TECHNICAL FIELD OF THE INVENTION The present invention relates to iron-fortified edible oil-and-water emulsions that combine improved bioavailability of the iron with excellent taste and good product stability. Examples of edible emulsions encompassed by the invention include spreads, margarine, mayonnaise and dressings. BACKGROUND OF THE INVENTION

Iron is an essential trace element in animal and human nutrition. It is a component of heme in hemoglobin and of myoglobin, cytochromes as well as of the catalytic region of many enzymes. The main role of iron is its participation in the transport, storage and utilization of oxygen.

Iron deficiency (sideropenia or hypoferremia) was and remains a common nutritional problem not only in the developing world but also in the industrialized countries. When loss of iron is not sufficiently compensated by adequate intake of iron from the diet, a state of iron deficiency develops over time. When this state remains uncorrected, it eventually leads to iron deficiency anemia (IDA). Before iron deficiency develops into IDA, the medical condition of Iron

Deficiency without anemia is called Latent Iron Deficiency (LID) or Iron-deficient erythropoiesis (IDE). IDA is a common anemia (low red blood cell or

hemoglobin levels). Iron deficiency causes approximately half of all anemia cases worldwide.

The recommended daily allowance for iron intake is from 10 to 27 mg per day, and is dependent on age and gender. Children, women of child-bearing age, and in particular pregnant women, women who wish to become pregnant and nursing mothers are in the group with higher requirements of iron. Iron compounds which are used or have been studied as iron fortificants in nutritional supplements and food products include ferrous sulfate, ferrous fumarate, ferrous folate, an iron dextran, ferric oxyhydroxide dextran, a chitosan derivative of iron, an oligosaccharide derivative of iron, ferrous acetyl salicylate, ferrous gluconate, ferrous pyrophosphate, carbonyl iron, ferric orthophosphate, ferrous glycine sulfate, ferrous chloride, ferrous ammonium citrate, ferric ammonium citrate, ferric ammonium tartrate, ferric phosphate, ferric potassium tartrate, ferric albuminate, ferric cacodylate, ferric hydroxide, ferric

pyrophosphate, ferric quinine citrate, ferric valerate, saccharated iron oxide, iron oxide, ferric chloride, ferrous iodide, ferrous nitrate, ferrous glycerophosphate, ferrous formate, an amino acid and iron salt, an iron salt of a protein

hydrolysate, ferrous lactate, ferrous tartrate, ferrous succinate, ferrous glutamate, ferrous citrate, ferrous pyrophosphate, ferrous choline isocitrate, ferrous carbonate, an iron-sugar-carboxylate chelate, ferrous sucrate malate, ferrous sucrate citrate, ferrous fructate citrate, ferrous sucrate ascorbate, ferrous fructate ascorbate, sodium iron EDTA (NaFeEDTA), and ferrous bisglycinate chelate. In general, water-soluble iron compounds have the highest relative

bioavailability of the conventional iron sources but frequently cause

unacceptable sensory changes or deleterious changes in food quality. Ferrous sulfate is the most commonly used, water-soluble iron fortificant and is found in infant formula, bread and pasta, and iron supplements. It can also be added to wheat flour when stored for short periods but may provoke fat oxidation and "off-flavors" in milk, wheat and other cereal flours stored for longer periods. Pestaner et al. have stated, "Ferrous sulfate is the cheapest, most toxic, and most frequently used iron supplement and has an elemental iron content of approximately 20%." [J. P. Pestaner, K. G. Ishak, F. G. Mullick, J. A. Centeno, Ferrous sulfate toxicity: a review of autopsy findings, Biolog. Trace Element Res 69: 191 -198, 1999] Ferrous sulfate is very soluble in water and aqueous solutions, dissolves to provide solutions having a strongly acid pH of about 2, and is described as a corrosive agent on related Material Safety Data Sheets.

A more expensive alternative to ferrous sulfate, NaFeEDTA, offers the advantages that it has equivalent bioavailability and prevents iron binding to iron absorption inhibitors, particularly phytate. Further, it does not catalyze fat oxidation in stored wheat flour. However, the EFSA Panel on Food Additives and Nutrient Sources advised in 2010 that the daily intake of EDTA from food should not exceed 1 .9 mg/kg bodyweight day. This maximum severely limits the scope for application of EDTA in products that are consumed by infants and children.

Compounds that are poorly soluble in water but soluble in dilute acid (e.g., ferrous fumarate, ferrous gluconate, ferrous saccharate) offer the advantages that they cause less organoleptic changes and may be selected to have a bioavailability that is comparable to that of ferrous sulfate. At present, ferrous fumarate is widely used to fortify infant cereals, and ferrous saccharate is added to chocolate drink powders. Ferrous bisglycinate, a more expensive alternative to the other ferrous salts, has exhibited equivocal iron bioavailability, and has a tendency to cause color reactions and catalyze fat oxidation.

Water-insoluble compounds that are poorly soluble in dilute acid are the least well absorbed of the iron fortificants. In general, this class of insoluble iron fortificants comprises ferric iron in a form which precipitates from aqueous solutions having a pH above 3.5 (e.g., ferric phosphate, ferric pyrophosphate) or fine particles of elemental iron (e.g., colloidal iron). In general, fortificants in this class offer the significant advantages that they have no distinctive taste and have lower tendencies to promote fat oxidation, but special strategies may be needed to enhance bioavailability to useful ranges. Finally, protoporphyrin-bound iron (heme-Fe) has been studied both as a dietary supplement and an additive in cereals for infants and children. Heme-Fe offers the advantages that uptake is high and predictable, but its intense color and concerns about contamination during its collection from bovine blood, together with technical difficulties in processing, residual contamination removal, and storage, deter broad use.

As a consequence of its widespread use, both in foods and in dietary

supplements, ferrous sulfate is currently the standard against which the bioavailability of other iron sources is compared. Among the conventional strategies that have been used to enhance the availabilities of other iron sources are:

• Particle size reduction: Micron ization significantly decreases particle size and enhances uptake in the intestine, through more rapid solubilization and other mechanisms.

• Addition of ascorbate: For over 50 years, it has been recognized that

ascorbate significantly enhances iron absorption. The primary activity of ascorbate is believed to be chemical reduction of iron from its ferric to its ferrous oxidation state, since intestinal absorption of ferrous iron is favored. Further, ferric iron is reduced at the surface of the intestinal cells by the enzyme ferri-reductase (also known as Dcytb), which in turn requires ascorbate for its function. Finally, ascorbate may also enhance iron availability by preserving its solubility through metal chelation for uptake via the divalent metal transporter DMT-1 and/or through transport of the chelate via the ascorbate-transporter.

• Addition of organic acids: Studies have shown that non-chelated lactic, citric, malic and tartaric acids effected increases in iron absorption and non- chelated oxalic acid significantly reduced uptake in a rat model of iron availability. • Addition of amino acids: The effects of amino acids have been studied in humans and in rat models. In both humans and rodents, cysteine enhanced iron absorption. Further, in vitro studies in CaCo-2 monolayers have shown that both cysteine and (reduced) cysteinyl glycine enhanced iron uptake. A significant benefit of cysteine and related thiols over ascorbate is that the former increased iron solubility at the pH of the intestinal lumen, whereas ascorbate must be combined with iron at pH 2 to reduce and solubilize the metal.

• Encapsulation in lipophilic materials: Application of a surface coating serves the dual purposes of masking adverse sensory changes that are associated with the un-encapsulated form and modifying uptake of the encapsulated material. Encapsulation may also prevent degradative interactions between the encapsulated material and its environment during long-term storage. Typical coating materials include hydrogenated oils, maltodextrins, modified cellulosics, and pH-responsive coatings (e.g., poly(meth)acrylates). This strategy for enhancement of iron availability has been employed both to provide iron in dried infant formula and dried infant cereals and in dietary supplements.

• Combinations of these approaches: To date, the most widely studied of the combination approaches is one in which a micronized iron source has been encapsulated (e.g., Taiyo Sun Active. RTM., an iron supplement available from Taiyo International Food Company).

Iron fortification of food products - especially of food products that are frequently consumed - provides an ideal vehicle for reducing the occurrence of iron deficiency in the general populations as well as in specific consumer groups. Oil-and-water emulsions such as spreads, margarines, mayonnaise and dressings are examples of food products that may be suitably be used as a vehicle for iron supplementation. However, fortification of oil-and-water emulsion with known iron compounds is associated with significant drawbacks. The use of water-soluble iron

compounds has an adverse impact on taste and product stability, especially if the fat component contains appreciable levels of oxidisable (poly)unsaturated fatty acids. Water-insoluble iron compounds have the disadvantage that they their bioavailability is relatively low and that they have a tendency to form a sediment.

US 2009/0061068 describes an iron-containing additive in the form of iron containing nanopartides having a particle size of 5 to 1 ,000 nanometer, wherein the nanopartides are stabilised by means of a biopolymer. The US application mentions application of the additive in beverages, (dry) soups, fat spreads, (yoghurt or protein) drinks, dressings or cereal products like bread. Example 3 describes the preparation of such an additive by separately preparing a solution of sodium pyrophosphate decahydrate and biopolymer in demineralized water and solution of ferrous sulfate heptahydrate in demineralized water. Next, the iron (II) solution was added to the pyrophosphate-biopolymer solution with vigorous stirring. The reaction self-terminated after formation of iron (II) pyrophosphate nanopartides. The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.

US 2009/0023686 describes a process for preparing a water-soluble solid ferric pyrophosphate citrate chelate composition comprising:

• combining a citrate ion source, a pyrophosphate ion source, and a ferric ion source in water to form a solution;

• adding an organic solvent in a volume sufficient to precipitate a solid ferric pyrophosphate citrate chelate composition from the resulting solution; and

• isolating the solid ferric pyrophosphate citrate chelate composition, said chelate composition having 2% or less phosphate by weight. These chelate compositions are expected to exhibit advantageous

biocompatibility as compared to conventional ferric pyrophosphates, ferric salts, ferric polysaccharide complexes and ferrous salts. US 2013/0330459 describes the preparation of an aqueous soluble ferric pyrophosphate concentrate comprising the steps of:

a) adding 0.1 to 5 weight/volume % ferric pyrophosphate to water,

b) adding 0.15 to 50 weight/volume % citrate salt to the dispersion from (a), c) heating the resulting solution from (b) until complete dissolution of ferric

pyrophosphate.

The concentrate so obtained can be used to supplement beverages with soluble and bio-available iron in the form of ferric pyrophosphate.

SUMMARY OF THE INVENTION

The inventors have discovered a way to achieve iron-fortification of edible oil- and-water emulsions that combines high bioavailability of the iron with excellent product taste and good product stability. More particularly, the inventors have found that this goal can be achieved by incorporating on the one hand cationic iron and on the other hand an excess amount of polyphosphate anions. Here an excess amount of polyphosphate anions means that the absolute value of the total negative charge contributed by the (fully deprotonated) polyphosphate anions is significantly higher than the total positive charge contributed by the cationic iron and the following divalent cations: Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ .

Accordingly, one aspect of the invention relates to an edible oil-and-water emulsion comprising:

• 10 - 85 wt% of fat;

• 5 - 90 wt% of water;

· 0.4 - 100 μιτιοΙ per ml of water of cationic iron selected from ferric iron (Fe ³⁺ ), ferrous iron (Fe ²⁺ ) and combinations thereof; • 0 - 25 μηηοΙ per ml of water of divalent cations (M ²⁺ ) selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ and combinations thereof;

• 0.5 - 1 ,000 μιτιοΙ per ml of water of polyphosphate anion selected from

pyrophosphate (P2O7 ^4" ), triphosphate (P3O10 ^5" ), tetraphosphate (P4O13 ^6" ) and combinations thereof ; and

• 0 -20 wt% of one or more other ingredients;

wherein: (4x[P ₂ O7 ⁴ -]+5x[P ₃ Oio ⁵ -]+6x[P4Oi3 ⁶ -]) / (2x[Fe ²⁺ ]+3x[Fe ³⁺ ]+2x[M ²⁺ ]) > 1 .2;

[X] representing the molar concentration of compound X in the edible emulsion, and [M ²⁺ ] representing the total molar concentration of the divalent cation(s) M ²⁺ .

Although the inventors do not wish to be bound by theory, it is believed that in the aqueous phase of the present oil-and-water emulsion the cationic iron is present as a polyphosphate complex that includes both strongly coordinated and loosely associated polyphosphate anions. The loosely associated polyphosphate anions are believed to aid dissolution and absorption of the iron. In order to produce iron-phosphate complexes containing loosely associated polyphosphates, polyphosphate anions should be present in the emulsion in an excess amount. Such an excess amount is not achieved by dispersing, for instance, iron pyrophosphate (Fe ^(lll) 4P6O2i) in water. In the latter case, the ratio 4x[P ₂ O ₇ ⁴ -]/3x[Fe ³⁺ ] equals 1 .0.

In case the emulsion contains Ca ²⁺ , Mg ²⁺ , Zn ²⁺ and/or Cu ²⁺ cations (divalent cations), more polyphosphate anion is required to form the desired iron- polyphosphate complex, probably because a fraction of the polyphosphate cations is competitively bound by these divalent cations.

Rao et al. ("Studies on the effect of inorganic polyphosphates on dietary ionisable iron and the solubility of other minerals in vitro"; Nutrition Reports International; 1984, Vol. 29, no.4, p. 941 -948) have reported that sodium tripolyphosphate, sodium trimetaphosphate and tetra sodium pyrophosphate when added to raw and cooked foods at 1 % could increase dietary ionisable iron significantly. The authors investigated the effect of polyphosphates on the ionisable content of wheat flour, bengalgram flour, baked chapathi and cooked rice.

The iron-fortified of the present invention offers the additional advantage that it is very easy to manufacture. Thus, the invention further provides a process of preparing the aforementioned edible emulsion, said process comprising addition of (i) an iron salt selected from ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferric orthophosphate, ferric pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and combinations thereof and (ii) a polyphosphate salt of an alkali metal, said polyphosphate being selected from pyrophosphate,

triphosphate, tetraphosphate and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to an edible oil-and-water emulsion comprising:

• 10 - 85 wt% of fat;

• 5 - 90 wt% of water;

• 0.4 - 100 μιτιοΙ per ml of water of cationic iron selected from ferric iron (Fe ³⁺ ), ferrous iron (Fe ²⁺ ) and combinations thereof;

· 0 - 25 μιτιοΙ per ml of water of divalent cations (M ²⁺ ) selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ and combinations thereof;

• 0.5 - 1 ,000 μιτιοΙ per ml of water of polyphosphate anion selected from

pyrophosphate (P2O7 ^4" ), triphosphate (P3O10 ^5" ), tetraphosphate (P4O13 ^6" ) and combinations thereof; and

· 0 -20 wt% of one or more other ingredients; wherein: (4χ[Ρ ₂ θ7 ⁴ -]+5χ[Ρ ₃ Οιο ⁵ -]+6χ[Ρ4θΐ3 ⁶ -]) / (2x[Fe ²⁺ ]+3x[Fe ³⁺ ]+2x[M ²⁺ ]) > 1 .2;

[X] representing the molar concentration of compound X in the edible emulsion and [M ²⁺ ] representing the total molar concentration of the divalent cation(s) M ²⁺ .

The term "fat" as used herein, unless indicated otherwise, refers to a lipid material that may be solid, semi-solid or liquid at room temperature (20°C). The term "oil-and-water emulsion" as used herein refers to a composition comprising a fat phase and an aqueous phase. The oil-and-water emulsion may be water-continuous and/or oil-continuous.

The term "polyphosphate salt" as used herein encompasses anhydrous polyphosphate salts as well as hydrated polyphosphate salts.

In accordance with one embodiment, the edible oil-and-water emulsion of the present invention is an oil-in-water emulsion. Examples of edible oil-in-water emulsions that are encompassed by the present invention include mayonnaise and dressings. The fat contained in the oil-in-water emulsion preferably is liquid at 20°C. Typically, the fat content of the oil-in-water emulsion is in the range of 5-85 wt.%, more preferably in the range of 10-83 wt.% and most preferably in the range of 30-81 wt.%. The aqueous phase of the oil-in-water emulsion preferably has a pH in the range of 2.0 to 6.0, more preferably in the range of 2.5 to 5.5 and most preferably in the range of 3.0 to 5.0.

In accordance with another embodiment, the edible oil-and-water emulsion is a water-in-oil emulsion. Examples of water-in-oil emulsion encompassed by the present invention include spreads and margarine, including liquid margarine. The fat contained in the water-in-oil emulsion preferably has a solid fat content of at least 10% at 20°C. The solid fat content of fat at 20°C can suitably be determined by pulse NMR spectroscopy (ISO method 8292-1 :2008). The fat content of the water-in-oil emulsion typically is in the range of 18-85 wt.%, more preferably in the range of 28-83 wt.% and most preferably in the range of 35-81 wt.%.

The aqueous phase of the water-in-oil emulsion preferably has a pH in the range of 2.5 to 8.0, more preferably in the range of 3.0 to 7.0 and most preferably in the range of 4.0 to 6.0.

The fat contained in the present emulsion preferably is selected from

triglycerides, diglycerides, monoglycerides, phospholipids and combinations thereof. Typically, said fat contains at least 50 wt.%, more preferably at least 80 wt.% and most preferably at least 90 wt.% triglycerides.

According to a preferred embodiment, the present emulsion contains 0.8 - 50 μιτιοΙ cationic iron per ml of water. Even more preferably, the emulsion contains 1 .4 - 30 μιτιοΙ per ml of water, most preferably 2 - 20 μιτιοΙ per ml of water.

Preferably, the ratio (4χ[Ρ ₂ θ7 ⁴ -]+5χ[Ρ3θιο ⁵ -]+6χ[Ρ4θΐ3 ⁶ -]) /

(2x[Fe ²⁺ ]+3x[Fe ³⁺ ]+2x[M ²⁺ ] is at least 1 .3. Even more preferably, this ratio is at least 1 .5, more preferably at least 2.0, even more preferably at least 3.0 and most preferably at least 4.0

In one embodiment of the present invention the emulsion contains at least 0.4 μιτιοΙ ferric iron per ml of water. Even more preferably the ferric iron content is at least 0.8 μιτιοΙ per ml of water, most preferably at least 2 μιτιοΙ per ml of water. In case the present emulsion contains such a significant amount of ferric iron the concentrations of ferric iron and polyphosphate anions preferably meet the following condition: (4χ[Ρ ₂ θ7 ⁴ -]+5χ[Ρ3θιο ⁵ -]+6χ[Ρ ₄ Οΐ3 ⁶ -]) / (3x[Fe ³⁺ ]+2x[M ²⁺ ]) > 1 .3.

Even more preferably, the latter ratio is at least 1 .5, more preferably at least 2.0, even more preferably at least 3.0 and most preferably at least 4.0. In accordance with another embodiment, the emulsion contains at least 0.4 μιτιοΙ ferrous iron per gram of water. Even more preferably the ferrous iron content is at least 0.8 μιτιοΙ per ml of water, most preferably at least 2 μιτιοΙ per ml of water. The emulsion that contains a significant amount of ferrous iron preferably contains ferrous iron and polyphosphate anions in concentration that meet the following condition:

(4x[P2O7 ⁴ -]+5x[P3Oio ⁵ -]+6x[P ₄ Oi3 ⁶ -])/(2x[Fe ²⁺ ]+2x[M ²⁺ ]) > 1 .3.

Even more preferably, the latter ratio is at least 1 .5, more preferably at least 2.0, even more preferably at least 3.0 and most preferably at least 4.0. The polyphosphate anion is preferably contained in the emulsion in a

concentration of 1 - 500 μιτιοΙ per ml of water, more preferably of 2 - 350 μιτιοΙ per ml of water and most preferably of 4 - 250 μιτιοΙ per ml of water.

The concentration of divalent cations (M ²⁺ ) in the present emulsion preferably does not exceed 20 μιτιοΙ per ml of water. More preferably, the cationic calcium content does not exceed 15 μιτιοΙ per ml of water, most preferably it does not exceed 10 μιτιοΙ per ml of water.

The cationic calcium content of the present emulsion preferably does not exceed 20 μιτιοΙ per ml of water. More preferably, the cationic calcium content does not exceed 10 μιτιοΙ per ml of water, most preferably it does not exceed 5 μιτιοΙ per ml of water.

The molar ratio of M ²⁺ to cationic iron in the emulsion typically does not exceed 1 :1 . More preferably said molar ratio does not exceed 1 :2, even more preferably ratio does not exceed 1 :3 and most preferably it does not exceed 1 :4. Besides the fat, water, cationic iron, optional cationic calcium and

polyphosphate anion, the present emulsion can contain up to 20 wt% of one or more other ingredients. Examples of other ingredients that can be present in the emulsion include biopolymers (e.g. proteins and/or polysaccharides), carbohydrates, acids, minerals, vitamins, colourings, flavourings, preservatives, anti-oxidants and emulsifiers (other than monoglycerides, diglycerides and phospholipids). Preferably the emulsion contains not more than 14 wt.%, more preferably not more than 10 wt.% of the one or more other ingredients.

The present emulsion may suitably be prepared by adding ferrous iron in the form of a water soluble salt and by adding a polyphosphate salt that is not an iron salt. Examples of ferrous salts that may be employed include ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrous tartrate, ferrous succinate and ferrous saccharate.

Accordingly, in a preferred embodiment, the emulsion contains one or more anions selected from sulphate, gluconate, lactate, bisglycinate, fumerate, tartrate, succinate, saccharate and combinations thereof, and at least 0.4 μιτιοΙ per ml of water of ferrous iron. According to a particularly preferred

embodiment, the concentrations of ferrous iron and the latter anions meet the following equation:

0.5 <

([sulphate]+0.5x[gluconate]+0.5x[lactate]+[bisglycinate]+[fu merate]+[tartrate]+ [succinate]+0.5x[saccharate])/[Fe ²⁺ ] < 1 .1 .

In a preferred embodiment, the emulsion contains a significant amount of pyrophosphate and/or triphosphate relative to the amount of cationic iron and cationic calcium. Accordingly it is preferred that the polyphosphate and cationic iron concentrations meet the following condition: (4χ[Ρ2θ7 ^4" ]+5χ[Ρ3θιο ^5" ]) / (2x[Fe ²⁺ ]+3x[Fe ³⁺ ]+2x[M ²⁺ ]) > 1 .2. More preferably, the latter concentration ratio exceeds 1 .5, even more preferably the ratio exceeds 2 and most preferably it exceeds 4.

According to a particularly preferred embodiment the emulsion contains a significant amount of pyrophosphate relative to the amount of cationic iron and cationic calcium. Thus, it is preferred that the pyrophosphate and cationic iron concentrations meet the following condition: 4χ[Ρ2θζ ^4" ] /

(2x[Fe ²⁺ ]+3x[Fe ³⁺ ]+2x[M ²⁺ ]) > 1 .2. The emulsion of the present invention preferably comprises not more than a very small amount of EDTA, e.g. less than 1 μιτιοΙ EDTA per ml of water. More preferably, the emulsion contains not more than 0.2 μιτιοΙ EDTA per ml of water, most preferably, the emulsion does not contain EDTA. Another aspect of the present invention relates to the use of the edible oil-and- water emulsion as defined herein in the treatment or prevention of iron deficiency.

A further aspect of the invention relates to a process of preparing the edible oil- and-water emulsion as defined herein before, said process comprising addition of

(i) an iron salt selected from ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferric orthophosphate, ferric pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and combinations thereof; and

(ii) a polyphosphate salt of an alkali metal, said polyphosphate salt being selected from pyrophosphate salt, triphosphate salt, tetraphosphate salt and combinations thereof. The polyphosphate salt employed in the present process is preferably selected from sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate and combinations thereof. According to a particularly preferred embodiment, the polyphosphate salt used is a pyrophosphate salt. Even more preferably, polyphosphate salt is selected from tetrasodium pyrophosphate (Na4P2O7), tetrapotassium pyrophosphate (K ₄ P2O7), disodium pyrophosphate (Na2H2P2O7), dipotassium pyrophosphate (K2H2P2O7) and combinations thereof.

The iron salt employed in the present process is preferably selected from ferric pyrophosphate, ferrous sulphate and combinations thereof. More preferably, the iron salt is ferric pyrophosphate. The ferric pyrophosphate is preferably added in the form of a powder having a weight averaged mean particle size of less than 25 μιτι, more preferably of less than 15 μιτι and most preferably in the range of 0.1 -10 μιτι.

Yet another aspect of the invention relates to the use of a water-soluble pyrophosphate to increase bioavailability of ferric or ferrous iron in oil-and-water emulsions. According to a particularly preferred embodiment, this use

comprises separate addition of the water-soluble pyrophosphate and of an iron salt selected from ferric salt and ferrous salt. The invention is further illustrated by the following non-limiting examples. EXAMPLES

Example 1

Bioaccessibility of ionic iron from two different kitchen margarines was assessed using an in vitro assay.

The term ' bioaccessible iron' refers to the fraction of an iron dose which is - after dissolution and digestion steps - in the ionic form and capable to pass a low molecular weight (MW) cut-off filter (also referred to as dialyzable iron).

A suspension containing 37.5 mmol ferric iron was prepared by dispersing micronized iron pyrophosphate (FePP) into milli-Q water. In addition, a pyrophosphate solution was prepared by dissolving sodium pyrophosphate (NaPP) in milli-Q water. The formulations of these stock solutions are shown in Table 1 .

Table 1

1 Fe4(P2O7)3*XH2O (Fe content 24.9 wt.%), ex Dr. Paul Lohmann

2 Na4P2O7, ex BK Guilini

In order to assess the influence of added pyrophosphate on bioaccessibility of iron from a kitchen margarine fortified with FePP, the mixtures shown in Table 2 were tested in an in vitro bioaccessibility assay. Table 2

keuken light, the Netherlands) Bioaccessibility was assessed using the following procedure:

All glassware was incubated overnight in 10 % (v/v) HNO3. On the day of the experiment all glassware was washed 5 times with milli-Q water to remove HNO3. Margarine samples were transferred into 100 ml dissolution vessels in the dissolution apparatus (type II, VanKel VK700) and milli-Q water, FePP suspension and NaPP solution were added (see Table 2). The pH in the vessel was adjusted to 2.0 with HCI. Subsequently, 10 ml pepsin solution (0.5 mg/mL in 0.1 M HCI) was added to each vessel, yielding a 90 ml dispersion of margarine in simulated gastric fluid at pH 2.0. After 60 minutes incubation at 37 °C with mixing at 100 rpm, samples were taken for total iron determination and for simulation of the intestinal phase in an Erlenmeyer flask.

For the simulation of the intestinal phase, a dialysis membrane (Spectra/Por 7 MWCO 8000) filled with a water solution of NaHCO3 was placed in the

Erlenmeyer flask. The amount of sodium bicarbonate present in the dialysis membrane was sufficient to adjust the simulated digestion to pH 7.5. After 30 minutes incubation in a water bath at 37 °C and continuous shaking (100 rpm) to raise the pH gradually, a mix of pancreatin (0.4 mg/mL) and bile acids solution (1 .25 mg/mL) was added to the flask. The flask was further incubated with the dialysis membrane for another 2 hours in the same water bath at 37 °C with continuous shaking (100 rpm). Hereafter, the dialysis membrane was removed and the content of the membrane (dialysate) was stored in aliquots for determination of ionic dialyzable iron and iron uptake experiments. Bioaccessibility of iron is calculated by means of the following

(Dialyzable Ionic iron / 0.4)

wherein:

• 0.4 represents the dilution factor that compensates for the dilution that

occurred when going from gastric phase to intestinal phase

• dialysable ionic iron is the ionic iron (mg/kg) present in the dialysate

• total Fe is the total iron concentration (mg/kg) after gastric simulation.

The results from the bioaccessibility test are shown in Table 3

Table 3

Example 2

Bioaccessibility of ionic iron from two different mayonnaises was assessed using an in vitro assay. In order to assess the influence of added pyrophosphate on bioaccessibility of iron from a mayonnaise fortified with FePP, the mixtures shown in Table 4 were tested in the in vitro bioaccessibility assay described in Example 1 . Table 4

¹ Commercially available mayonnaise with a fat content of 25% (Hellmann's light, UK)

2 Same as in Example 1

The results from the bioaccessibility test are shown in Table 5

Table 5

In addition, samples 1 and 2 were subjected to an In-vitro iron uptake test, using human colonic adenoma carcinoma (Caco-2) cells, to determine bioavailable iron.

'In vitro bioavailable iron' refers the fraction of an iron dose which is - after dissolution and digestion steps - in the ionic form and capable to enter cells to trigger a response (ferritin formation).

The following procedure was followed to determine In vitro bioavailable iron'.

Caco-2 cells were seeded in 12-wells plates (Brand) at a density of 2 ^* 10 ⁵ cells per well. The cells were grown in Dulbecco's modified Eagle's medium with 4.5 g/L glucose and L-glutamine (Bio-Whittaker), supplemented with 20% (v/v) heat-inactivated fetal bovine serum (Gibco), 1 % (v/v) penicillin/streptomycin (Bio-Whittaker) and 1 % (v/v) non-essential amino acids (Gibco). The cells were maintained at 37°C in an incubator with a 5% CO2 / 95% air atmosphere at constant humidity; the medium was changed every 2-3 days. The cells were cultured for 21 days, so that they can form a monolayer of differentiated cells that resembles that of the intestinal mucosa. At this point, cells were used for iron uptake experiments.

On the day of experiment, the dialysate samples were thawed and diluted (3 ml dialysate with 0.6 ml milli-Q water and 0.4 ml 10 times concentrated Minimum Essential Medium+ (MEM+)). MEM+ concentrated media was prepared by mixing PIPES (100 mmol/L), 5 % penicillin/streptomycin solution (Bio- Whittaker), hydrocortisone (20 Mg/L), insulin (50 mg/L), selenium (50 pg/L), triiodothonine (340 pg/L), epidermal growth factor (50 mg/L), 10 % non essential amino acids (Bio-Whittaker), NaHCO3 (22 mg/L), NaOH (for pH correction to pH 7.0), powder MEM (Gibco) and milli-Q water to a final volume of 100 mL. The concentrated MEM+ as well as the diluted samples was sterile filtered (0.22 μιτι). As positive control, a sample was prepared with 5 μΜ FeSO4 in MEM+. The negative control was plain 1 x MEM+. After washing the cells twice with 1 mL of 1 x MEM+, 1 mL of the diluted samples and control was applied.

Exactly 48 hours after the start of the incubation of the dialysates and the control in the incubator, the cell monolayers were harvested for ferritin and protein measurements. For this, the medium covering the cells was removed carefully and the cells were washed twice with 1 mL "rinse" solution (containing NaCI (140 mM), KCI (5 mM) and PIPES (10 mM) adjusted to pH 7.0 with NaOH (4 M); osmolarity 0.301 Osmol/kg). The "rinse" solution was then aspirated and 250 μί of ice-cold milli-Q water were added. The plate was wrapped in parafilm and sonicated on ice in a water bath at 4°C for 15 minutes. After sonication the cells were scraped and collected in vials. The samples were stored at -20°C until further use. Ferritin was measured using a commercial ELISA kit (H-ferritin (human) ELISA kit, 96 assays, Abnova, Taipei city, Taiwan, Catalogue number: KA021 1 ) according to the manufacturer's description (version 4). Wavelengths 620, 450 and 405 nm were read according to the Radim protocol (KP33IW - ferritina iema well - M108 - Rev08 - 10/2007).

Total protein was measured with the Bradford assay (Bradford reagent, Sigma- Aldrich) using immunoglobulin G (0 - 0.7 mg/ml, Bio-Rad Protein standard 1 (IgG)) as standard. Cell lysates were 10 times diluted prior analysis and 250 μΙ Bradford reagent was added to 20 μΙ diluted sample/standard. Assay was performed at 595 nm.

Caco-2 cell iron uptake results were expressed as ng of ferritin per mg of total protein. The results of this iron uptake test are summarised in Table 6

Table 6

Example 3

Analyses were conducted to determine the stoichiometry of

(Fe ³⁺ )pyrophosphates complexes in aqueous environment.

Materials and methods:

1 D ³¹ P ( ¹ H-decoupled) NMR spectra were recorded with a ZGIG pulse sequence on a Bruker Avance DRX 600 NMR spectrometer, equipped with a 10-mm broadband probe. The probe was tuned to detect ³¹ P resonances at 242.94 MHz ( ¹ H 600.13 MHz). The internal probe temperature was set to 303 K. 128 scans were collected in 32K data points with a relaxation delay of 15 seconds and an acquisition time of 0.85 seconds. The data were processed in TOPSPIN software version 3.1 pi 5 (Bruker BioSpin GmbH, Rheinstetten, Germany). An exponential window function was applied to the free induction decay (FID) with a line-broadening factor of 1 Hz prior to the Fourier

transformation. Manual phase correction and baseline correction were applied to all spectra. Sample preparation:

Two titration experiments with NaPP were performed at pH 2.5: one with a constant amount of FePP (66 mg Fe/L) and one without Fe (blank). All solutions were subsequently taken to neutral pH where some precipitation occurred, in particular at low NaPP equivalents. 800 μΙ_ of the prepared solution was added to 3200 μΙ_ D2O. This mixture was then stirred for 10 minutes. 3 ml_ of this mixture was transferred to a 10-mm NMR tube.

Measurements & Results:

For both titrations, twelve different NaPP concentrations were measured, making for a total of 24 samples. All 24 experiments showed a singlet in the ³¹ P- NMR spectrum, with only minor pH-induced chemical shifts. This singlet was assigned to pyrophosphate (PP). Throughout the spectra, these signals were integrated and normalized. The blank titration experiment showed slightly higher PP-amounts than the FePP-experiment: this is expected because an FePP- complex is formed, which broadens the PP-signal beyond detection due to its paramagnetic effect.

The difference between the blank and FePP experiments should be an indication as to the amount of PP that is tightly bound in complex with Fe. The measured differences are summarized in Table 7. It can be seen that the difference approaches 3 equivalents. To check whether the observed differences (between blank and FePP titrations) can indeed be attributed to spectral perturbation by paramagnetic iron, ICP-MS analysis was performed to check the amounts of Fe in the system. Also these results are depicted in Table

Table 7

It can be seen that Fe is indeed present in the system, reaching its weighed in value (61 mg Fe/kg) at 6-8 equivalents of NaPP. At low NaPP equivalents not all added iron is recovered, but at higher values all added iron is dissolved. This confirms our interpretation of the differences between titration of the blank and FePP solution.

Conclusions

The ³¹ P-NMR analysis of the two titration experiments shows that approximately 3 pyrophosphate molecules are associated to Fe ³⁺ on a timescale long enough to extinguish the PP-signal in the ³¹ P-NMR spectrum. Even though maximum solubility of Fe ³⁺ is achieved around 6-8 NaPP equivalents, this effect cannot be solely explained due to complexation of Fe ³⁺ with PP-ligands. Ionic strength possibly adds to increased solubility.