabecular and cortical structures are shown in Figure 4 H and Figure 4 I.

[0201] For the trabecular bone area, bone volume and tissue volume fraction (BV / TV) and trabecular bone mineral density (Tb.BMD, mg HA / ccm) were evaluated. The results are shown in Figure 4 A and Figure 4 B. Cortical bone volume (Ct.BV, mm) was measured at the midshaft of the femur. 3 ), front and rear diameters and inner and outer diameters (see Figure 4 J) The results are shown in Figure 4 C. Figure 4 D and Figure 4 In E.

[0202] To test the biomechanical properties of the bone, we performed three-point bending tests as described previously (Turner CH, Burr DB. Bone 1993; 14: 595–608). Load was applied in compression mode at a nominal deformation rate of 2 mm / min until fracture. Load-displacement curves were recorded during the test. Yield force and stiffness results are shown in Figure 4 F and Figure 4 G in.

[0203] In summary, the results showed that mice supplemented with lactoferrin-osteopontin coacervate complexes showed improved bone development, growth and strength compared to mice supplemented with different forms of lactoferrin-osteopontin. For example, mice supplemented with lactoferrin-osteopontin coacervate complexes had significantly higher trabecular BV / TV, trabecular bone mineral density and cortical bone volume.

[0204] Implementation

[0205] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paragraphs).

[0206] 1. Complex coacervates containing lactoferrin and osteopontin.

[0207] 2. The complex coacervate according to paragraph 1, wherein the complex coacervate comprises protein in an amount of 5% w / w to 50% w / w, preferably in an amount of 15% w / w to 40% w / w and more preferably in an amount of 25% w / w to 35% w / w.

[0208] 3. A complex coacervate according to any one of paragraphs 1 and 2, wherein the complex coacervate comprises water in an amount of 50% w / w to 95% w / w, preferably in an amount of 60% w / w to 85% w / w and even more preferably in an amount of 65% w / w to 75% w / w.

[0209] 4. A complex coacervate according to any of the preceding paragraphs, wherein the complex coacervate has a diameter of at least 500 nm in the shortest dimension, preferably at least 600 nm in the shortest dimension, more preferably at least 700 nm in the shortest dimension and more preferably at least 900 nm in the shortest dimension.

[0210] 5. The complex coacervate according to any one of the preceding paragraphs, wherein the complex coacervate has a zeta potential in the range of -15 mV to +15 mV, preferably in the range of -10 mV to +10 mV and more preferably in the range of -8 mV to +5 mV.

[0211] 6. A method for preparing a complex coacervate according to any one of the preceding paragraphs, wherein the method comprises the following steps:

[0212] a. providing a separate aqueous solution comprising lactoferrin and osteopontin,

[0213] b. mixing the separate aqueous solutions comprising lactoferrin and osteopontin at a pH of 4 to 6, preferably at a pH of 4.5 to 5.5, more preferably at a pH of 4.8 to 5.2, even more preferably at a pH of 5, and wherein the separate aqueous solutions comprising lactoferrin and osteopontin are adjusted such 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.

[0214] 7. The method according to paragraph 6, wherein in step b., the ionic strength in the mixed aqueous solution is not higher than 30 mM, preferably not higher than 20 mM, more preferably not higher than 10 mM, more preferably not higher than 5 mM and even more preferably not higher than 0.2 mM of added salt, preferably added inorganic salt, more preferably added NaCl.

[0215] 8. The method according to any one of paragraphs 6 and 7, wherein in step b., the separate aqueous solution comprising lactoferrin and osteopontin is adjusted so that the total protein concentration is in the range of 2% w / v to 8% w / v, preferably in the range of 4% w / v to 6% w / v.

[0216] 9. A composition comprising the complex coacervate according to any one of paragraphs 1 to 5.

[0217] 10. The composition according to paragraph 9, 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.

[0218] 11. The composition according to any of paragraphs 9 and 10, wherein the composition is an infant formula, a first 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 supplement.

[0219] 12. A complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin for use in the treatment or prevention of metabolic disorders, in particular overweight, obesity, prediabetes or diabetes and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis.

[0220] 13. A method for treating or preventing metabolic disorders, in particular overweight and obesity, prediabetes or diabetes, and / or inflammatory diseases, in particular sepsis or necrotizing enterocolitis, by administering to a subject a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition comprising a complex, preferably a complex coacervate comprising lactoferrin and osteopontin.

[0221] 14. A complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin for use in promoting bone development, growth, strength and / or healing or in preventing and / or treating bone diseases.

[0222] 15. A method for promoting bone development, growth, strength and / or healing or preventing and / or treating bone diseases by administering to a subject a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin or a composition containing a complex, preferably a complex coacervate, comprising lactoferrin and osteopontin.

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Claims

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

2. The complex coacervate for use according to claim 1, wherein the complex coacervate comprises protein in an amount of 5% w / w to 50% w / w, preferably in an amount of 15% w / w to 40% w / w and more preferably in an amount of 25% w / w to 35% w / w.

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

4. A complex coacervate for use according to any preceding claim, wherein the complex coacervate has a diameter in the shortest dimension of at least 500 nm, preferably at least 600 nm in the shortest dimension, more preferably at least 700 nm in the shortest dimension and even more preferably at least 900 nm in the shortest dimension.

5. The complex coacervate for use according to any preceding claim, wherein the complex coacervate has a zeta potential in the range of -15 mV to +15 mV, preferably in the range of -10 mV to +10 mV and more preferably in the range of -8 mV to +5 mV.

6. The complex coacervate for use according to any preceding claim, wherein the complex coacervate is administered in the form of a composition.

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

9. The complex coacervate for use according to any preceding claim, wherein the complex coacervate is administered orally.

10. The complex coacervate for use according to any preceding claim, wherein the complex coacervate is administered at a dose of 100 mg / kg / day to 10000 mg / kg / day, preferably at a dose of 500 mg / kg / day to 5000 mg / kg / day and more preferably at a dose of 1000 mg / kg / day to 2000 mg / kg / day.

11. A complex coacervate for use according to any of the preceding claims, wherein the complex coacervate increases bone growth and / or bone strength, preferably wherein the complex coacervate increases one or more bone parameters selected from the following: trabecular bone volume and tissue volume fraction (BV / TV), bone mineral density (BMD), bone mineral content (BMC), cortical bone volume (Ct.BV), inner and outer diameters, anterior-posterior diameter, bone yield force and bone stiffness.

12. The complex coacervate for use according to any preceding claim, 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 complex coacervate for use according to any preceding claim, wherein the subject is a teenager, adolescent, child or infant, preferably wherein: (i) the subject is born prematurely or has a low birth weight or experiences intrauterine growth retardation; (ii) the subject suffers from growth retardation due to malnutrition or experiences a disease such as anorexia, Crohn's disease and / or celiac disease; and / or (iii) the subject is growth-retarded due to treatment with a drug that causes malabsorption, anorexia and / or metabolic bone disease.

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

15. The complex coacervate for use according to any preceding claim, wherein the subject is a human being, or wherein the subject is an animal, preferably wherein the subject is a human being.

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