Stable isotonic lyophilized protein formulation

LACTOFERRIN

This application claims the priority of New Zealand Application Serial No. 518 121, filed April 3, 2002.

BACKGROUND

Lactoferrin is an 80kD iron-binding glycoprotein present in most exocrine fluids including tears, bile, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus, seminal fluid and milk. It is one of the major constituents of the secondary specific granules of circulating polymorphonuclear neutrophils. The richest source of lactoferrin is mammalian milk and colostrum.

Lactoferrin circulates at a concentration of 2 - 7 mg / ml.

Multiple biological functions have been postulated, including regulation of iron metabolism, immune function, and embryonic development. Lactoferrin has antimicrobial activity against a range of pathogenic organisms, including Gram positive and Gram negative bacteria, yeast and fungi. The antimicrobial effect of lactoferrin is based on its ability to bind iron, which is essential for the growth of pathogens. Lactoferrin also inhibits the replication of various susceptibility of some bacterial lysozyme by binding to the lipopolysaccharide component of the bacterial membranes.

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EP 1,116,490 A1 describes the use of lactoferrin to improve liver function.

US Patent No. 5,932,259 describes the use of a basic protein fraction isolated from milk as a bone strengthening agent.

US Application No. 2002/0004073 discloses food supplements for human consumption comprising colostrum and lactoferrin for use in inhibiting infection and enhancing tissue repair and healing.

JP 2000281586 discloses the use of an iron / lactoferrin composition for use in bone strengthening wherein the composition comprises an iron / lactoferrin complex having at least 3 iron atoms per lactoferrin molecule.

Kanyshkova et al Lactoferrin and its biological function; Biochemistry 66 (1) 2001, pages 1-7 discloses that lactoferrin has two iron binding sites.

According to the present invention there is provided a lactoferrin polypeptide for use in the treatment or prevention of osteoporosis, wherein said polypeptide is at least 65% pure dry weight lactoferrin and wherein said lactoferrin polypeptide is not contains more than two metal ions per lactoferrin molecule.

Preferably, said lactoferrin polypeptide contains two metal ions per molecule.

Advantageously, said lactoferrin polypeptide contains iron, copper, chromium, cobalt, manganese, zinc, or magnesium ions.

More preferably, said lactoferrin polypeptide contains iron ions.

Conveniently, said polypeptide is human or recombinant lactoferrin.

Lactoferrin polypeptide is preferably at least 75%, more preferably at least 85% and even more preferably at least 95% pure.

The invention also provides a composition comprising said nutraceutical lactoferrin polypeptide of the invention, together with a pharmaceutical composition comprising said lactoferrin polypeptide of the invention.

Advantageously, the compositions of the present invention further comprise another bone enhancing agent.

Conveniently, said bone strengthening agent is selected from calcium, zinc, magnesium, vitamin C, vitamin D, vitamin E and vitamin K2.

RESUME

This invention relates to lactoferrin, which is capable of stimulating the growth of bone resorption inhibition.

a skeleton polypeptide and the

Specifically, the present invention provides a pure lactoferrin polypeptide containing no more than two (i.e., 0.1, or preferably 2) metal ions per molecule. A pure polypeptide is a polypeptide free from other biological macromolecules, and at least 65% (e.g. at least 70, 75, 80, 85, 90, 95 or 99%) pure by dry weight. The purity of a polypeptide may be measured by any appropriate standard method, for example by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The lactoferrin polypeptide may be a naturally occurring polypeptide, a recombinant polypeptide, or a synthetic polypeptide. Variants of a wild type lactoferrin polypeptide (e.g., a wild type lactoferrin polypeptide fragment containing at least two (e.g. 4, 6, 8, 10, 20, 50,100, 200, 300, 400, 500, 600, 700) amino acids, or a recombinant protein containing a lactoferrin polypeptide sequence) that maintain the biological activity of a wild type lactoferrin polypeptide are within the scope of the invention. A lactoferrin polypeptide of the invention may have a mammalian origin, for example from human or bovine milk. The metal ion bound to the polypeptide may be an iron ion (as in a naturally occurring lactoferrin polypeptide), a copper ion, a chromium ion, a cobalt ion, manganese ion, zinc ion, or an ion. of magnesium.

The lactoferrin polypeptide of the invention may be used to stimulate skeletal growth (e.g., by promoting proliferation of osteoblasts and chondrocytes) and inhibit bone resorption (e.g., inhibit osteoclast development). A preparation of a lactoferrin polypeptide of the invention (for example lactoferrin, isolated from bovine milk) may contain polypeptides of a single species, for example, each molecule having two iron ion bonds. It may also contain polypeptides of different species, for example some non-ionic binding molecules and others each binding to one or two ions; some molecules of each bond of an iron ion and others bonded to copper ions, some molecules of each are a biological active lactoferrin polypeptide (full length or shorter than full length) containing 0, 1, or 2, metal ions and others, each being a fragment (same or different) of the polypeptide, or all molecules of each being a fragment (same or different) of a full-length lactoferrin polypeptide containing 0, 1 or 2 metal ions. For example, a mixture of full-length lactoferrin polypeptides and various full-length lactoferrin polypeptide fragments may be prepared from a hydrolyzate, for example, a partial digestion such as a digested proteinase, whole-body lactoferrinated polypeptide. Otherwise, it may be obtained by mixing full-length lactoferrin polypeptides with various fragments of full-length lactoferrin polypeptides (e.g., synthetic fragments). A mixture of several full-length lactoferrin polypeptide fragments, on the other hand, may be prepared, for example, by complete digestion (i.e. no full length polypeptides remain after digestion) of full-length lactoferrin polypeptides, or by different fragment mixtures. lactoferrin polypeptide fragments.

The invention further provides a nutritional composition, which may be milk, a juice, a soda, an energy bar, or a dietary supplement. The composition contains a nutraceutical lactoferrin polypeptide of the invention or a mixture of polypeptide fragments and the polypeptide in an amount greater than naturally occurring. Lactoferrin has been shown to stimulate osteoblast and chondrocyte proliferation and inhibit osteoclast development. Thus, a nutraceutical composition of the present invention is useful for the prevention and treatment of bone diseases such as osteoporosis and arthritis or osteoarthritis. The composition may further include nutraceutical in a suitable amount of another bone augmenting agent, such as calcium, zinc, magnesium, vitamin C, vitamin D, vitamin E, vitamin K2, or a mixture thereof.

Further, the present invention provides a pharmaceutical composition containing a lactoferrin polypeptide of the invention or a mixture of polypeptide fragments and the polypeptide and a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition also includes another bone strengthening agent. The invention also encompasses the use of a lactoferrin polypeptide or a mixture of the polypeptide and polypeptide fragments described above for the manufacture of a medicament for the prevention and treatment of bone diseases.

The present invention provides a method of preventing and treating bone related diseases (e.g., stimulating skeletal growth and inhibiting bone resorption). The method includes administering to a subject in need thereof an effective amount of a lactoferrin polypeptide of the invention or a mixture of polypeptide and polypeptide fragments. 0 The method may further concurrently include administering to a subject an effective amount of another bone augmenting agent.

Details of one or more embodiments of the invention are set forth in the accompanying description. Other features, objects and advantages of the invention will be apparent from the detailed description, and from the claims.

DETAILED DESCRIPTION

The present invention is based on the unexpected discovery that lactoferrin stimulates osteoblast and chondrocyte proliferation and inhibits osteoclast development. Thus, it is useful for the prevention and treatment of bone diseases.

The lactoferrin polypeptide of the invention is a pure polypeptide containing no more than two metal ions per molecule. Practically, the ion / lactoferrin ratio measurement for a lactoferrin preparation can be in the range 0

2.5. It can be isolated from a natural source (e.g., mammalian milk), or produced using genetic engineering techniques or chemical synthesis techniques well known in the art. The following is an exemplary procedure for isolating lactoferrin from bovine milk:

Fresh skimmed milk powder (7 L, pH 6.5) is passed through a 300 ml column of Milli Q water-balanced S-Sepharose Fast Flow at a flow rate of 5 ml / min at 4 ° C . Unbound protein is washed with 2.5 bed volumes of water and the bound protein stepwise diluted with approximately 2.5 bed volumes each of 0.1 M, 0.35 M and sodium chloride 1. The MA lactoferrin eluting as a discrete pink band in 1 M sodium chloride is collected as a single fraction and dialyzed against Milli Q water followed by dry freezing. The lyophilized powder is dissolved in 25 mM sodium phosphate buffer pH 6.5 and subjected to S-Sepharose Fast Flow chromatography with a 1 M sodium chloride gradient in the above buffer and a flow rate of 3 mL / min. . Lactoferrin containing fractions of sufficient purity as determined by gel electrophoresis and reverse phase HPLC are combined, dialyzed and lyophilized. Final purification of lactoferrin is performed by gel filtration on potassium Sephacryl 300 in 80 mM phosphate, pH 8.6, containing 0.15 M potassium chloride. The selected fractions are combined, dialyzed against Milli-Q water and lyophilized. . The purity of this preparation is greater than 95% as indicated by HPLC analysis and the spectral ratio values ​​(280 nm / 465 nm) of ~ 19 or less for the iron-saturated lactoferrin form.

Iron saturation is obtained by the addition of a 2: 1 molar excess of 5mM ferric nitrilotriacetate (Foley and Bates (1987) Analytical Biochemistry 162, 296-300) to a 1% solution of 50mM Tris purified lactoferrin, pH 7.8 containing 10 mM sodium bicarbonate. Excess ferric nitrilotriacetate is dialyzed against 100 volumes of Milli Q water (twice renewed) for a total of 20 hours at 4 ° C. The charged iron (holo-) lactoferrin is then lyophilized.

Iron-free (apo-) lactoferrin is prepared by dialysis of a solution of 1% of the highly purified lactoferrin sample in water against 30 volumes of 0.1 M citric acid, pH 2.3, containing 500 mg / l disodium EDTA , for 30 h at 4 ° C (Massons and Heremans (1966) Protides of the Biological Fluids 14, 115-124). Citrates and EDTA are then removed by dialysis against 30 volumes of Milli Q water (once renewed) and the solution colorless resultant lyophilized.

The lactoferrin polypeptide of the invention may contain an iron ion (as in the naturally occurring lactoferrin polypeptide) or a non-iron metal ion (e.g. a copper ion, a chromium ion, a cobalt ion, a manganese ion). (zinc ion, or a magnesium ion). For example, lactoferrin isolated from bovine milk may be iron-depleted and then charged with another type of metal ion. For example, copper loading may be achieved according to the same iron loading method described above. To charge lactoferrin with other metal ions, the method of Ainscough, et al. ((1979) Inorganic Chimica Acta 33, 149-153) may be used.

In preparing a lactoferrin polypeptide of the invention, the polypeptides may be from a single species or from different species. For example, the polypeptides may each contain a different number of iron ions or different species of metal ions, or the lengths of the polypeptides may vary, for example some are full length polypeptides and some are fragments and the fragments may each represent a specific technique of a full body polypeptide. Such a preparation may be obtained from a natural source or by mixing different lactoferrin species. For example, a lactoferrin mixture of different lengths may be prepared by proteinase digestion (full or partial) of lactoferrin full length polypeptides. The degree of digestion may be controlled according to methods well known in the art, for example by manipulating the amount of proteinase or incubation time. Complete digestion yields a mixture of lactoferrin fragments of various total polypeptide lengths; a partial digest yields a mixture of full length lactoferrin polypeptides and various fragments.

of polypeptide polypeptide lactoferrin polypeptide or a mixture of polypeptide fragments and the above described polypeptide is used to prepare a nutraceutical composition of this invention for the prevention and treatment of bone related diseases. Examples of such disorders include, but are not limited to, osteoporosis, osteoarthritis or rheumatoid arthritis, hepatic osteoarthritis, osteomalacia, rickets, cystic fibrous osteitis, renal osteoarthritis, osteopenia, fibrogenesis-imperfect ossium, hyperparathyroidism, secondary parathyroidism, hyperparathyroidism, hyperparathyroidism, , sarcoidosis, renal glucocorticoid-induced osteoporosis, idiopathic hypercalcemia, Paget's disease, and osteogenesis imperfecta. The nutraceutical composition may be a dietary supplement (e.g., a capsule, a mini-bag, or a tablet), or a food product (e.g., milk, juice, soft drink, an herbal tea bag, or herbal products). confectionery). The composition may also include other nutrients, such as a protein, carbohydrate, vitamins, minerals or amino acids. The composition may be in a form suitable for oral use, such as a tablet, hard or soft capsule, an aqueous or oil suspension, or a syrup, or in a form suitable for parenteral use, such as non-aqueous propylene glycol solution, or a buffered aqueous solution. The amount of the active ingredient in the nutraceutical composition depends to a large extent on the specific need of a subject. The amount also varies, as recognized by those skilled in the art, depending on the route of administration, and possible co-use of other bone augmenting agents.

Still in accordance with the object of the present invention is a pharmaceutical composition containing an effective amount of a lactoferrin polypeptide or a mixture of the polypeptide and polypeptide fragments described above, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to prevent and treat bone related disorders described above. The pharmaceutical composition may further include an effective amount of another bone augmenting agent. 0 A pharmacologically acceptable carrier includes a dispersing solvent medium, a coating, an antifungal antibacterial agent, and an isotonic absorption-retarding agent. An effective amount is the amount required to confer therapeutic effect. The interrelationship of dosages for animals and humans (based on milligrams per square meter of body surface area) is described by Freireich et al. (1966) Cancer Chemother. Rep. 50: 219. Body surface area can be roughly determined from the height and weight of the subject. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses also vary, as recognized by those skilled in the art, depending on the route of administration, excipient use, and the like.

The lactoferrin polypeptide of the invention or a mixture of polypeptide fragments and the polypeptide may be formulated into pharmaceutical forms for different administration routes using conventional methods. For example, it may be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules may contain conventional pharmacologically acceptable materials, such as gelatin or cellulose. Tablets may be formulated according to conventional procedures by compressing mixtures of lactoferrin polypeptide or a mixture of polypeptide fragments and the polypeptide with a solid carrier and a lubricant. Examples of solid carriers include starch and bentonite sugar. Lactoferrin polypeptide or a mixture of polypeptide and polypeptide fragments may also be administered in the form of a hard shell tablet or a capsule containing a binder, for example lactose or mannitol, a conventional filler, and an agent. of tablet formation. A pharmaceutical composition may be administered parenterally. Examples of parenteral dosage forms include aqueous solutions, 5% isotonic or glucose saline of the active agent, or other well known pharmacologically acceptable excipients. Cyclodextrins, or other solubilizing agents known to those of skill in the art, may be used as pharmaceutical excipients for delivery of the therapeutic agent.

The effectiveness of a composition of the present invention can be evaluated both in vitro and in vivo. See, for example, the examples below. Briefly, the composition can be tested for its ability to promote osteoblast and chondrocyte proliferation in vitro. For in vivo studies, the composition can be injected into an animal (e.g., a rat) and its effects on bone tissues are then evaluated. Based on the results, an appropriate dosage range and route of administration can be determined.

The specific examples given are to be construed as illustrative only and not limiting the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. Lactoferrin favors the proliferation of primary rat osteoblasts.

Osteoblasts were isolated by 20-day calvary rat fetal collagenase digestion, as previously described by Lowe and co-contributors (Lowe, et al. (1991) Journal of Bone Mineral Research and 6, 1277-1283). Calvariae were dissected aseptically, and the frontal and parietal bones were stripped of their periosteum. Only the bone-free central portions of the suture tissue were collected. Calvaria were treated twice with buffered saline phosphate (PBS) containing 3 mM EDTA (pH 7.4) for 15 minutes at 37 ° C in a water bath. with agitation. After washing once with PBS, the calves were treated twice with 3 ml of 1 mg / ml collagenase for 7 minutes at 37 ° C. After discarding the supernatants from digestions with I and II, the calvaria were further treated twice with 3 ml of 2 mg / ml collagenase (30 min, 37 ° C). Digestion III and IV supernatants were pooled, centrifuged and the cells were washed in Dulbecco's Modified Eagle's Medium (DME) with 10% fetal calf serum (FCS), suspended in DME / 10% FCS and vials. 75 cm³. Cells were incubated in 5% CO₂ and 95% air at 37 ° C. Confluence was reached within 5-6 days, at which time the cells were subcultured. After trypsinization using trypsin-EDTA (0.05% / 0.53 mm), cells were washed in minimal essential medium (MEM) with 5% FCS and resuspended in fresh medium and then seeded at 5 x 10 4 cells. / ml in 24 - well plates (0.5 ml cell suspension per well, ie 1.4 x 104 cells / cm²). The osteoblast character of these cells was established by demonstrating high levels of activity and osteocalcin acaline phosphatase production [as described by Groot et al. (1985) Cell Biol Int. Res. 9, 528], and a parathyroid hormone-sensitive adenylate cyclase response and prostaglandins [as described by HermannErlee, et al. (1986) Ninth International Conference on Calcium Regulating Hormones and Bone Metabolism, p 409].

Proliferation studies (cell count and thymidine incorporation) were performed on both active and non-active growing cell populations. To produce the active growing cells, confluent subpopulations (24 h after subculture) were placed in fresh MEM containing 1% FCS and a lactoferrin sample. To produce non-active growth cells, confluent subpopulations were placed in serum free medium with 0.1% bovine serum albumin plus a lactoferrin sample. Cell numbers were analyzed at 6, 24 and 48 hours after addition of lactoferrin samples (i.e. purified holo-and apo-lactoferrin lactoferrin) prepared as described above. Cell numbers were determined after removing well cells by trypsin / EDTA exposure (0.05% / 0.53 mM) for about 5 minutes at 37 ° C. Counting was performed in a hemocytometer chamber. [3H] Actively growing and non-actively cells graving thymidine was assessed by pulsing the cells with [3 H] thymidine (1 mCi / well) two hours before the end of incubation. The experiment was terminated at 6, 24 or 48 hours by washing the cells in MEM medium containing cold thymidine followed by the addition of 10% trichloroacetic acid. The precipitate was washed twice with ethanol: ether (3: 1) and the WDLS dehydrated at room temperature. The residue was redissolved in 2 M KOH at 55 ° C for 30 min, neutralized with 1 M HCl, and an aliquot counted for radioactivity. For both cell counts and thymidine incorporation, each experiment at each time point was performed at least four times using different experimental groups consisting of at least six wells.

The mitogenic response of the purified lactoferrin sample was found to be very potent as shown by a significantly increased rate of osteoblast cell proliferation (e.g., increased incorporation of thymidine into growing cell DNA). The potent osteogenic response observed above was compared to that of insulin-like growth factor 1 (IGF-1), a well-recognized osteoblast mitogen. IGF-1 demonstrated a maximal effect of 1.25-fold control on the same osteoblast cell culture system, while the effect of lactoferrin was 2.26-fold greater than the control for the highest dose tested (10 mg / ml).

Lactoferrin favors chondrocyte proliferation

Chondrocytes were isolated by cartilage removal (complete depth slices) from the tibial and femoral surfaces of sheep under aseptic conditions. The slices were placed in Dulbecco Modified Eagles (DME) media containing 5% SFB (v / v) and antibiotics (penicillin 50 g / l, streptomycin 50 g / l and neomycin 100 g / l) and cut very thin with a scalpel . Tissues were removed and incubated at 37 ° C first with pronase (0.8% w / v for 90 minutes) followed by collagenase (0.1% w / v for 18 hours) to complete the digestion. Cells were isolated from digestion by centrifugation (10 minutes at 1300 rpm), resuspended in 5% DME / FBS, passed through a 90 Fm pore size nylon mesh screen to remove any fragments digested, and recentrifuged. The cells were then washed and resuspended twice in the same medium, seeded into 75 cm 2 flasks containing DME / 10% FBS, and incubated in 5% CO2 / 95% air at 37 ° C. ⁰ C. Confluence was reached within 7 days, at which time the cells were subcultured. After trypsinization using trypsin-EDTA (0.05% / 0.53 mm), cells were washed in 5% DME / FBS and resuspended in fresh medium, and then seeded in 24-well plates (5 x 10 4 cells / mL, 0.5 mL / well). Measurement of thymidine incorporation was performed on growth-arrested cell populations as for the osteoblast cell cultures described above. Lactoferrin has been shown to stimulate chondrocyte proliferation at concentrations above 0.1 mg / ml.

Lactoferrin favors the proliferation of osteoblasts in organ culture

Neonatal rat organ culture has been previously described (Cornish, et al. (1998) Am J Physiol 274, E827-E833).

Briefly, neonatal two-day-old rats were injected subcutaneously with radiolabelled 45Ca. Three days later, the calvarias were excised and meshed in petri dishes containing 0.1% albumin / Mean 199 bovine serum. Lactoferrin was added, and the calvaria were incubated for 48 hours. Four hours before the end of the incubation period, [3 H] -thymidine was added. The experiment was terminated, and 45 Ca release and thymidine incorporation were measured. Lactoferrin has been shown to stimulate DNA synthesis, which reflects the proliferation of osteoblast lineage cells.

Signs of lactoferrin via MAP kinase in osteoblasts

The methodology has been described previously (Gray, et al. (2001) Endocrinology 142,1098-1106). Specifically, rat primary osteoblasts were prepared as described above seeded in 6-well tissue culture plates at an initial density of 5 x 104 cells / ml in 5% FCS MEM, and cultured to 80-90% confluence. Following overnight serum deprivation, cells were treated at room temperature with lactoferrin in MEM / 0.1% experiments designed to determine the

BSA. In the effects of signal transduction inhibitors on p42 / 44 MAP induced lactoferrin phosphorylation kinase, cells were pretreated with the inhibitor for 30 min prior to the addition of lactoferrin. After treatment for the indicated time period, the average treatment was aspirated, the cells were washed in ice cold PBS and then scraped in ice cold HNTG lysis buffer (50 mM HEPES, pH 7.5.150 mM NaCl, 1% Triton, 10 % glycerol, 1.5 mM MgC12, 1 mM EDTA), containing a protease and phosphatase inhibitor cocktail (1 mM PMSF, 1 mg / ml peptatin, 10 mg / ml leupeptine, 10 mg / ml aprotinin 1 nM sodium vanadate, 500 The lysates were briefly vortexed, centrifuged at 13,000 rpm at 4 ° C, and then stored at 70 ° C until analyzed. The protein content of cell lysates was measured using the DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of all cell lysate (30-50 mg) were subjected to 8% SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted and overnight at 4 ° C with an anti-phospho-p42 / 44 MAP kinase antibody. (1: 1000). As a control for protein loading, the same filters were cleaned and punctured again with an antibody against the total p42 / 44 MAP kinase (1: 400). Incubation with the HRP-conjugated secondary antibody was for 1 h at room temperature. and the membranes were analyzed by ECL. Inoculation tests were repeated at least three times.

Lactoferrin has been shown to induce MAP kinase phosphorylation in P42 / P44 osteoblasts at a dose and time-dependent concentration of 1-100 mg / ml.

Lactoferrin stimulates bone growth in vivo

The rat model used in these studies has been previously described (Cornish, et al. (1993) Endocrinology 132, 13591366). Injections (0 mg, 0.04 mg, 0.4 mg and 4 mg) of lactoferrin were administered daily for 5 days and the animals were sacrificed one week later. Bone formation was determined by fluorescent labeling of newly formed bone. Bone resorption and bone mass indexes were determined by conventional light microscopy assisted by image analysis software. Local injection of lactoferrin into adult rats resulted in increased skullcap bone growth, with significant increases in bone area after only five injections, and the animals were sacrificed one week later. Bone formation was determined by fluorescent labeling of newly formed bone.

Application 1

Set of 14 to 17% solids yoghurt, whether or not containing fruit, may be prepared as follows:

Heat skimmed milk powder medium (between 109-152 g) and stabilizer ALACO (100 g) are reconstituted with approximately 880 ml of water at 50 ° C. Anhydrous milk fat (20 g) is then added and mixed for 30 mins The mixture is then heated to 60 ° C, homogenized at 200 bar, and then pasteurized at 90 ° C. After cooling to 40-42 ° C, the starting mixture and the lyophilized protein preparation described above (up to 50 mg of 95% purity lactoferrin, or an equivalent amount from a less highly purified source) is

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An alternative method for preparing the same set of yogurts is by dry mixing the indicated amount of lactoferrin or the amount indicated as a dosage rate in the dried milk solids prior to their use in the yogurt formulation.

Application 2

Dry mixtures of skimmed milk powder or skimmed milk or calcium hydroxide and dry frozen lactoferrin preparations may provide formulations based on milk or compositions which may be used as either functional foods or functional food ingredients. Such compositions may be used as reconstituted milks, powdered milk ingredients, dairy desserts, functional foods, cheese, butter or beverages and nutraceuticals or dietary supplements. Mixing the dry ingredients in milk powder: calcium: active lactoferrin ratios between 90: 9.5: 0.5 and 94: 5.95: 0.0001 providing suitable compositions for such uses.

Application 3

Mixtures of milk powder, calcium, and the rich ingredient lactoferrin may be used as bone health foods, functional bone health food ingredients, or as a food ingredient for the supply of bone health nutrients. in a range of healthy foods.

For these compositions, the calcium and protein concentrations of the compositions must be adjusted to the required permissible nutritional limits. Commercially available ingredients such as milk powder usually contain between 300 and 900 mg of calcium per 100 g of powder, depending on their sources. A source of calcium may be added to the powder to increase the calcium content of up to 3% by weight of milk powder ingredient as a mixture. The level of commercially available milk or dairy-based protein ingredient and protein powders vary, depending on the type of ingredient, the method of manufacture, the intended use.

and its powder be used as healthy drinks.

The milk ingredient usually contains between 12% and 92% protein. Examples that are commercially available are skim and whole milk powder, food grade caseins, caseinates, powdered milk protein concentrate, spray dried or retained microfiltrated ultrafiltrate powder, and isolate the protein from milk products. The lactoferrin rich preparation may be incorporated into a mixture of protein and calcium to provide nutritional milk powders which may be ingredients in foodstuffs and

These mixtures provide the ingredients suitable for use in the preparation of yoghurt and yogurt drinks, acidic beverages, powdered milk ingredient mixtures, pasteurized dairy liquid products, dairy products, cultured UHT milk products, acidified dairy drinks, combination products. milk and malt-based cereals, milk, milk and combination soy products. For such uses, the mixture may have a composition wherein the calcium content is between 0.001% and 3.5% (w / w), the protein composition is between 2% and 92%, and lactoferrin as osteoblast proliferating agent is added at levels between 0.000001% and 5.5^!

OTHER WAYS OF CARRYING OUT

All features described herein may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same or similar purpose. Thus, unless expressly indicated otherwise, each feature is disclosed in only one example of a generic series of equivalent or similar features.