OA17461A - Fortified savoury food concentrate - Google Patents

Abstract

The present invention relates to a savoury food concentrate comprising iron salt and a process to produce the same. It is therefore an aim of the present invention to provide a glutamate containing savoury food concentrate which comprises an iron salt, wherein the amount of off-color which appears upon storage of the food concentrate is reduced, preferably wherein off-coloring is absent. It is therefore a further object of the present invention to improve the bioavailability of iron in food concentrates. It was found that a composition comprising an iron salt and a further non-iron phosphate salt, provides reduced discolouration in food concentrate compositions comprising glutamate, and also provides improved bioavailability of the iron.

Description

Fortified savoury food concentrate

The présent invention relates to a savoury food concentrate comprising iron sait and a process to produce the same.

Background ofthe invention

Savoury food concentrâtes, like seasoning cubes are a well known concentrate food product which is normally dissolved before consumption. It is normally dissolved in water or a dish, in this way giving rise to a bouillon, a soup, a sauce, a gravy or a seasoned dish. A savoury food concentrate cube normally comprises sait and often glutamate, which, among other functions, contribute to the taste impact on the food product which results upon dilation of the bouillon cube. A bouillon cube has been recognised in the field as a relatively cheap and convenient way to provide iron to the population. This is especially relevant for a population which suffers from a lack of iron in its diet, as can be observed in several developing countries. Iron-fortified bouillon cubes hâve been described în the art.

It is especially desired by présent day consumera that the iron is not only présent in a composition, but is also taken up by the body; this is generally referred to in the art as bioavailability.

W02009/068378 discloses a bouillon cube comprising, based on the weight of the bouillon cube: 30-70 percent wt. NaCI, 10-45 percent wt. monosodium glutamate, and at least one iron compound of the group of ferrie sodium EDTA, reduced iron, ferrous lactate, ferrie citrate, ferrie pyrophosphate, ferrous sulphate monohydrate, ferrie ammonium citrate brown, in such an amount that the bouillon cube comprises an amount of Fe^z+ and/or Fe³⁺ taken together of from at least 0.01 percent wt. and less than 2 percent wt, based on the weight of the bouillon cube. This document aims at réduction of off-color in the food product resulting after dilution of the fortified cube.

W02010/086192 discloses a dry savoury food concentrate comprising NaCI, an iron ion selected from the group consisting of Fe2*and Fe³⁺ and mixtures thereof, which iron ion is derived from an added iron compound which is dissolvable in an aqueous solution. This document aims at réduction of staining of the cooking pot during cooking when a fortified food concentrate is used.

The problem of off-color related to iron fortification not only appears on the level of the cooking pot and the ready-to-eat food product which results upon dilution of the food concentrate, for example a seasoning cube, but can also be présent in the food concentrate itself. Such an appearance of off-colorîng may be observed after a certain time of storage, which can dépend on relative air humidity and température during transport and storage in the shop and by the consumer at home. It may be perceived that especially in tropical or subtropical areas the risk for off-coloring effects is higher than in moderate climates. It is however also the sub-tropical and tropical area where fortifîed products can show their highest benefit to the population. Off-coloring can be observed for example in the form of dark spots or stains on the surface of the food concentrate, e.g. on the surface of a seasoning cube.

The respective mechanisms which underlie these iron-related off-coloring effects are not clear. It was observed that the appearance of off-color on the food concentrate, was significant when glutamate was présent. Without willing to be bound to theory, the presence of glutamate appears to hâve a négative influence on the appearance of a iron-fortified food concentrate.

It is therefore an aim of the présent invention to provide a glutamate containing savoury food concentrate which comprises an iron sait, wherein the amount of off-color which appears upon storage of the food concentrate is reduced, preferably wherein off-coloring is absent.

It is therefore a further object of the présent invention to improve the bioavailability of Iran in food concentrâtes.

It was surprisingly found that a composition comprising an Iron sait and a further non-lron phosphate sait, provides reduced discolouration in food concentrate compositions comprising glutamate, and also provides improved bioavailability ofthe Iron.

Summary of the invention

Accordingly, the présent invention provides a savoury food concentrate comprising:

• NaCI, • glutamate, • iron sait, further comprising • phosphate sait, not being an iron phosphate.

In a second aspect, the invention provides a process to provide a savouryfood concentrate according to the invention, the process comprising the steps of:

a) preparing a mixture comprising • NaCI, • iron sait, • glutamate, and further comprising • phosphate sait, not being an iron phosphate,

b) packaging.

In a third aspect the invention provides the use of a concentrate according to the invention for preparing a bouillon, a soup, a sauce, a gravy or a seasoned dish.

In a fourth aspect the invention provides the use of a phosphate sait to prevent discolouration of food concentrâtes comprising an iron sait in the presense of glutamate.

In a fifth aspect the invention provides the use of a phosphate sait to enhance the bioavailability of iron in a food composition.

Detailed description ofthe invention

The présent invention provides a savouryfood concentrate comprising NaCI, glutamate, iron sait, and a phosphate sait, not being an iron phosphate.

NaCI

The présent invention relates to a savoury food concentrate. The savoury character of the food concentrate is at least partly created by the presence of NaCI. The concentrate of the invention therefore comprises NaCI, preferably in an amount of from 10 to 70 wt%, preferably of from 30 wt% to 70 wt%, more preferably of from 40 wt% to 65 wt%, even more preferably of from 45 wt% to 60 wt%, based on the weight of the food concentrate. It can be preferred that in addition to NaCI, optionally a potassium sait is présent, like for example KCI, for example to replace part of the NaCI by potassium sait. Potassium sait, preferably KCI, can preferably be présent in a ratio potassium sait, preferably KCI, to NaCI of from 1:10 to 1:1, preferably of from 1:10 to 1:2.

Glutamate

The savoury food concentrate of the invention comprises glutamate. Although glutamate contributes to the savoury character of the food concentrate, without wîllîng to be bound to theory, it appeared that the presence of glutamate is associated with off-coloring in the iron5 fortified food concentrate. The type and intensity of off-coloring can dépend on the type and amount of iron sait présent in the food concentrate and the time and circumstances of storage. In general, the off-coloring results in a darker appearance of the food concentrate, that can go from brownish, to dark brown, even towards black color of the food concentrate. Especially iron sulphate can provide off color in the form of dark speckles, developing towards 10 general dark coioring after prolonged storage time. In general, the amount of off color tends to increase with increased storage time.

Preferably, the invention relates to a food concentrate wherein glutamate comprises one of the group consisting of mono sodium glutamate, potassium glutamate, glutamic acid and mixtures thereof. More preferably, glutamate comprises mono sodium glutamate. Glutamate 15 is preferably présent in an amount of from 0.5 to 45 wt%, more preferably in an amount of from 1 to 35 wt%, even more preferably in an amount of from 5 to 30 wt%, most preferably in an amount of from 7 to 20 wt%, based on the weight of the food concentrate. Preferably the savoury food concentrate comprises monosodium glutamate, which is preferably présent in an amount of from 0 to 45 wt%, more preferably in an amount of from 1 to 35 wt%, even more 20 preferably in an amount of from 5 to 30 wt%, most preferably în an amount of from 7 to 20 wt%, based on the weight of the food concentrate.

Iron sait

The savoury food concentrate ofthe invention comprises iron sait. The iron sait preferably 25 comprises, more preferably is, an iron sait selected from the group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and mixtures thereof.

Even more preferably, the iron sait comprises, even more preferably is an iron sait selected 30 from the group consisting of iron phosphate, ferrous sulphate and mixtures thereof. Iron phosphate is a term known to the skilled artisan and comprises the group of salts comprising one or more iron atoms and one or more phosphate groups. It comprises for example ferrie orthophosphate, ferrie pyrophosphate, or ferrous orthophosphate.

Even more preferably, the iron sait comprises, even more preferably is, ferrie pyrophosphate 35 or ferrous sulphate or a mixture thereof.

Even more preferably the iron sait comprises ferrie pyrophosphate, even more preferably is ferrie pyrophosphate.

Iran sait îs preferably présent in an amount of from 0.03 to 2 wt%, more preferably of from

0.07 to 1 wt%, based on the weight of the food concentrate.

Preferably, the food concentrate comprises an iron sait selected from the group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succînate, ferrous saccharate, ferrous orthophosphate and mixtures thereof in an amount of from 0.03 to 2 wt%, 10 more preferably of from 0.07 to 1 wt%, based on the weight of the food concentrate. It can be preferred that an iron sait is selected from this list, to be présent in the food concentrate, which iron sait individually can be présent in an amount of from 0.03 to 2 wt%, more preferably of from 0.07 to 1 wt%, based on the weight of the food concentrate.

More preferably, the food concentrate comprises an iron sait selected from the group consisting of ferrie pyrophosphate, ferrous sulphate and a mixture thereof in an amount of from 0.03 to 2 wt%, more preferably of from 0.07 to 1 wt%, based on the weight of the food concentrate.

Even more preferably, the food concentrate comprises ferrie pyrophosphate in an amount of from 0.03 to 2 wt%, more preferably of from 0.07 to 1 wt%, based on the weight of the food 20 concentrate.

Phosphate sait

The savouryfood concentrate ofthe invention comprises a phosphate sait. The phosphate sait is not an iron-phosphate sait, as iron-phosphate sait is separately categorised in this 25 description under ‘iron sait'. Indeed, in case the iron sait is an iron phosphate, the food concentrate comprises in addition to said iron phosphate another phosphate sait that is not an iron phosphate.

The phosphate sait preferably comprises a phosphate sait, more preferably is a phosphate 30 sait, selected form the group consisting of orthophosphate sait, diphosphate sait, triphosphate sait, polyphosphate sait and mixtures thereof. The phosphate sait preferably comprises a sait selected from the group consisting of Na-phosphate sait, K-phosphate sait, Ca-phosphate sait and Mg-phosphate sait. More preferably, the phosphate sait comprises Na-phosphate sait. Preferably Na-phosphate salts comprise Na3PO4, Na2HPO4, NaH2PO4, sodium pyrophosphate, sodium triphosphate, sodium polyphosphate and mixtures thereof.

Preferably, K-phosphate salts comprise K3PO4, K2HPO4, KH2PO4, potassium pyrophosphate, potassium triphosphate, potassium polyphosphate and mixtures thereof. The phosphate sait preferably comprises a sait selected from the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, calcium phosphate, magnésium phosphate, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate, sodium polyphosphate, potassium polyphosphate and mixtures thereof. More preferably, the phosphate sait comprises a sait selected from the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate and mixtures thereof. Most preferably, the phosphate sait comprises Na-pyrophosphate, The phosphate sait is preferably Na-pyrophosphate.

Phosphate sait, not being an iron phosphate, preferably Na-phosphate sait, is preferably présent in an amount of from 0.03 to 20 wt%, more preferably from 0.07 to 10 wt%, even more preferably of from 0.1 to 5 wt%, even more preferably of from 0.2 to 2 wt%, based on the weight of the food concentrate.

It is especially preferred if the iron sait comprises ferrie pyrophosphate and the phosphate sait comprises sodium pyrophosphate.

For stability, the molar ratio of iron sait, preferably iron pyrophosphate, to phosphate sait (not being an iron-phosphate), preferably Na-pyro phosphate, is preferably of from 0.1 to 10 (i.e from 1:10 to 10:1), preferably of from 0.25 to 5 (i.e. from 1:4 to 5:1), most preferably of from 0.5 to 2 (i.e. from 1:2 to 2:1).

For improved bioavailability, the molar ratio of phosphate sait to ionic iron, the ratio is preferably of from 1:1 to 20:1, more preferably 1:1 to 10:1, still more preferably 1:1 to 6:1, wherein it is noted that the effect is difficult to measure at a ratio of less than 1:1 and that above 10:1 the effect is constant and does not further împrove; however, the addition of more phosphate is also not detrimental to the bioavailability.

Binder

The food concentrate of the invention can comprise a binder, for example to maintain the desired texture and/or shape of the food concentrate. A binder is especially preferred when the food concentrate is in the form of a cube or tablet. Preferably, the binder comprises one selected from the group consisting of fat, polysaccharide, sugar, and mixtures thereof. Sugar preferably comprises monosaccharîde. Polysaccharide preferably comprises starch, gums or mixtures thereof.

Eat

The food concentrate of the invention preferably comprises fat. This is preferred especially when the food concentrate is a cube or tablet, such as a bouillon cube or tablet, a soup cube or tablet or a seasoning cube or tablet. Fat can function as a binder. Fat preferably comprises a fat selected from the group consisting of chicken fat, beef fat, vegetable fat, pork fat and mixtures thereof. Fat is preferably présent in an amount of from 1 to 35 wt%, preferably of from 5 to 30 wt%, more preferably of from 10 to 20 wt%. Especially if the food concentrate is an extruded food concentrate, like for example an extruded cube or tablet, also known in the field as ‘pasty’ cube, the fat is preferably présent in an amount of from 10 to 35 wt%, more preferably of from 15 to 30 wt%, most preferably of from 17 to 25 wt%. Especially if the food concentrate is a pressed cube or tablet or a roller-formed cube or tablet, the fat is preferably présent in an amount of from 1 to 20 wt%, preferably of from 5 to 15 wt%, most preferably of from 7 to 12 wt%. However, it might be preferred that the food concentrate has a low fat level, for example of lower than 15 wt%, for example of between 0.1 and 15 wt%., more preferably of lower than 10 wt%, for example of between 0.1 and 10 wt%„ even more preferably lower than 5 wt, for example of between 0.1 and 5 wt%, or lower than 2 wt% for example of between 0.1 and 2 wt%, or even lower than 1 wt%., for example of between 0.1 and 1 wt%. These low fat Ievels might be preferred for example in case of low-fat cubes or tablets or in case of water-based granules or powders, for example bouillon-, soup-, or seasoning granules or powder.

Starch

The food concentrate may preferably comprise starch. Starch is preferably présent in an amount of from 0.1 to 15 wt%, 2 to 15 wt%, more preferably of from 5 to 12 wt%, most preferably of from 7 to 10 wt%. Starch can function as a filler. It may further contribute to the mouth feel of the food product resulting upon dilution of the food concentrate. Starch is preferably selected from the group consisting of corn starch, tapioca starch, pea starch, potato starch and mixtures thereof. The starch is preferably non-gelatinised starch. It might be preferred however, that starch comprises gelatinised starch, especially when the starch is used as a binder, a preferred amount of non-gelatinised starch, based on the weight ofthe food concentrate, can be of from 0.1 to 10 wt%.

Water

The food concentrate of the présent invention is preferably a dry food concentrate. Although some water can be présent in the food concentrate, it comprises preferably less than 10 wt% of total water, more preferably less than 5 wt%, for example from 1 to 10 wt% or from 2 to 5 wt% of total water, based on the weight of the food concentrate. Total water includes water which may be présent in the other ingrédients of the food concentrate.

The water activity of the food concentrate is preferably of between 0.1 and 0.6, more preferably of between 0.15 and 0.4, most preferably of between 0.2 and 0.3.

As is commonly understood in the art, salts as described in the présent document may, at least partly, dissolve in the food concentrate, especially when water is présent. “Sait, like NaCI, glutamate, iron sait and phosphate sait includes the dissolved or partly dissolved or dissociated form of these respective salts. For example, the part of the total amount of added NaCI, which turns into the dissolved form in the food concentrate, contributes to the total amount of NaCI in the food concentrate.

Taste imparti no components

It can be preferred that the food concentrate comprises plant particles, for example particles from herbs or from vegetable. The plant particles are preferably présent in an amount of from 0.1 wt% to 20 wt%, more preferably of from 0.5 wt% to 10 wt%, even more preferably of from to 5 wt%, or they can be preferred to be présent in an amount of from 0.5 to 2 wt% (dry weight of the plant particles based on weight of the food concentrate).

The food concentrate of the présent invention preferably comprises flavours. Flavours can be présent for example in an amount of for example 0.1 wt% to 10 wt%, more preferably of from 0.5 wt% to 8 wt%, (dry weight of the flavours based on weight of the food concentrate). Flavours are preferably selected from the group consisting of vegetable flavour, méat flavour and mixtures thereof. Méat flavour preferably comprises flavours selected from the group consisting of chicken flavour, fish flavour, beef flavour, pork flavour, lamb flavour, and mixtures thereof. Vegetable flavour preferably comprises spices.

The food concentrate might contain extract of beef. The food concentrate may contain extract of yeast, altematively to, or in addition to extract of beef. Extract of beef and extract of yeast can individually be présent in an amount of from 1 to 10 wt%, preferably in an amount of from to 7 wt%, based on the weight of the food concentrate. It can be preferred that the total amount of beef extract and yeast extract taken together is présent in an amount of from 1 to wt%, preferably in an amount of from 2 to 7 wt%, based on the weight of the food concentrate.

The savoury food concentrate is preferably in the form of a cube, a tablet, a granule, or a powder. It is more preferably a cube or a tablet, most preferably a pressed cube or tablet. A cube or tablet is preferably between 2 and 30 grams, preferably of between 3 and 20 grams, more preferably of between 4 and 12 grams. A cube and tablet are terms used in this description inter-exchangeable, as common in the field, and do not refer necessarily to geometrically defîned structures. It can be preferred however that the cube is a regular figure wherein height, width and height hâve the same size. It can be preferred that the tablet is an oblong wherein the longest dimension (length) and the second-longest dimension (width) 1:1 to 1:3, preferably of between 1:1 to 1:2. The longest dimension (length) and the shortest dimension (height) preferably relate to each other as 1:2 to 1:6.

The savoury food concentrate is preferably a concentrate selected from the group consisting of a bouillon concentrate, a soup concentrate, a gravy concentrate, a sauce concentrate and a seasoning concentrate.

Preferably, the présent invention relates to a savoury food concentrate in the form of a cube or a tablet comprising glutamate, NaCI, iron sait and a phosphate sait, wherein • NaCI is présent in an amount of from 10 to 70 wt%, • Glutamate is présent in an amount of from 0.5 to 45%, • Iron sait is présent in an amount of from 0.03 to 2 wt%, and wherein the iron- sait is selected from the group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and mixtures thereof, preferably wherein the iron- sait is selected from the group consisting of iron phosphate, ferrous sulphate and mixtures thereof, most preferably wherein the iron sait is selected from the group consisting of ferrous sulphate, ferrie pyrophosphate and mixtures thereof.

• phosphate sait, not being an iron phosphate, is présent in an amount of from 0.03 to 20 wt%, and wherein the phosphate sait is selected from the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, calcium phosphate, magnésium phosphate, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate, sodium polyphosphate, potassium polyphosphate and mixtures thereof.

The savoury food concentrate is preferably packaged. It can be packaged for example in a wrapper, a sachet, a box or a tub.

Process

In a further aspect, the présent invention relates to a process to provide a food concentrate according to the invention, the process comprising the steps of:

a) preparing a mixture comprising:

• NaCI, • glutamate • iron sait, preferably selected from the group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and mixtures thereof,, • phosphate sait, not being an iron phosphate, wherein the phosphate sait îs preferably selected from the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, calcium phosphate, magnésium phosphate, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate, sodium polyphosphate, potassium polyphosphate and mixtures thereof.

b) packaging, to resuit in a food concentrate.

In step a) a mixture is provided comprising NaCI, iron phosphate and phosphate sait. The mixture can further comprise fat, water, starch, flavour,

NaCI is preferably added to the mixture of step a) in an amount of from 30 wt% to 70 wt%, more preferably of from 40 wt% to 65 wt%, even more preferably of from 45 wt% to 60 wt%, based on the weight of the resulting food concentrate.

Glutamate is preferably added in an amount of from 0.5 to 45 wt%, more preferably in an amount of from 1 to 35 wt%, even more preferably in an amount of from 5 to 30 wt%, most preferably in an amount of from 7 to 20 wt%, based on the weight of the resulting food concentrate. Preferably glutamate is added in the form of monosodium glutamate. Mono sodium glutamate is preferably added in an amount of from 0.5 to 45 wt%, more preferably in an amount of from 1 to 35 wt%, even more preferably in an amount of from 5 to 30 wt%, most preferably in an amount of from 7 to 20 wt%.

Iron sait is preferably added to the mixture of step a) in an amount of from 0.03 to 2 wt%, more preferably of from 0.07 to 1 wt%, based on the weight of the resulting food concentrate. Fe-pyro phosphate and/or ferrous sulphate is preferably added in an amount of from 0.03 to 2 wt%, more preferably of from 0.07 to 1 wt%, based on the weight of the resulting food concentrate.

For stability, Iron sait, preferably iron phosphate and/or ferrous sulphate, more preferably iron pyrophosphate and/or ferrous sulphate, and phosphate sait, not being an iron-phosphate, preferably Na-pyro phosphate, are preferably added to resuit in a molar ratio of iron phosphate, preferably iron pyrophosphate, to phosphate sait, not being an iron-phosphate, preferably Na-pyro phosphate, of from 0.1 to 10, preferably of from 0.25 to 5, most preferably of from 0.5 to 2, as présent in the resulting food concentrate.

For improved bioavailability, the molar ratio of phosphate sait to ionic iron, the ratio is preferably of from 1:1 to 20:1, more preferably 1:1 to 10:1, still more preferably 1:1 to6:1, wherein it is noted that the effect is difficult to measure at a ratio of less than 1:1 and that above 10:1 the effect is constant and does not further improve; however, the addition of more phosphate is also not detrimental to the bioavailability.

Phosphate sait, not being an iron phosphate, preferably Na-phosphate sait, is preferably added in an amount of from 0.03 to 20 wt%, more preferably from 0.07 to 10 wt%, even more preferably of from 0.1 to 5 wt%, even more preferably of from 0.2 to 2 wt%, based on the weight of the resulting food concentrate.

Water can be added to the mixture of step a). If added, it is preferably added in an amount of from 0.5 to 4 wt%, preferably in an amount of from 1 to 3 wt%, based on the weight of the total resulting food concentrate.

If fat is présent, it is preferably added to the mixture of step a) in the total amount of from 1 to 35 wt%, preferably of from 5 to 30 wt%, more preferably of from 10 to 20 wt%. Especially if the food concentrate is an extruded food concentrate, like for example an extruded cube, also known in the field as ‘pasty’ cube, the fat is preferably added in a total amount of from 10 to 35 wt%, more preferably of from 15 to 30 wt%, most preferably of from 17 to 25 wt%.

Especially if the food concentrate is a pressed cube or a roller formed cube, the fat is preferably added in a total amount of from 1 to 20 wt%, preferably of from 5 to 15 wt%, most preferably of from 7 to 12 wt%,

The process according to the invention may further comprise the step of shaping the mixture resulting from step a). Shaping preferably comprise compressing or extruding the mixture of step a) to resuit in a cube or tablet. These technologies are known in the art and can be carried out for example by a press from Fette™ or Bonals™, to obtain a hard cube, also known as pressed cube or by an extrader from Corazza™, to obtain an extruded cube, also known as soft cube or as pasty cube. The compressing can altematively be carried out by roller compaction. Shaping can comprise roller formation. These shaping techniques hâve been described in the art.

Shaping may comprise one of granulation, agglomération, or roller compaction, for example in case granules or pellets are desired. These technologies are known in the art. Granulation is preferably carried out in a basket granulator, as known in the art.

Accordingly, the process of the présent invention preferably comprises shaping, wherein shaping comprises a technique selected from the group consisting of compression, extrusion, roller compactîng, granulation, agglomération and mixtures thereof.

The process of the invention further comprises the step of packaging. The mixture resulting from step a) or the shaped mixture, in case the process comprises the step of shaping the mixture resulting from step a), is packaged, for example to allow transportation and/or dosing of the product. Preferably packaging comprises packaging in a packaging selected from a wrapper, a jar, a box, a tub, a sachet and mixtures thereof.

The présent invention further relates to the use of a concentrate according to the invention for preparing a bouillon, a soup, a sauce, a gravy or a seasoned dish.

The invention describes a savoury food concentrate comprising glutamate, NaCI, iron sait and further comprising a phosphate sait, wherein said phosphate sait is different from an iron phosphate. By the présent invention the appearance of off-color in glutamate containing savoury food concentrâtes which are fortified with an iron sait can be significantly reduced.

The invention will now be exemplified by the following, non limiting Examples:

Examples

Materials and Methods - Corn starch ‘C*GEL LM03411' was obtained from Cargill UK Ltd.

Yeast extract ‘Gistex Xtra Light’ was obtained from DSM Food Specialties (min.weight MSG

3.0 weight%). Ferrous sulphate, ferrie pyrophosphate and iron phosphate were obtained from

Dr. Paul Lohmann GmbH KG. Sodium pyrophosphate was obtained from BK Guilini.

Trisodium citrate was obtained from Merck. Sodium dicalcium EDTA was obtained from Akzo. Sodium polyphosphate was obtained from Riedel-de-Haen. Ail other phosphates were obtained from Sigma Aldrich.

Préparation of seasoning cubes - Ail the materials, with the exception of the palm stéarate and the iron sait are weighed together in a plastic jar and mixed with a mixer (Kenwood Chef Premier KMC650) for 1 minute at speed setting 4. The palm stéarate is molten by placing a container containing it in a hot water bath and is added to the mixture when liquid, after which the mixture is mixed for 1 minute at speed setting 6. The iron sait is added and again the mixture is mixed for 1 minute at speed setting 6. 4 Grams of this mixture at a time are transferred in the pressing block of an Instron press (Instron 5567) and the cube is pressed at 5 kN. This procedure is repeated for each cube.

Test procedure - Off-color formation is analysed in an accelerated off-color test. Two cubes are put on a plastic holder and placed in a 100 ml glass jar. 1 Gram of water is added in the jar in such a way that the cube does not corne into direct contact with the water. This procedure simulâtes typical storage conditions of commercial products, where the water content of seasoning cubes increases over time, but in an accelerated fashion. The jars are closed with an lid and placed in an oven at 40°C for the accelerated test. Results are given after three weeks. For example, after 3 weeks comparative samples containing ferrous sulphate are very dark, and comparative samples with iron pyrophosphate show significant discoloration. The off-color formation was représentative for discolouration observed after longer storage times in real-life.

Colour measurements - Off-color formation was analysed by a color measurement as known in the art. Ail colour measurement hâve been performed by using a DigiEye Imaging system from VeriVide Ltd. From photographs under controlled and calibrated conditions the L*a*b* values were determined. The colour différence ΔΕ was determined using the formula: ΔΕ = square root of ((LZ-Lo*)² + (a^-ao*)² + (b^-bo*)² ). Where L/, a^ and b/ are the colour values for the sample, and the Lq*. a₀* and b₀* are the values for the reference relative to which the colour value is expressed. In the examples mentioned hereafter ail colour différences are expresses as ΔΕ relative to the formulation without any iron added. A high ΔΕ value represents a relatively high amount of off-color. Ail values given represent average of duplo measurements.

Example 1

In this experiment, a seasoning cube without iron - composition 1 (table 1 ) - was compared to a seasoning cube with FeSO4, iron pyrophosphate and a combination of iron pyrophosphate and 1 équivalent of sodium pyrophophate (composition 2, 3 and 4 in table 1 ). In each case, the total iron content was kept constant and the formulation was adapted in the amount of sodium chloride.

Ingrédient	Compar. comp. 1 (no iron sait)	Compar. comp. 2 (Fe pyrophosphate)	Comp. 3 (Iron pyrophosphate + Na pyrophosphate)
NaCI	53	52.6	52.2
Sucrose	15.5	15.5	15.5
Corn starch	5.7	5.7	5.7
Palm stéarate	7	7	7
MSG	14	14	14
Yeast	3	3	3
Herbs and spices	1.8	1.8	1.8
Ferrie pyrophosphate		0.4	0.4
Sodium pyrophosphate			0.4
total	100	100	100

Table 1: compositions of Example 1; ail numbers in weight%. Comp.=composition. Compar. Comp.= comparative composition.

The seasoning cubes were prepared, tested in an accelerated test and color measurements were performed, as described in the Materials and Methods section above. The results are given in table 2 in L*a*b* and corresponding ΔΕ value relative to the reference sample without iron.

L* a* b*

Compar. comp.

62.6 11.8 58.1

Delta E

Ô

Compar. comp. 2	55.17	12.4	47.1	13.3
composition 3	61.6	11.6	53.8	4.4

Table 2: L*a*b* values of the seasoning cubes measured in the accelerated test after 21 days. Delta E values relative to composition 1.

Off color was observed as a darker, brownish appearance of the cube. The addition of sodium pyrophosphate sîgnifîcantiy reduces the off-colour formation of the cube after storage.

Example 2

Following the same procedures as described in Example 1, ferrie pyrophosphate in combination with various phosphates were tested. The compositions are given in table 3, the results in table 4.

Ingrédient	Compar.	Compar.	Comp. 3	Comp. 4	Comp. 5
Comp. 1	Comp. 2
NaCI	53.0	52.6	52.3	51.9	52.4
Sucrose	15.5	15.5	15.5	15.5	15,5
Corn starch	5.7	5.7	5.7	5.7	5.7
Palm stéarate	7.0	7.0	7.0	7.0	7.0
MSG	14.0	14.0	14.0	14.0	14.0
Yeast	3.0	3.0	3.0	3.0	3.0
Herbs and spices	1.8	1.8	1.8	1.8	1.8
Ferrie pyrophosphate		0.4	0.4	0.4	0.4
Sodium dihydrogen phosphate			0.3
sodium triphosphate				0.7
Sodium polyphosphate					0.2
total	100.0	100.0	100.0	100.0	100.0

Table 3: compositions of Example 2; ail numbers in weight%. Comp.com position. Compar. Comp.= comparative composition.

	L*	a*	b*	Delta E
Compar. Comp.1	62.6	11.8	58.1	0.0

Compar. Comp. 2	55.2	12.4	47.1	13.3
Comp. 3	59.4	10.6	49.0	9.7
Comp. 4	64.2	10.4	54.0	4.6
Comp. 5	62.4	10.6	55.0	3.3

Table 4: L*a*b* values of the seasoning cubes measured in the accelerated test after 21 days. Delta E values relative to composition 1.

Off color was observed as a darker, brownish appearance of the cube. As can be seen from the results, the addition of the phosphates significantly limited the colour formation in the samples: sodium dihydrogen phosphate decreases the colour formation in the cube with ferrie pyrophosphate significantly, while triphosphate and polyphosphate decrease it to an even larger extent.

Example 3

Following the same procedures as described in Example 1, various îron salts in combination with phosphates were tested. The compositions are given in table 5, the results in table 6,

Ingrédient	Compar. Comp. 1	Comp. 2	Comp. 3	Comp, 4	Comp.5
NaCI	53.0	52.4	52.5	52.5	52.6
Sucrose	15.5	15.5	15.5	15.5	15.5
Corn starch	5.7	5.7	5.7	5.7	5.7
Palm stéarate	7.0	7.0	7.0	7.0	7.0
MSG	14.0	14.0	14.0	14.0	14.0
Yeast	3.0	3.0	3.0	3.0	3.0
Herbs and spices	1.8	1.8	1.8	1.8	1.8
Ferrie phosphate		0.2	0.2	0.2	0.2
Trisodium phosphate			0.3
Disodium hydrogenphosphate				0.3
Sodium dihydrogenphosphate					0.2
Sodium pyrophosphate		0.4
total	100.0	100.0	100.0	100.0	100.0

Comp.=composition. Compar. Comp.= comparative composition.

Table 5: compositions of Example 3; ail numbers in weight%.

	L*	a*	b*	Delta E
Compar. Comp. 1	62.6	11.8	58.1	0.0
Comp, 2	61.3	11.1	54.1	4.3
Comp. 3	57.9	11.2	49.4	9.9
Comp. 4	57.7	11.1	48.6	10.7
Comp. 5	57.2	11.6	48.0	11.5

Table 6: L*a*b* values of the seasoning cubes measured in the accelerated test after 21 days. Delta E values relative to composition 1.

Off color was observed as a light brownish appearance of the cube. As can be seen from the results, ail samples showed less discoloration than the samples with ferrie pyrophosphate (see Example 1 ). The combination of ferrie phosphate with sodium pyrophosphate gave the least discoloration.

Example 4

Following the same procedures as described in Example 1, the effect of FeSO4 in combination with sodium pyrophosphate was tested. The compositions are given in table 7, the results in table 8.

Ingrédient	Compar. Comp. 1	Compar. Comp. 2	Comp. 3	Compar. Comp, 4	Comp. 5
NaCI	53.0	52.8	52.4	52.6	52.2
Sucrose	15.5	15.5	15.5	15.5	15.5
Corn starch	5.7	5.7	5.7	5.7	5.7
Palm stéarate	7.0	7.0	7.0	7.0	7.0
MSG	14.0	14.0	14.0	14.0	14.0
Yeast	3,0	3.0	3.0	3.0	3.0
Herbs and spices	1.8	1.8	1.8	1.8	1.8
Ferrous sulphate		0.2	0.2
Ferrous lactate hydrate				0.4	0.4
Sodium pyrophosphate			0.4		0.4
total	100.0	100.0	100.0	100.0	100.0

Table 7: compositions of Example 3; a I numbers in weight%. Comp.=composîtion. Compar. Comp.= comparative composition.

	L*	a*	b*	Delta E
composition 1	62.6	11.8	58.1	0.0
composition 2	36.7	5.2	8,0	56.8
composition 3	58.1	10.9	48.6	10.6
composition 4	52.5	11.2	43.8	17.5
composition 5	55.5	12.3	47.4	12.8

Table 8: L*a*b* values of the seasoning cubes measured in the accelerated test after 21 days. Delta E values relative to composition 1.

As can be seen from the results, ferrous sulphate on its own (comparative composition 2) gave severe discolouration. Off color was observed as dark speckles and darker color of the cube. Also ferrous lactate on its own resulted in off color formation, (comparative composition 4). Off color was observed in this case as a darker, brownish appearance of the cube. The presence of sodium pyrophosphate (composition 3 and 5) resulted in a significant 10 réduction of off color formation.

Comparative example 5

As a comparative exampie, the use of sodium citrate and sodium calcium EDTA was tested in combination with ferrie pyrophosphate and compared to ferrie pyrophosphate in combination with sodium pyrophosphate. The procedure as described in Example 1 was followed. The compositions are given in table 9 and the results in table 10.

Ingrédient	Compar. comp. 1	Compar. comp. 2	Compar. comp. 3	Compar. comp. 4
NaCI	53.0	52.6	52.1	50.7
Sucrose	15.5	15.5	15.5	15.5
Corn starch	5.7	5.7	5.7	5.7
Palm stéarate	7.0	7.0	7.0	7.0
MSG	14.0	14.0	14.0	14.0
Yeast	3.0	3.0	3.0	3.0
Herbs and spices	1.8	1.8	1.8	1.8
Ferrie pyrophosphate		0.4	0.4	0.4
Ingrédient	Compar. comp. 1	Compar. comp. 2	Compar. comp. 3	Compar. comp. 4

Disodium calcium EDTA			0.5
Trisodium citrate				1.9
total	100.0	100.0	100.0	100.0
Table 9: composil	ions of Example 3; ail numbers in weight%. Comp.=composition. Compar.

Comp.= comparative composition.

	L*	a*	b*	Delta E	Off color
Compar. comp. 1	62.6	11.8	58.1	0.0
Compar. comp. 2	55.17	12.4	47.1	13.3	brown ish
Compar. comp. 3	47.6	13.1	20.9	40.1	dark brown
Compar. comp. 4	50.0	11.5	40.2	21.9	dark brown

Table 10; L*a*b* values of the seasoning cubes measured in the accelerated test after 21 5 days. Delta E values relative to composition 1.

As can be seen from the results, the use of the two most commonly used séquestrants did not give similar improvement of the colour stability of the seasoning cube: in fact, in both cases much more discolouration was observed as compared with the sample with ferrie pyrophosphate only, demonstrating the unique properties of phosphate salts, as exemplified in the previous examples 1 to 4.

Example 6: Bioavailability

An example composition of bouillon cubes containing FeSO4 made at Unîlever R&D

Vlaardingen was made according to table 11 below. is presented in Table 1. The mix is produced in a quantity large enough to ensure homogeneity and from the mix a spécifie weight (nominal 4000 mg) is pressed in a dye to a cube approximately 1 x 1 x 1 cm (5 kN Instron press force).

Table 11: Sample composition of test cubes with FeSO4 (weight %)

Sample name:	HBR120013	HBR120014
ingrédient:	Whole mix	Whole mix + FeSO4
NaCI	51.2	51.2

sugar	15	15
corn starch	5.6	5.6
fat	7.0	7.0
MSG	14	14
l&G	0.2	0.2
Yeast	3	3
Curcuma	0.35	0.35
onion	0.8	0.8
garlic	1	1
bayleaf	0.1	0.1
Fe(ll)SO4		0.65

Table 12: Sample composition H BR sériés of test cubes with FePP (weight %)

Experiment sample compositions	HBR12059-02
sample name	Full formulation + FePP + NaPP
ingrédient:	target amount	weighed actual (g)
NaCI	129.42	129.43
Sugar	37.92	37.96
com starch	14.16	14.16
Fat	17.69	17.66
MSG	35.39	35.38
l&G	0.51	0.58
Yeast	7.58	7.59
Curcuma	0.88	0.88
Onion	2.02	2.05
Garlic	2.53	2.54
Bayleaf	0.25	0.25
micronized Fe (lll)Pyrophosphate (Fe4P6021)( FePP)	0.95	0.96

The amount of sait (NaCI) was corrected when Iran pyrophosphate (FePP) and Sodium pyrophosphate (NaPP) were added, the total weight of one cube remains 4 g.

Two consecutive sets of experiments hâve been executed within URDV wîth these example formats to explore the effect of NaPP on in-vitro Iron bioavaîlability further.

The first set of experiments focused on in-vitro bioavaîlability of iron of several bouillon cubes formats for the Nigérian market in order to rank prototypes to be used in an in-vivo study.

The experimental protocol included both dissolution / digestion and iron uptake experiments using human colonie adenoma carcinoma (Caco-2) cells. Gastrointestinal dissolution / digestion tests are simple tests to détermine the ability of a compound to dissolve in gastrointestinal fluid. This method is well known by the skilled person. The experiments are based on simulating gastrointestinal digestion, by exposure of food products and meals to gastric-like conditions (low pH and gastric enzymes) foliowed by intestinal-like conditions ( neutre lization of pH and incubation with pancreatic enzymes and bile salts). Subsequently, the digested meal or food product is dialysed and the soluble/ionic/dialyzable fractions (containing solutés and small molécules) are collected for détermination of iron content.

For non-heme iron this set-up first solubilises ail iron at pH 2.0, mîmicking the gastric conditions. Then, during the intestinal phase at neutral pH, some of the iron will form complexes with components from the (food) matrix. The ionic iron diffuses into a dialysis membrane (< 8000 g/mol) till equilibrium is reached while the complexed iron - with exception of low molecular weight soluble complexes - remains on the outside of the membrane. The ionic iron solution is collected from the membrane pouch and used for analysis to calculate the “dialyzable iron fraction”, or for further iron uptake experiments with intestinal cells.

To simulate the absorption of iron by intestinal cells after the gastrointestinal digestion, the model uses in-vitro gastrointestinal digestion techniques (see above) coupled with uptake of iron by Caco-2 cell monolayers. After 21 days of culture under adéquate conditions, Caco-2 cells form a monolayer that can be used as model of the intestinal epithelium in absorption experiments. If exposed to iron in the ionic form, Caco-2 cells synthesise ferritin as response to iron uptake. The ferritin amount is proportional to the iron content in the cell culture medium. Ferritin can be measured via a commercially available ELISA. Therefore the measurement of cellular ferritin is a good indicator of the iron absorbed by the cell (ref: Glahn, R.P., Lee, O.A., Yeung, A. et al.; Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model; Journal of Nutrition; 1998,

Vol. 128, no.9, p. 1555-1561).

Many published studies indicate that these combined digestion and cell models are useful to understand and rank iron uptake from food or meal formats. An international expert panel (ref:

Fairweather-Tait, S.J., Lynch, S., Hotz, C. et al.; The usefulness of in vitro models to predîct the bioavailability of iron and zinc: A consensus statement from the HarvestPIus expert consultation; International Journal for Vitamin and Nutrition Research; 2005, Vol. 75, no.6, p. 371-374) has reviewed several in-vitro méthodologies and concluded that the combined use of digestion and Caco-2 cells are a suitable approach in this field. This in-vitro approach was more recently critically reviewed and the conclusion of the expert panel confirmed (ref: Sandberg, A.S.; The use of Caco-2 cells to estîmate fe absorption in humans - a critical appraisal; International Journal for Vitamin and Nutrition Research; 2010, Vol. 80, no.4-5, p. 307-313).

For ranktng purposes of prototypes it is enough to perform only the dissolution / digestion part which predicts the amount of bioavailable iron which is available to be taken up by the intestinal cells. However, we hâve chosen to détermine the in-vitro bioavailability of iron in the bouillon cubes in the first sériés with the complété setup since the combination of iron and NaPP was never tested in the iron uptake experiments and we would like conformation of the iron bioavailability results.

Materials & Methods

Products (first set of experiments)

Bouillon cubes were manually prepared and the amount of iron and sodium pyro-phosphate added to the base formulation (see above) are shown in Table 3. Total of NaCI is corrected for the FePP and NaPP amount to remain at a total weight of each cube = 4 g.

Table 13 Bouillon cubes prepared to study the in-vitro bioavailability of iron from bouillon cubes - sériés 1 (the amount of iron per cube was set to 3.5 mg, 15% of the RDA, assuming 3 cubes would be shared in a meal for 5p).

Bouillon code	Description
HBR 12061- 1	No Fe
HBR 13003-1	FeSO4(15% RDA Fe)
HBR 12061-12	FeSO4 + 1eq NaPP (15% RDA Fe)
HBR 13003-2	2xFeSO4 (30% RDA Fe)
HBR 12061-2	Micronized FePP (15% RDA Fe)

HBR 12059-16	Micronized FePP + 0.25eq NaPP (15% RDA Fe)
HBR 12059-2	Micronized FePP + 1eq NaPP (15% RDA Fe)
HBR 12045-14	Micronized 2xFePP (30% RDA Fe)
HBR 12050-12	Micronized 2xFePP + 2x NaPP (30%RDA Fe)

To préparé bouillon with an iron level high enough to détermine at the end of the dissolution / digestion experiments, three bouillon cubes ofthe same batch were added to 400 ml boiled hot milli-Q water in a plastic beaker. The bouillon cubes were dissolved for 5-10 minutes using an incubator of 50 °C at 150 rpm. The obtained bouillon was gently shaken and divided over 4 plastic containers, 3 used for dissolution / digestion experiments and 1 for total iron détermination. The intact cubes where also analysed for their total iron content.

Protocol s

Total Iron détermination

The total iron content of the cubes was determined with open acid extraction method TE460 followed by Inducted Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). Total iron în the start bouillons was determined with open acid extraction method TE460 and with microwave destruction (TE461 ) followed by ICP-AES as above. Total iron in the samples after 15 the gastric phase and after the intestinal phase (dialysates) was determined with extraction method TE461 followed by ICP-AES.

Ionic iron détermination

Ionic iron (sum of Fe2+ and Fe3+) was determined with the Ferrozine method as described by Viollier et al. (Viollier, E., Inglett, P.W., Hunter, K.; The ferrozine method revisited: Fe (ll)/(lll) détermination in natural waters.; Applied Geochemistry; 2000, Vol. 15, no.6, p. 785790).

Samples were acidîfled (50 μΙ 37% HCl added to 1000 μΙ dialysate) and ovemight stored at 4°C.

Standard (FeSO4*7H20) was dissolved in 0.5 M HCl and the concentration was corrected for Fe content (20.09 %). A standard sériés of 0.06 - 30 mg/l Fe diluted in 0.5M HCl was analysed and the linear function was used to calculate the amount of Fe in the dialysates.

Reducing agent (150 μΙ of 1.4 M hydroxylamîne in 2 M HCl) was added to 100 μΙ sample or standard. After mixing, the solution was allowed to stand for 30 minutes at room température before ferrozine solution (100 μΙ of 0.01 M 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'disulfonic acid monosodium sait hydrate in 0.1 M ammonium acetate) and buffer (150 μΙ of 5

M ammonium acetate adjusted to pH9.5 with ammonium hydroxide (28-30%)) were added. The solution was mixed and 300 μΙ was transferred to a micro plate and absorption at À562 nm was read. The ferrozine-iron complex is stable for a least one hour.

Dissolution / Digestion

Bîoavailability is defined as the fraction of the total amount of iron présent in the cube which goes into solution (as ionic iron) and hence has the potential to be absorbed. Bîoavailability of the iron présent in the fortified bouillons was determined.

A short description of the in-vitro dissolution / digestion is as follows:

Ail glassware was incubated overnight in 10 % (v/v) H N 03. On the day ofthe experiment ail glassware were washed 5 times with milli-Q to remove HNO3. With exception of the bouillon product without added iron, ail bouillon products (80 ml) were transferred into 100 ml dissolution vessels in the dissolution apparatus (USP dissolution apparatus type II, VanKel VK700) and the pH was adjusted to 2.0. Subsequently, pepsin (0.5 g/l) was added to each vessel, yielding a 90 ml solution of bouillon in simulated gastric fluid ai pH 2.0. After 60 minutes incubation at 37 °C with mixing at 100 rpm, samples of the products were taken for total iron détermination and 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 présent in the dialysis membrane was able 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 ofthe membrane (dialysate) was stored in aliquots for détermination of ionic dialyzable iron and iron uptake experiments.

Average osmolarity of the bouillon samples after dissolution / digestion was 0.280 ± 0.010

Osmol/kg. Average pH after the intestinal phase was 7.2 ± 0.2.

Calculation

BiDavaiîability of iron is calculated by using Equation 1:

%bioavailable iron= (ionic dialysable iron)/(total Fe) *100% (Equation 1)

Iron uptake experîments in Caco-2 cells ln-vitro iron bioavailability was determined. However the used medium to dilute the dialyses samples for Caco-2 cell experîments was changed since the osmolarity of the samples with addition of 10 times concentrated medium was too high and resulted in loose cells.

Short description of the iron uptake experîments in Caco-2 cells is as follows:

Caco-2 cells were seeded in 12-wells plates at a density of 2*105 cells per well (passage 15). The cells were grown in Dulbecco’s modified Eagle's medium with 4.5 g/L glucose and Lglutamine (Bîo-Whittaker), supplemented with 20 % (v/v) heat-inactîvated foetal bovine sérum (Gibco), 1 % (v/v) penicillin/streptomycin (Bio-Whîttaker) 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 atmosphère 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 experîments.

On the day of experiment, dialysis fluids were thawed and diluted (3 ml dialysis fluid + 0.4 ml 10 times concentrated Customized Minimum Essential Medium+ (CMEM+) + 0.6 ml mQ). Average osmolarity of the samples as applied to the cells was 0,3386 ± 0.0161 Osmol/kg. Customized 10 times concentrated MEM+ (Osmolarity: 1.3 Osmol/kg) was prepared by mixîng 20% (v/v) MEM Amino Acids solution (50x, Sigma-Aldrich), 10 % (v/v) non essential amino acids (Bio-Whittaker), 10% (v/v) MEM Vitamin Solution (100*, Sigma-Aldrich), 5 % (v/v) penicillin / streptomycin solution (Bio-Whittaker), L-glutamine solution (20 mM), glucose (100 g/l), CaCl2*2H2O (18 mM), MgSO4*7H2O (8.14 mM),NaH2PO4 (9.3 mM), PIPES (100 mM), NaHCO3 (15.8 g/L), KOH (4 M for pH correction to pH 7.0), hydrocortisone (40 mg/L), insulin (100 mg/L), sélénium (50 pg/L), tri-iodotrionine (340 pg/L), epidermal growth factor (200 pg/L) and milli-Q to a final volume of 100 mL. The concentrated CMEM+ was stérile filtered (0.22 pm) and stored in aliquots at -20°C.

The diluted dialysate samples were stérile filtered (0.22 pm). As positive control, a sample was prepared with 5 pM FeSO4 in MEM+ (osmolarity 0.289 Osmol/kg). After washing the cells twice with 1 mL of 1 x MEM+, 1 mL of the diluted dialysate samples and control was applied in duplicate. Dialysates of day 1,2 and 3 (5 products) were applied to different 12wells plates. On a fourth 12-wells plate was product 6 applied. The positive control was applied to each plate.

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 pL of ice-cold milli-Q 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 Eppendorf tubes. The cell lysâtes 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 cîty, Taiwan, Catalogue number; KA0211) according to the manufacturées 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 lysâtes were 10 times diluted prior analysis and 250 pl Bradford reagent was added to 20 pl diluted sample/standard. This assay was performed at 595 nm.

Caco-2 cell iron uptake results were expressed as nanogram (ng) of ferritin per mg of total protein.

Results and Discussion

Total iron content

The total iron content in the bouillon cubes, start bouillon and after the gastric phase was determined. The results are listed in Table 14. The start bouillon is the dissolved bouillon (3 cubes / 400 ml) as used in the dissolution vessel. The iron content after the gastric phase (8/9 dilution) is also determined. From these figures the solubility at pH 2.0 is calculated (Table

14). At pH 2.0 the solubility of FeSO4 is somewhat better then the solubility of FePP, respectively 106 ± 1.9% and 94 ± 3.7%, but on average ail iron is dissolved.

The total iron content in the dialysates ofthe intestinal phase was also determined and compared to the ionic iron content obtained with a ferrozine assay. The two methods show similar results (Table 15), meaning that ail iron that passed the dialysis membrane is ionic iron. The large error bars for 30% RDA FeSO4 are due to the sample of the first dissolution / digestion experiment (day 1 ). The amount of iron found in the dialysate of day 1 resulted in roughly two times the amount of iron as measured for the second and third experiment. This is measured in both assays.

Table 14. Total iron content determined in bouillon cubes, start bouillon and after gastric phase using ICP-AES

Product	Fe in cube (mg/kg)	Fe in start bouillon (mg/kg)	Fe after gastric phase* (mg/kg)	Solubility (%) atpH 2.0
No Fe	4.5	n.d.	n.d.	n.a.
FeSO4(15% RDA)	760	24.8+0.35	23.4±0.60	106
FeSO4 + 1eq NaPP (15% RDA)	665	26.4±0.57	24.5±0.90	104
2xFeSO4 (30% RDA)	1570	47.1±1.41	45.9±0.07	108
FePP (15% RDA)	790	21.5±0.99	17.9±3.20	94
FePP + 0.25eq NaPP (15% RDA)	1080	22.3±0.64	18.8±1.40	90
FePP+1eq NaPP (15% RDA)	895	22.7±0.00	20.2i2.42	99
2xFePP (30% RDA)	1610	46.1 ±0.42	39.5+1.69	94
2xFePP + 2eq NaPP (30%RDA)	1650	53.0±0.35	43.1 ±5.40	91

* Products are 8/9 diluted at the end ofthe gastric phase compared to the start bouillon.

n.d. = not determined; n.a. = not applicable

Table 15. Comparison of Dialyzable ionic iron content analysed using ICP-AES (total iron) and ferrozine (ionic iron) methods. Mean ± SD (n=3 dissolution / digestion experiments).

Bouillon format	ICP-AES (mg Fe/kg dialysate)	Ferrozine (mg Fe/kg dialysate)
FeSO4 (15% RDA)	1.33 ±0.06	1.38 ±0.12
FeSO4 + 1eq NaPP (15% RDA)	3.20 ± 0.30	3.06 ±0.17
2 x FeSO4 (30% RDA)	2.20 ± 0.79	2.14 ±0.75
FePP (15% RDA)	0.43 ± 0.06	0.40 ± 0.09
FePP + 0.25eq NaPP (15% RDA)	0.70 ±0.17	0.68 ±0.17
FePP + 1eq NaPP (15% RDA)	1.60 ±0.17	1.59 ±0.04
2 x FePP (30% RDA)	0.50 ±0.10	0.47 ±0.11
2 x FePP + 2eq NaPP (30% RDA)	2.87 ± 0.25	2.71 ±0.18

Bioavailability (dissolution data)

Bioavailability is defined as the fraction, or percentage, of the total amount of iron présent in the cube which goes into solution (as ionic iron). Only unbound ionic iron is potentially absorbed by the intestinal cells. The bioavailability of iron form the bouillon formats is shown în Table 16.

Table 16. Bioavaîlable ionic iron (%) of the first bouillon sériés, mean ± SD, n = 3. The amount of iron per cube was set to 3.5 mg, 15% ofthe RDA, assuming 5 cubes would be shared in a meal for 3p. Statistic analysis was performed with JMP One way ANOVA with student T-test (comparison for ail pairs, p< 0.05), different letters indicate significant différences.

sample description	mean(%)	St Dev	statistical analysis
FeSO4 (15% RDA; ref)	14.24	0.97	c.d
FeSO4 + 1eq NaPP (15% RDA)	32.65	2.66	a
2xFeSO4 (30% RDA)	11.92	4.40	d,e
FePP (15% RDA)	6.30	2.12	f.g
FePP + 0.2eq NaPP (15% RDA)	9.24	1.82	e.f
FePP + 0.7eq NaPP (15% RDA)	19.79	0.49	b
2xFePP (30% RDA)	3.19	0.73	g
2xFePP + 2eq NaPP (30%RDA)	16.74	1.56	b,c

FeS04 (14.2%, shows a better bioavailability than FePP (6.3%), the factor is about 2.

Surprisingly however, an increase of the bioavailable ionic iron (%) is seen for bouillon containing FePP + NaPP as well as for bouillon containing FeSO4 + NaPP.

Without wishing to be bound by a theory, this increase seems to be dose-dependent for the amount of NaPP added to the bouillon. More NaPP added to bouillon cubes containing FePP resuit in higher bioavailable iron (%).

In-vitro iron (Caco-2 cell data)

In-vitro iron is defined as the fraction of bioavailable ionic iron which is capable to enter cells to trigger a physiological cell response. In-vitro iron is expressed by the ferritîn formation by Caco-2 cells. Ferritîn is the naturel intra-cellular storage protein for ionic iron and hence ferritîn formation is proportional, but not linear, to cell iron (6). The ferritîn formation by Caco-2 cells induced by ionic iron présent in the dialysates of the first bouillon sériés is shown in Table 17.

Bouillons without NaPP with 15 and 30% RDA of a Fe source, give similar amounts of ferritîn formation. This could be explained by equal amounts of Fe présent in their dialysates (Table 15). Addition of 1 équivalent NaPP to the cubes results in an enhancement of iron response in the cells.

When we compare the bioavailability results (Table 16) with the in-vitro bioavailability results (Table 17) indeed, a similar trend is seen between the plots of both methods. This means that also for bouillon cubes containing NaPP, ranking could be performed on base of bioavailability results.

Table 17. Ferritîn formation (ng ferritin/mg protein, mean ± SD, n = 3) by human colonie adenoma carcinoma (Caco-2) cells in response to the ionic iron from dialysates obtained from the in-vitro dissolution / digestion of iron. Statistical analysis was performed with JMP One way ANOVA with student T-test (comparison for ail pairs, p< 0.05). Different letter indicate significant différence, na = not available.

	mean	SD	statistical analysis
FeSO4 solution, 5 micromol/L, reference	49.40	15.32	--
FeSO4 (15% RDA; ref)	9.62	5.30	c,d
FeSO4 + 1eq NaPP (15% RDA)	61.20	12.18	b

2xFeSO4 (30% RDA)	11.38	no SD, n = 1	c,d
FePP (15% RDA)	7.03	2.09	d
FePP + 0,2eq NaPP (15% RDA)	Na	na	—
FePP + 0.7eq NaPP (15% RDA)	34.59	9.64	c
2xFePP (30% RDA)	8.11	no SD, n = 1	c,d
2xFePP + 2eq NaPP (30%RDA)	88.84	28.08	A

Conclusions

Addition of NaPP to FePP containing food formats tested has a positive, and apparently

NaPP dose related effect, on the in-vitro bioavailability of iron. Surprisingly, addition of 1 5 équivalent NaPP shows also enhancement of bioaccessable iron and in-vitro bioavailability for the tested iron source FeSO4.

Example 7: Iron to phosphate ratio

In this example the effect of the phosphate to iron ratio on in-vitro bioaccessibility is determined at different levels of iron added to a cube format and at different molar ratio's, a sériés of cube formats were prepared and pressed into cubes as indicated above. An experimental design was chosen to cover a range of sodium pyrophosphate to iron pyrophosphate (NaPP/FePP) molar ratio’s at different levels, see Table 8.

Table 18. Experimental design used to détermine effect of different levels of FePP (Xaxis) and NaPP (Y-axis) formulated into a bouillon cube format (nominal weight 4000 mg; having the same composition as in Example 6) on the in-vitro bioaccessible iron.

code	sample description	mg Fe per cube of 4 g	mg NaPP per cube of 4 g
00	(00) JNJ13037-01	25.1	641
+-	(+-) JNJ13037-02	9.1	1089
-+	(-+) JNJ13037-03	41.2	188
0A	(0A) JNJ13037-04	47,8	640
++	(++) JNJ13037-05	41.1	1093
a0	(a0) JNJ13037-07	25.1	1.4
0a	(0a) JNJ13037-08	2.5	641
A0	(A0) JNJ13037-09	25.1	1278
—	(-) JNJ13037-10	9.3	189
00	(00) JNJ13037-11	25.2	641
00	(00) JNJ13037-01	25.1	641

Table 18a Second sériés of samples to détermine effect of different levels of FePP (Xaxis) and NaPP (Y-axis) formulated into a bouillon cube format (nominal weight 4000 mg) on the in-vitro bioaccessible iron.

Bouillon cube	Code	FePP (mg)	NaPP (mg)	EQ NaPP/Fe
1 - JNJ13048-05	low Fe 0 Eq NaPP	5	0	0.25
2 - JNJ13048-01	high Fe 0 Eq NaPP	218	0	0.25
3-JNJ13049-01	low Fe 0.18 Eq NaPP	5	1	0.42
4-JNJ13048-02	high Fe 0.18 Eq NaPP	217	47	0.43
5 - JNJ13049-02	low Fe 5 Eq NaPP	5	29	5.08
6 - JNJ13048-03	high Fe 5 Eq NaPP	217	1279	5.22
7 - JNJ13049-03	low Fe 15 Eq NaPP	5	86	14.67
8-JNJ13048-04	high Fe 7.2 Eq NaPP	218	1863	7.47
9 - JNJ13049-04	low Fe 25 Eq NaPP	5	148	25.22
10 -JNJ13049-05	low Fe 35 Eq NaPP	5	207	35.14
11 -JNJ13049-06	low Fe 45 Eq NaPP	5	267	45.32

It is noted that the molar ratio of NaPP/lron is defîned as équivalents, so the relative amount of NaPP over iron becomes independent of the Iron form. This to exclude the impact of Iran coming from a sait low in Iron content (e.g. iron-gluconate) or a sait high in Iran content (e.g.

ferrous sulphate, dried) or even to iron in its pure form (elemental- iron). The experimental details are similar to the first sériés (above).

Bioaccessibility (dissolution)

The effect of the different levels can be seen in Table 19. Please note that each data point not 15 only reflects the molar ratio in équivalents of NaPP over Iran, but also represents different absolute levels of iron added to the cube format (according to Table 18 and 18a). Despite this wide range in compositional variation, the data shows that already at very low ratio’s of NaPP/Fe the positive effect on bioavailable ionic iron is significantly présent.

Table 19 Bioavailable ionic iron (%) at different levels of FePP and NaPP in a cube format (4000 mg). KO = Knorr Olympus formulation. Sample codes between brackets (e.g. (0A), refers to the sample codes as in experiment in Table 18.

sample description

Ratio PP/Fe

% Bioavailable ionic iron

Stdev

low Fe 0 Eq NaPP	0.25	28.8	1.9
high Fe 0 Eq NaPP	0.25	2.7	0.3
KO (aO)JNJ13037-07	0.26	4.7	0.2
FePP (15% RDA)	0.30	6.3	2.1
2xFePP (30% RDA)	0.30	3.2	0.7
low Fe 0.18 Eq NaPP	0.42	19.1	2.2
high Fe0.18Eq NaPP	0.43	5.4	0.1
FePP + 0.25eq NaPP (15% RDA)	0.50	9.2	1.8
FeSO4 + 1eq NaPP (15% RDA)	0.97	32.6	2.7
FePP + 1eq NaPP (15% RDA)	1.07	19.8	0.5
2xFePP + 2x NaPP (30%RDA)	1.09	16.7	1.6
KO (-+) JNJ13037-03	1.21	22.0	0.9
KO (0A) JNJ13037-04	3.06	49.3	5.4
KO (-) JNJ13037-10	4.52	52.2	7.2
low Fe 5 Eq NaPP	5.08	46.9	4.8
high Fe 5 Eq NaPP	5.22	59.6	4.4
KO (00)JNJ13037-11	5.60	62.5	12.6
KO (00) JNJ13037-01	5.61	69.2	7.1
KO (++) JNJ13037-05	5.83	68.3	3.6
high Fe 7.2 Eq NaPP	7.47	70.2	0.2
KO (A0)JNJ13037-09	10.95	63.2	8.1
low Fe 15 Eq NaPP	14.67	62.4	6.2
low Fe 25 Eq NaPP	25.22	62.5	3.4
KO (+-) JNJ13037-02	25.40	82.7	4.7
low Fe 35 Eq NaPP	35.14	60.6	3.4
low Fe 45 Eq NaPP	45.32	61.1	5.2
KO (0a)JNJ13037-O8	54.78	30.5	4.2

The table above shows that the percentage of bioavailable iron does not substantially increase above a ratio of phosphate to Iron ion of 10:1. Good bioavailability is obtained at a ratio of 1:1 or more. However the level of bioavailable iron remains high above 10:1, The best results are obtained at a ratio of between 1:1 and 6:1. The KO(Oa) value is likely to be an outlier, because that was measured on a very low concentration of Iron and a high NaPP concentration in the composition, causing an increased measuring error.

Y

Without wishing to be bound by a theory, it is thought that 6 pyrophosphate molécules may be arranged around one ionic iron atom to form a stable, soluble complex with an overall négative charge, existing at a neutral pH. This is further remarkable, as for other ionic iron forms, the solubîlity dépends strongly on the pH. At neutral pH in aqueous Systems, iron is not in solution but forms insoluble (poly)hydroxides.

Claims (15)

1. Savouryfood concentrate comprising:

• NaCI, • glutamate, • iron sait, further comprising • phosphate sait, not being an iron phosphate.

2. Food concentrate according to claim 1, wherein iron sait comprises one of the group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisgiycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and mixtures thereof.

3. Food concentrate according to any one of the preceding claims, wherein the phosphate sait comprises a sait selected form the group consisting of orthophosphate sait, diphosphate sait, triphosphate sait, polyphosphate sait and mixtures thereof.

4. Food concentrate according to any one of the preceding claims, wherein the phosphate sait is one of the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, calcium phosphate, magnésium phosphate, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate, sodium polyphosphate, potassium polyphosphate and mixtures thereof.

5. Food concentrate according to any one of the preceding claims, wherein the iron sait comprises one of the group consisting of ferrie pyrophosphate, ferrous sulphate and mixtures thereof, and the phosphate sait comprises Na-pyrophosphate.

6. Food concentrate according to any one of the preceding claims, wherein the glutamate is selected from one of the group consisting of monosodium glutamate, potassium glutamate, glutamic acid and mixtures thereof.

7. Food concentrate according to any one of the preceding claims, wherein the molar ratio of iron sait to phosphate sait, not being an iron phosphate, is between 1 and 10.

8. Food concentrate according to any one of the preceding claims, wherein the iron sait is présent in an amount of from 0.03 to 2 wt%, based on the weight of the food concentrate.

9. Food concentrate according to any one of the preceding claims, wherein the phosphate sait is présent in an amount of from 0.03 to 20 wt%, based on the weight of the food concentrate.

10. Food concentrate according to any one of the preceding claims, wherein the food concentrate is in the form of a cube or a tablet.

11. Food concentrate according to any one of the preceding claims, wherein the savoury food concentrate is in the form of a cube or a tablet and wherein • NaCI is présent in an amount of from 10 to 70 wt%, • iron-salt is présent in an amount of from 0.03 to 2 wt%, based on the weight ofthe food concentrate, and wherein the iron-salt is one ofthe group consisting of ferrous sulphate, ferrous gluconate, ferrous lactate, ferrous bisglycinate, ferrous fumerate, ferrie orthophosphate, ferrie pyrophosphate, ferrous tartrate, ferrous succinate, ferrous saccharate, ferrous orthophosphate and mixtures thereof, preferably wherein the iron- sait is selected from the group consisting of iron phosphate, ferrous sulphate and mixtures thereof, preferably is one of the group consisting of ferrie pyrophosphate, ferrous sulphate and mixtures thereof.

• phosphate sait, not being an iron phosphate, is présent in an amount of from 0.03 to 20 wt%, based on the weight of the food concentrate, and wherein this phosphate sait preferably is one of the group consisting of Na3PO4, Na2HPO4, NaH2PO4, K3PO4, K2HPO4, KH2PO4, sodium pyrophosphate, sodium triphosphate, potassium pyrophosphate, potassium triphosphate and mixtures thereof, • the glutamate is selected from one of the group consisting of monosodium glutamate, potassium glutamate, glutamic acid and mixtures thereof, preferably comprises mono sodium glutamate, • wherein the molar ratio of iron phosphate to phosphate sait, not being an iron phosphate, is between 1 and 10.

12. Process to provide a food concentrate according to any one of claims 1 to 11, the process comprising the steps of:

a) preparing a mixture comprising • NaCl, • iron sait, • glutamate, and further comprising

5 ♦ phosphate sait, not being an iron phosphate,

b) packaging.

13. Process according to claim 12, further comprising the step of shaping the mixture resulting from step a).

14. Process according to claim 13, wherein the shaping comprises a technique selected from

10 the group consisting of compression, extrusion, roller compacting, granulation, agglomération and combinations thereof.

15. Use of a concentrate according to any one of claims 1 toi 1 for preparing a bouillon, a soup, a sauce, a gravy or a seasoned dish.