WO2009047087A1

WO2009047087A1 - Iron-containing composition and the process to prepare such

Info

Publication number: WO2009047087A1
Application number: PCT/EP2008/062243
Authority: WO
Inventors: Arjen Bot; Marie N. Dobenesque; Sylvie C. Marchesseau; Sana Raouche
Original assignee: Unilever N.V.; Unilever Plc; Hindustan Unilever Limited
Priority date: 2007-10-12
Filing date: 2008-09-15
Publication date: 2009-04-16
Also published as: ZA201001862B; BRPI0816603A2; RU2010119048A; EP2194794A1; CN101820767A; MX2010003715A

Abstract

A composition comprising micelles, which micelles comprise both ϰ-casein and β- casein, and which micelles comprise in its inner structure iron. A method to prepare such by reacting a dissolved iron with a ϰ-casein and β-casein-containing aqueous composition, at a low temperature and an iron concentration of at least 2 mmol/l. Optionally, such composition may be dried.

Description

IRON-CONTAINING COMPOSITION AND THE PROCESS TO PREPARE SUCH.

Field of the invention

The present invention relates to a composition comprising casein micelles, which 5 micelles contain iron, as well as a process to prepare such.

Background of the invention

Fortification of foodstuffs with iron can be desired from a health point of view and/or for preventing anemia, and/or for promoting, maintaining or enhancing brain function or L O brain development.

Examples of foodstuffs which can be the carrier of such iron are ready-to-drink liquids or rehydratable liquids. Such compositions can contain iron as a dissolved salt like ferrous sulphate (for aqueous compositions) or a dissolvable salt like ferrous fumarate L 5 (for e.g. rehydratable aqueous compositions like instant drinks), or e.g. bound in a complex, such as with EDTA or with a protein.

EP 1166653 discloses iron-containing protein (e.g. of α-casein) complexes with good freeze-thaw stability, by including a specific sugar compound. Such proteins may be > 0 isolated from milk, but such is burdensome. Alternatively, one may use synthetic α- casein, but such is not food grade generally.

There is a desire for a relatively easy method to fortify a foodstuff with iron, which foodstuff should be foodgrade and preferably (already when not fortified, as a base to

>5 be fortified) have a healthy image with a large group of consumers. Preferably, the foodstuff to be fortified should already be "label-friendly" prior to fortification, to reduce the chances of the product to be regarded as a medicament rather than as a foodstuff. The method should allow to make an iron-fortified liquid or drink. Also, the method should enable a fairly high iron load, e.g. when the product is a fortified liquid product

30 which contains iron in an amount of at least 2 mmol/l, preferably at least 4 mmol/l, more preferably at least 8 mmol/l, yet more preferably at least 13 mmol/l. It is also desired that such foodstuff is stable (i.e. such that the appearance does not change dramatically) against heating to temperatures of at least 80⁰C (e.g. to enable preservation like pasteurisation and possibly even sterilisation) and that such product can be dried (e.g. by drying by heat) to yield e.g. a rehydratable particulate matter (rehydratable to an iron-fortified drink or liquid, and/or be suitable as an iron-fortified 5 product that can be added to aqueous products).

Preferably (as a large target group is living in the developing parts of the globe) the raw materials should not be very expensive.

L O Furthermore, there is a need for a concentrated or dry ingredient which can be used to fortify a foodstuff (including e.g. aqueous compositions and water-continuous or oil- continuous emulsions) with otherwise similar requirements as set out above.

Preferably, in the above the iron is in a form such that it is bioavailable: i.e. taken up L 5 by the body of the consumer (human or animal), e.g. as can be ascertained with being easily dissolvable at pH 2 and body temperature or by a model system of intestinal cells. It is additionally preferred that the composition according to the invention does not promote oxidation of unsaturated fats in the same manner as dissolved iron (Fe ²⁺ or Fe ³⁺ ions), but less. > 0

It is also preferred that the iron is in such a format that it allows formulation in a food additive or food product together with PUFA's and/or (tri)glyceride esters thereof, and/or formulation with fatty acids DHA and/or EPA and/or (tri)glycehde esteres thereof, with low tendency to fatty acid oxidation.

Summary of the invention

It has now been found that above formulated objectives may (at least in part) be achieved by a composition comprising micelles comprising κ-casein and β-casein (preferably κ-casein being present in the micelles in an amount of at least 5% by 30 weight, based on the total amount of protein present in the micelles, and β-casein being present in the micelles in an amount of at least 15% by weight, based on the total amount of protein present in the micelles), which micelles comprise within said micelles iron, in an amount of at least 20 mmol iron / kg micelle, preferably in an amount of at least 40 mmol iron / kg micelle.

Such product can be prepared using a process for preparing an iron-containing

5 composition, said process comprising the step of: bringing together in an aqueous composition a dissolved iron salt and dispersed casein micelles, which casein micelles comprise both κ-casein and β-casein, characterised in that for a time stage of at least the first 10 minutes (preferably at least 15 minutes) after the bringing together the iron and casein micelles the pH of said

L O aqueous composition is between 4 and 8 and for at least said time stage the temperature of said aqueous composition is kept below a temperature T (in ⁰C) determined by the relation:

T = 25 - 3 x log(concentration casein micelles in weight %) - 0.6 x (concentration of Fe in mmol/l),

L 5 with the proviso that T is 15°C or lower, and that the concentration iron in the aqueous composition at the start of bringing together the iron and the casein micelles is at least 2 mmol/l, more preferably at least 3 mmol/l, even more preferably at least 4 mmol/l, most preferably at least 10mnnol/l. For compositions close to regular milk, the concentration of casein micelles would equal the dry matter in milk, being 10%,

> 0 resulting in the equation T = 22 - 0.6 x (concentration of Fe in mmol/l) because log(10)=1.

In the process according to the present invention, it is preferred that that the concentration casein micelles in said aqueous composition is between 0.1 and 25% >5 (by weight, based on said aqueous composition), more preferably between 5 and 15 % (weight %, based on the total composition), and even more preferably is between 8 and 13% (weight %, based on the total composition).

In the process of the invention, it is preferred that κ-casein is present in said micelles 30 in an amount of at least 5% by weight, based on the total amount of protein present in the micelles, and β-casein is present in said micelles in an amount of at least 15% by weight, based on the total amount of protein present in the micelles) Preferably, the above referred first 10 minutes (the "time stage" referred to above) lasts at least 15 minutes, more preferably at least 20 minutes, or even lasts at least 30 minutes, but generally less than 24 hours, more likely less than 12 hours.

5

Detailed description of the invention

For the present invention it was surprisingly found that ordinary milk (or milk prepared from reconstituted milk powder, skim milk powder, and so on, as long as casein micelles are present) can be fortified with high loads of iron, and it is even more

L 0 surprising that once the initial stage of "loading" the micelles present in the milk with iron is done, that such iron-fortified milk is stable, also to heating, as long as the loading occurs at a low temperature. Needless to say, milk is a cheap product, and has a healthy image with many consumers, and (when offered in a dried format) is easily reconstitutable. Also diluted or concentrated milk may be used in the process

L 5 according to the invention.

The micelles used in the present invention are normally present in (e.g. cow's) milk, but also in (skim)milk powder, buttermilk powder, and the like. Such micelles are formed in the cow, and contain (when directly taken from the cow) other minerals in > 0 these micelles, such as colloidal calcium phosphate (CCP) comprising calcium.

Without wishing to be bound by theory, it is believed that by following the process according to the present invention (i.e. by contacting micelles containing κ-casein and β-casein with a dissolved iron salt in an aqueous environment, at the specified

>5 concentrations, pH and temperature) part of the calcium normally present in such micelles is exchanged for iron. The iron in the present process can be in the form of dissolved salts of Fe²⁺ or Fe ³⁺. What was found is that by the process according to the present invention, far more iron can be bound by casein micelles than when the iron is complexed on the outside of other milk proteins (e.g. α-lactalbumin, α-casein,

30 whey protein), which is one of the conventional ways of fortifying milk with iron. In the process according to the invention, it is preferred that the concentration iron in the aqueous composition at the start of bringing together the iron and the casein micelles is at most 40 mmol/l, preferably at most 35 mmol/l, more preferably at most 30 mmol/l. The actual amount preferred depends also on the concentration of the 5 casein micelles selected: with higher casein micelle concentration also iron concentrations are possible which are on the high limits, and similarly for using low levels of casein micelles (then lower concentrations of iron are desired.

It was found that in the process of manufacture, by reducing the pH to a range of 4.8- L O 7, preferably 5.2-6.8 (for at least the first 10 minutes after the bringing together the iron and casein micelles), the reaction of exchanging calcium for iron can be speeded up. Hence, it is preferred that for the process according to the present invention, the pH of the aqueous environment containing the micelles comprising κ-casein and β- casein is lowered to something of the above ranges, after the bringing together of the L 5 iron and casein micelles. After the reaction between iron and the casein micelles (or possibly also during, as the pH drops during the course of the process) the pH is preferably raised again to the original or other pH, e.g. about neutral, e.g. 6.5-7.5.

In the process according to the present invention, it is preferred that the pH of the _ 0 aqueous composition during said time stage of at least the first 10 minutes (and other preferred time intervals referred to above) is between 4.8 and 7, more preferably between 5.2 and 6.8.

Lowering the pH (e.g. to achieve the pH to be between 4 and 8, or other preferred pH >5 range referred herein) is preferably achieved by carbonation, e.g. by pressurising the composition with CO2, but traditional acidulants (e.g. citric acid, lactic acid, or by fermentation by means of e.g. lactic acid bacteria) can be used as well. Raising the pH afterwards can, when carbonation through pressurising with CO2 is followed, be achieved by depressurising the fortified micelle composition, but adding alkaline or 30 buffer agents can be used as well. Hence, it may be preferred that in the process according to the present invention, for cases in which the pH is reduced to below 6 (either through addition of the iron-solution or through other means), such pH is adjusted back to 6.5-7.5 after the time interval referred to above.

Also, it was found that the casein micelles do not aggregate (curdle), when the 5 micelles are loaded with iron, using the process of the invention (i.e. using the prescribed temperature and pH). It was also found that the lower the temperature (especially but not exclusively during the pH lowering to speed up the reaction, as set out above) the more iron can be contained in the micelles (provided that the sample is not frozen). For example, at 20⁰C the micelles may hold an amount of e.g. 2 mmol/l of L O iron, and at 5°C the micelles may hold up to about 30 mmol/l. Hence, it is preferred that the temperature of the aqueous composition during said time stage of at least the first 10 minutes is between -0.5⁰C and 15°C, preferably between 0⁰C and 10⁰C, more preferably between 1 °C and 5°C.

L 5 It was also found, that once the iron is inside the micelles, the composition is fairly stable, i.e. can be heated to e.g. about 80⁰C without curdling and/or iron loss. Such stability allows for e.g. heat preservation and/or drying the product, to for example a rehydratable powder or an ingredient for another food product.

_ 0 In view of the above, and depending on the intended use, it may be preferred that in the process herein, after the bringing together of the iron with the casein micelles the resulting iron-containing micelles are subjected to a drying operation, to result in a particulate product having a moisture content of less than 10%, preferably less than 5% (by weight, based on the composition after said drying operation).

In the process according to the present invention, the micelles are preferably obtained from milk, preferably cow's milk, but which may also have been subjected first to a drying step. Hence, in the present invention, the aqueous composition preferably comprises milk and/or rehydrated milk powder, or at least a casein-micelle containing 30 fraction thereof. The rehydrated milk powder may be e.g. skim milk powder, butter milk powder, or other casein-micelle containing dried milk product. Also a semi- concentrated or diluted milk may be used, but preferably the concentration of the casein micelles in the aqueous composition is between 0.1 and 25% (by weight, based on said aqueous composition), more preferably between 5 and 15 % (weight %, based on the total composition), and even more preferably is between 8 and 13% (weight %, based on the total composition).

5

In the process as set out above, the dissolved iron salt preferably comprises a solution comprising one of: ferrous sulphate, ferrous fumarate, ferrous chloride, ferric chloride, iron EDTA, or mixtures thereof.

L O In the compositions as can be prepared using the process as set out above, it is preferred that the micelles of κ-casein and β-casein are present in a concentration of at least 5 % (preferably at least 8%) by weight, based on the total composition. Such amount may be higher after e.g. using concentrated milk or removing part of the water before, during or after the reaction of micelles with iron. The reaction products

L 5 obtained may also be subjected to a water removal step, e.g. drying by heat. This may lead e.g. to a composition wherein the amount of moisture is below 10%, preferably below 5%, by weight based on the total composition. Such composition may be rehydrated again to a liquid, e.g. upon consumption, but such dry (or mostly dry) product may also be used as an iron-containing ingredient in the manufacture of iron-

> 0 containing foodstuffs and products.

Next to iron, another class of compounds believed to be contributing to promoting, maintaining or enhancing brain function or brain development comprise omega-3 fatty acids and/or omega-6 fatty acids, which herein preferably have 20-30, more preferably

>5 20-24 carbon atoms. These omega-3 fatty acids and/or omega-6 fatty acids can be in the form of free fatty acid, C1 to C6 alkyl esters thereof, glycehdes (including mono-, di- and tri- glycehdes) thereof, phospholipid esters thereof or mixtures thereof, all of which are herein collectively referred to as "omega-3 fatty acids and/or omega-6 fatty acids". Such omega-3 fatty acids and/or omega-6 fatty acids can be obtained e.g. from

30 marine oils such as fish oils, fish liver oils, and algae, and some specific vegetable oils like borage. Preferred omega-3 fatty acids and/or omega-6 fatty acids in the present invention comprise DHA (docosahexaenoic acid) and/or EPA (eicosapentaenoic acid). In the present invention, the omega-3 fatty acids and/or omega-6 fatty acids are preferably present in a weight ratio iron to omega-3 fatty acids and/or omega-6 fatty acids of between 0.03 and 3.

5

It may be preferred that the composition according to the present invention further comprising omega-3 fatty acids and/or omega-6 fatty acids, in an amount such that the weight ratio omega-3 fatty acids and/or omega-6 fatty acids : micelles comprising κ-casein and β-casein is between 0.16 and 1.

L O

The composition may further contain other polyunsaturated fatty acids than the omega-3 fatty acids and/or omega-6 fatty acids referred to above (e.g. linoleic acid).

EXAMPLES

L 5 Iron-containing milk preparations were prepared, along the processing as set out below. The milk was prepared by reconstituting 106 g of commercial skim milk powder with 694 g water. Such milk preparation contains casein micelles.

Experiment 1 (control) ( Fe³⁺ at 40 mmol/l without carbonation)

> 0 To a batch of 500 g milk (magnetically stirred), which was held at room temperature, was dropwise added (under stirring) 80 ml of a FeCb solution (0.25 M). Simultaneously, 32.5 ml of a KOH solution (1 mol/l) was added to counterbalance the pH drop caused by adding the FeCb solution. This addition took 17 minutes. The pH at start was around 6.6, and did not drop below 6.2, and in the end was 6.6 again. The >5 milk sample showed aggregation and was not stable.

Experiment 2 ( Fe³⁺ at 20 mmol/l, without carbonation)

To a batch of 500 g milk (magnetically stirred), which was held at a temperature of 3 to 4°C, was dropwise added (under stirring) 40 ml of a FeCb solution (0.25 M). 30 Simultaneously, 16 ml of a KOH solution (1 mol/l) was added to counterbalance the pH drop caused by adding the FeCb solution. This addition took 17 minutes. The pH at start was around 6.6, and did not drop below 6.2, and in the end was 6.6 again. This solution was kept at a temperature of 3 to 4°C, after which the temperature was brought up to 20⁰C. The total amount of iron added was 10 mmol, of which by analysis it turned out that 0.9 mmol was present in the serum, which would leave ((10- 0.9)/556)^*(800/106) = 0.12 mol/kg micelle to be present in the dry matter of the casein 5 micelles.

Experiment 3 (Fe³⁺ at 20 mmol/l, with carbonation)

To a batch of 500 g milk (magnetically stirred), which was held at a temperature of 3 to

4°C, was dropwise added (under stirring) 40 ml of a FeCb solution (0.25 M).

L O Simultaneously, 16 ml of a KOH solution (1 mol/l) was added to counterbalance the pH drop caused by adding the FeCb solution. This addition took 17 minutes. The pH at start was around 6.6, and did not drop below 6.2, and in the end was 6.6 again. This solution was kept at a temperature of 3 to 4°C, after which the temperature was brought up to 20 ⁰C.

L 5 The fortified milk was poured into a jacketed stainless steel refrigerated vessel. Milk samples were pressurised while stirring (Micropump Corporation, Vancouver, Canada) by CO2 stepwise injection at 4 ± 1 °C. At time t=0, CO2 was injected to a pressure of 2-10⁵ Pa and incremented by 1 -10⁵ Pa each 3 min until the pH dropped to 5.5. The pressure was kept constant during 15 min at 4°C. The reactor was then

> 0 depressurised. To achieve degassing, milk samples were collected and kept for 2 h at 30⁰C under stirring and vacuum using a diaphragm pump. The pH of such carbonated milk samples returned consequently to their initial value (pH 6.63 ± 0.02). The total amount of iron added was 10 mmol, of which by analysis it turned out that 0.4 mmol was present in the serum, which would leave ((10-0.4)/556)^*(800/106) =

>5 0.13 mol/kg micelle to be present in the dry matter of the casein micelles.

Experiment 4 (Fe²⁺ at 20 mmol/l, without carbonation)

Experiment 2 was repeated using an FeC^ solution instead of a FeCb solution, everything else being the same. The total amount of iron added was 10 mmol, of 30 which by analysis it turned out that 1.6 mmol was present in the serum, which would leave ((10-1.6)/556)^*(800/106) = 0.11 mol/kg micelle to be present in the dry matter of the casein micelles. Experiment 5 (Fe²⁺ at 20 mmol/l, with carbonation)

Experiment 3 was repeated using an FeC^ solution instead of a FeCb solution, everything else being the same. The total amount of iron added was 10 mmol, of which by analysis it turned out that 1.0 mmol was present in the serum, which would leave ((10-1.0)/556)^*(800/106) = 0.12 mol/kg micelle to be present in the dry matter of the casein micelles.

Experiment 6

L O Bioavailability of iron from the iron-casein-micelles according to Examples 2-5 was measured by measuring the formation of ferritin in Caco-2 cells as described in the publications by Glahn RP et al, Caco-2 cell iron uptake from meat and casein digests parallels in vivo studies: Use of a novel in vitro method for rapid estimation of iron bioavailability, Journal of Nutrition 126:332-339 (1996) and Glahn RP et al, Caco-2 cell

L 5 ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion Caco-2 cell culture model, Journal of Nutrition 128:1555-1561 (1998). The ferritin in the cells was measured by an immuno-emzymic fluoromethc assay. The results in the table below show that in all cases the uptake of iron by the cells increases, showing that the iron from the casein micelles is bioavailable.

> 0

Table: difference between the amount of ferritin (in ng ferritin / mg protein) measured in Caco-2 cells exposed to an (20 mmol/kg) iron fortified milk compared to a non- fortified milk. The numbers between brackets indicate standard deviations.

> 5

Claims

1. Process for preparing an iron-containing composition, said process comprising the step of:

- bringing together in an aqueous composition a dissolved iron salt and dispersed casein micelles, which casein micelles comprise both κ-casein and β-casein, characterised in that for a time stage of at least the first 10 minutes (preferably at least 15 minutes) after the bringing together the iron and casein micelles the pH of said aqueous composition is between 4 and 8 and for at least said time stage the temperature of said aqueous composition is kept below a temperature T (in ⁰C) determined by the relation:

T = 25 - 3 x log(concentration casein micelles in weight %) - 0.6 x (concentration of Fe in mmol/l), with the proviso that T is 15°C or lower, and that the concentration iron in the aqueous composition at the start of bringing together the iron and the casein micelles is at least 2 mmol/l.

2. Process according to claim 1 , wherein the concentration casein micelles in the aqueous composition is between 0.1 and 25% (by weight, based on said aqueous composition).

3. Process according to claim 2, wherein the concentration of the casein micelles in the aqueous composition is between 5 and 15%, (by weight, based on said aqueous composition), preferably between 8 and 13%.

4. Process according to any of claims 1 to 3, wherein the pH of the aqueous composition during said time stage of at least the first 10 minutes is between 4.8 and 7, more preferably between 5.2 and 6.8.

5. Process according to any of claims 1 to 4, wherein the temperature of the aqueous composition during said time stage of at least the first 10 minutes is between -0.5⁰C and 15°C, preferably between 0⁰C and 10⁰C, more preferably between 1⁰C and 5°C.

6. Process according to any of claims 1 to 5, wherein the concentration iron in the aqueous composition at the start of bringing together the iron and the casein micelles is at most 40 mmol/l, preferably at most 35 mmol/l, more preferably at most 30 mmol/l.

7. Process according to any of claims 1 to 6, wherein the aqueous composition comprises milk and/or rehydrated milk powder.

8. Process according to any of claims 1 to 7, wherein the pH of the aqueous composition between 4 and 8 is achieved by carbonation of the aqueous composition.

9. Process according to any of claims 1 to 8, wherein the dissolved iron salt is a solution comprising one of: ferrous sulphate, ferrous fumarate, ferrous chloride, ferric chloride, iron EDTA, or mixtures thereof.

10. Process according to any of claims 1 to 9, wherein after the bringing together of the iron with the casein micelles the resulting iron-containing micelles are subjected to a drying operation, to result in a particulate product having a moisture content of less than 10%, preferably less than 5% (by weight, based on the composition after said drying operation).

11. Process according to any of claims 1 -10, wherein the concentration iron in the aqueous composition at the start of bringing together the iron and the casein micelles is at least 3 mmol/l, even more preferably at least 4 mmol/l, most preferably at least 10 mmol/l.

12. Composition comprising micelles comprising κ-casein and β-casein, which micelles comprise within said micelles iron, in an amount of at least 20 mmol iron / kg micelle, preferably in an amount of at least 40 mmol iron / kg micelle.

13. Composition according to claim 12, wherein κ-casein is present in the micelles in an amount of at least 5% (by weight, based on the total amount of protein present in the micelles) and β-casein is present in the micelles in an amount of at least 15% (by weight, based on the total amount of protein present in the micelles),

14. Composition according to claim 12 or 13, such that the micelles of κ-casein and β-casein are present in a concentration of at least 5 % (preferably at least 8%) by weight, based on the total composition.

15. Composition according to any of claims 12 to 14, comprising an amount of moisture of below 10%, preferably below 5%, by weight based on the total composition.

16. Composition according to any of claims 12 to 15, further comprising omega-3 fatty acids and/or omega-6 fatty acids in a weight ratio iron to omega-3 fatty acids and/or omega-6 fatty acids of between 0.03 and 3.