The present invention relates to a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, at most weight-10 % water, and being enriched with phytase.

Furthermore, the invention relates to the use of a ready-to-use food to deliver active phytase to humans, to correct deficiencies of mineral salts in undernourished humans.

Mineral nutrient (so-called micronutrient) deficiency continues to be highly prevalent in developing countries and is therefore an important public health issue. Iron deficiency (or "sideropenia") is the most common known form of nutritional deficiency. The direct consequence of iron deficiency is iron deficiency anemia. Mainly infants, children, adolescent girls and women, here especially pregnant and breast feeding women are affected. Of children, especially children from 6 months until 2 to 5 years of age are affected. In the first 6 months of life, infants obtain iron via breast milk. The iron status of the infant depends on the iron status of the mother before conception. After weaning, iron supply to the infant solely depends on the intake from food. In vegetarians, especially in poor countries, the main calorie intake stems from phytate-rich staples, which limits the mineral-including iron bioavailability. In infants, iron deficiency causes delayed brain and locomotor development and alters emotional development, which cannot be cured by later iron supplementation. Iron deficient infants are less self secure, thus less exploratory, and are less emotionally stable. Thus, iron deficiency in early infancy reduces the development options of children later in life. In adults, the main obvious impact of iron deficiency is lack of energy, thus reduced labour productivity, and increased maternal mortality. Because iron is essential for most plants and animals, a wide range of food can provide it. However, these foods are absorbed and processed differently by the body; for instance, iron from meat (heme iron source) is more easily broken down and absorbed than iron in grains (non-heme iron source), and minerals and chemicals in one type of food may inhibit absorption of iron from another type of food eaten at the same time.

Zinc deficiency (or "hypozincemia") is a condition where insufficient zinc is available for metabolic needs. In fact, one-third of the world population is at risk of zinc deficiency, ranging from 4 % to 73 % depending on the country. Zinc deficiency is the fifth leading risk factor for disease in the developing world. Providing micronutrients, including zinc, to humans is one of the four quick-win solutions to major global problems identified in the Copenhagen Consensus from an international panel of distinguished economists. Conservative estimates suggest that 25 % of the world's population is at risk of zinc deficiency. Populations that consume primarily plant based diets that are low in bioavailable zinc often have zinc deficiencies. Physiological states that require increased zinc include periods of growth in infants and children as well as in mothers during pregnancy. Zinc deficiency is connected with skin and hair problems and suboptimal immune function.

Calcium deficiency (hypocalcemia) is the presence of low serum calcium levels in the blood, usually taken as less than 2.1 mmol/L or 9 mg/dl, or an ionized calcium level of less than 1 .1 mmol/L (4.5 mg/dL). It is a type of electrolyte disturbance. In the blood, about half of all calcium is bound to proteins such as serum albumin, but it is the unbound, or ionized, calcium that the body regulates. If a person has abnormal levels of blood proteins, then the plasma calcium may be inaccurate. The ionized calcium level is considered more clinically accurate in this case. Magnesium deficiency refers to a lack of dietary magnesium below specified levels, which can result in numerous symptoms and conditions. This can generally be remedied by an alteration of diet or oral supplements. However intravenous supplementation is necessary for more severe cases. Symptoms of magnesium deficiency include: hyperexcitability, muscle weakness and tiredness. Severe magnesium deficiency can cause death from heart failure. Calcium as well as phosphorous are needed for healthy bones.

Phytic acid (or phytate when in salt form) is the principal storage form of phosphorus and minerals in many plant tissues, especially bran and seeds. Phytate is found within the hulls and germs of nuts, seeds, and grains. It is a strong chelator of important minerals such as calcium, magnesium, iron and zinc and can therefore contribute to mineral deficiencies in people whose diets rely on these foods for their mineral intake. In this way, phytate is an anti-nutrient.

Phytases are enzymes which split phytic acid (or phytate) in lower inosine phosphate esters, and phytogenic myo-inositol and orthophosphate and therefore increase the bioavailability of occluded minerals. Phytases are - like phytate - found within the hulls and germs of grains and are activated by food processing like soaking, dough processing, fermentation etc.: e.g. about 50 % of the phytate complexes are split during the fermentation process in the making of whole wheat meal bread with yeast. During the production of whole rye meal bread using sourdough even all phytate complexes are split. Besides the food processing techniques themselves also physical parameters like particle size of the flour as well as pH value affect the enzymatic degradation of phytate: finely ground grist, low pH and soaking times of several hours promote the liberalization of minerals.

It is known to the person skilled in the art that added phytase can be used during food processing to reduce the amount of phytate, in order to reduce its anti-nutritional effects, thereby improving the bioavailability of mineral salts. However, for some foods, this is unfortunately not usable since the conditions under which the food is processed are incompatible with the conditions under which the enzyme would exert its activity, or such modified food processing conditions would drastically increases the production cost of such processed foods.

As an alternative, phytase can be delivered to humans together with meals in the form of pills. This is however, not the preferred form since current trend is to avoid the multiplication of the number of pills to be ingested per day. A very typical form of supplying to humans phytase through food consists in adding a granulated form of the enzyme to beverages as disclosed in US 7,566,466 B2. Nevertheless, due to the inherent instability to temperature, light and oxygen of the phytase enzyme in liquid form the enzyme is often completely inactivated when reaching the stomach of the consumer where it should be active.

The goal of the present invention was to find a ready-to-use food in the form of a complete food or a nutritional supplement in which granulated phytase enzyme could be added and retain enzymatic activity until reaching its targeted place of activity in the stomach of a human thereby delivering the expected bio-available iron and zinc minerals.

The inventors of the present application now surprisingly found that a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, at most 10 weight-% water and enriched with phytase significantly improves the stability of phytase during storage time of the ready-to-use food thereby improving bioavailability of minerals (iron, zinc, calcium, magnesium, phosphorous) in humans.

Moreover, the addition of phytase to the ready-to-use food preparation allows to reduce downtime of production equipment, and to reduce cleaning costs during production. 1 phytase unit (also-called FTU) is the amount of phytase that liberates 1 μηιοΐβ phosphate from phytate per minute at pH 5.5 and 37 °C. It was not to be foreseen by the person skilled in the art that addition of phytase to this lipid rich ready-to-use food would trigger such an improvement in terms of phytase stability and consequently, subsequent improvement in mineral bio-availability in human. Therefore, in a first embodiment, the invention relates to a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, at most 10 weight-% of water, and being enriched with phytase.

The term "ready-to-use food" (RUF) comprises ready-to-use therapeutic food (RUTF), ready-to- use complementary food (RUCF), and ready-to-use supplementary food (RUSF). It is to be understood in a broad sense, it encompasses food designed for specific, usually nutritional, supplementary or complementary therapeutic purposes, and can be consumed without any additional food or beverage. It also encompasses concentrated food which can be consumed as such or added to a meal. However, dietary supplements in the form of tablets or capsules are excluded from this definition.

The term "phytase" as used herein denotes phosphatases which specifically split phytic acid in myo-inositol and orthophosphate. A distinction is drawn between 3-phytase and 6-phytase according to the carbon atom at which the orthophosphate is split off the phytic acid:

3-phytase (Enzyme EC 3.1.3.8):

myo-inositol hexakisphosphate + H ₂ 0 = l D-myo-inositol 1 ,2,4,5,6-pentakisphosphate + phosphate

6-phytase (Enzyme EC 3.1.3.26):

myo-inositol hexakisphosphate + H ₂ 0 = l D-myo-inositol 1 ,2,3,4,5-pentakisphosphate + phosphate

According to the present invention 3-phytase food grade, 6-phytase food grade, or mixtures thereof are especially preferred. The phytase of the present invention may come from plant and/or microbiological sources. A preferable plant source for phytase is malt. The fungal enzyme phytase from Aspergillus niger has been commercialized for use in animal feed. Genes encoding the enzymes have been cloned from many micro organisms, and respective enzymes can easily be produced and purified to industrial production scale.

The term enriched with Phytase enzyme means that exogenous phytase is added to the ready- to-use food in an amount of at least 5 FTU per one serving. A typical serving is around 100g and contains about 500 kcal in the case of a RUTF, and is around 20 g and about 100 kcal in the case of a RUSF. For the purpose of the present invention, a serving size is therefore comprised between 5 and 200 g and contains 20 to 500 kcal. Serving size is smaller for RUSF, while micronutrients and enzymes are included in a higher concentration.

The ready-to-use food of the present invention has a semi-pasty or pasty consistency with a water content by weight of less than 10 %. The preferred water content according to the present invention is less than 5 % by weight, even more preferred, less than 2 % by weight.

Thus the product based on the current invention helps provide children with the best possible start in life, helps boost children energy, supports skin and nail health, supports a strong immune function, helps boosting the immune system, supports bone health, supports healthy child development, helps keep children grow and thrive.

In another embodiment, the ready-to-use food of the present invention contains at least 25 % of its weight in the form of lipids, most preferred, at least 30 % of it weight in the form of lipids. In another embodiment, the ready-to-use food of the present invention contains at least 25 % of its weight in the form of lipids, at least 0.5 weight-% lecithin, at most 5 weight-% water, and enriched with phytase. More preferably, the ready-to-use food of the present invention contains at least 30 % of its weight in the form of lipids, at least 0.5 weight-% lecithin, at most 2 weight-% water, and enriched with phytase.

In yet another embodiment, the lipids are derived from oleaginous or legume seeds. Preferred oleaginous seeds originate from cocoa beans, almonds, walnuts, hazelnuts, peanuts, soy beans, coconuts, and pistachio nuts. Alternatively legume seeds are also used. Preferred legume seeds can be selected from chick peas, peas, black grams, lentils, alfalfa.

In another embodiment, the ready-to-use food of the present invention is enriched in phytase with 5 to 5000 FTU per one serving, preferably, 20 to 2000 FTU per one serving, most preferably, 50 to 1000 FTU per serving. Phytase is preferably in a powdered or granulated form and is first blended with all powdered ingredients and subsequently mixed with the lipid fraction of the ready-to-use food product. Alternatively, Phytase can also be added as a liquid oily dispersion, or formulated with e.g.: glycerol, sorbitol etc. Any phytase enzyme can be used for the present invention, but preferred phytase enzyme is EuphoVida 20000G available from DSM.

In another embodiment, the ready-to-use food of the present invention is enriched in phytase in a concentration comprised between 0.05 and 1000 FTU/g ready-to-use food. Preferably, the phytase concentration is comprised between 0.05 and 500 FTU/g, more preferably, the phytase concentration is comprised between 0.5 and 250 FTU/g, even more preferably, the phytase concentration is comprised between 5 and 150 FTU/g, most preferably, the phytase concentration is comprised between 10 and 100 FTU/g.

In another embodiment, the ready-to-use food of the present invention is a ready-to-use therapeutic food enriched in phytase in a concentration comprised between 0.5 and 500 FTU/g ready-to-use food. Preferably, the phytase concentration is comprised between 1 and 100 FTU/g, more preferably, the phytase concentration is comprised between 5 and 20 FTU/g.

In another embodiment, the ready-to-use food of the present invention is a ready-to-use supplementary food enriched in phytase in a concentration comprised between 1 and 1000 FTU/g ready-to-use food. Preferably, the phytase concentration is comprised between 10 and 500 FTU/g, more preferably, the phytase concentration is comprised between 50 and 250 FTU/g. In another embodiment, the ready-to-use food of the present invention contains at least one powdered product which provides proteins, and/or at least one product which provides carbohydrates, and/or at least one product which provides lipids. Most preferred proteins are skimmed-milk, powdered yoghurt, soy, rice, and whey protein isolates. Most preferred carbohydrates are carbohydrate bulking agents, sucrose, glucose, fructose, maltodextrin, skimmed milk, whey, or flour made of maize, wheat, millet, oats, rice, cassava, or potato starch, and/or at least one product which provides lipids.

In another embodiment, the ready-to-use food of the present invention is further enriched with nutrients, further enzymes, or mixtures thereof.

The term "nutrient" as used herein denotes physiologically essential components of the human diet such as vitamins, e.g., vitamin A, beta-carotene, vitamin B1 , Folic acid, Niacin, vitamin B12, vitamin B2, vitamin B6, vitamin E, vitamin C, Biotin, Pantothenates, vitamin K, vitamin D as well as derivatives and mixtures of these, as well as further minerals and trace elements such as iron, selenium, zinc, calcium, magnesium, and/or manganese. The term nutrient also denotes essential fatty acids, e.g.: Omega-3 fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), polyunsaturated omega-6 fatty acid such as Arachidonic acid.

According to the present invention it is advantageous if the amount of nutrients present in the ready-to-use food is sufficient to provide about 15 to 300 % of the RDA (Recommended Daily Allowance for an adult) in 1 g or one daily serving of the food. A typical ready-to-use food according to the present invention comprises the following ingredients with percentages expressed by weight based on the total weight of the ready-to-use food:

5 to 50 % of vegetable fats,

2 to 60 % of skimmed milk,

2 to 60 % of whey,

5 to 50 % of carbohydrate bulking agent,

0 to 15 % of sucrose,

0.05 to 10 % of emulsifier,

20 to 80 % of a lipid-rich oleaginous seed/ preferably as a paste,

0.002 % to 0.1 % phytase (at a specific activity of 20 000 FTU Ig).

Preferred composition of a ready-to-use food according to the present invention comprises the following ingredients with percentages expressed by weight based on the total weight of the ready-to-use food:

15 to 25 % of vegetable fats,

10 to 20 % of skimmed milk,

10 to 20 % of whey,

12 to 25 % of carbohydrate bulking agent,

6 to 8 % of sucrose,

0.05 to 3 % of emulsifier,

20 to 30 % of a lipid-rich oleaginous seed/ preferably as a paste,

0.05 % phytase (at a specific activity of 20 000 FTU Ig).

Carbohydrate bulking agents are understood as meaning hydrolysates of starch or starch derivatives which are assimilable by the organism, for example maltodextrin.

In another embodiment, the ready-to-use food of the present invention is characterised in that the residual phytase activity is at least 20 % of the initial activity after storage of the ready-to- use food supplemented with phytase for 1 year at room temperature. Preferably, the residual activity is at least 40 % of the initial activity.

The ready-to-use food of the present invention is not soluble in water, being a mixture consisting of pulverulent, granular or particulate phase which is anhydrous or has a very low water content, contained in a viscous lipid phase. Therefore, this mixture does not develop an appreciable osmotic pressure. The ready-to-use food of the present invention may be packed either in suitable single serving packages or in bigger, so called "multi serving packages".

The nutrients are usually added in a powdery form, even oily vitamins like vitamin A or vitamin E are preferably used as powdery product forms (e.g. as adsorbates, spray dried powders or beadlets which may contain further ingredients, like matrix components - e.g. hydrocolloids - antioxidants, plasticizers, and/or emulsifiers). Even more preferred are water-dispersible powdery product forms of these nutrients.

The efficacy of the composition of the present invention can be assessed as follows:

Stability of the Phytase activity in the ready-to-use food can be assessed using a method well established in the art (Animal feeding stuffs; Determination of phytase activity

ISO 30024:2009). Alternatively, indirect efficacy can be assessed by a mineral bioavailability study: absorption of iron can be assessed in vivo in humans using labeled Fe, and applying stable isotope measurement, allowing to assess iron absorption in human subjects as described in (B. Troesch et al., Am J Clin Nutr, 2009; 89: 539-44). This could be accompanied by a plasma response curve where the AUC (area under the curve) of the formulations with and without phytase is compared.

In yet another embodiment, the present invention provides the use of a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, at most 10 weight-% water, and enriched with phytase enzyme to deliver active phytase to humans. At least 5 FTU phytase units are delivered per serving, preferably, at least 20 FTU/serving, more preferably at least 50 FTU/serving, most preferably, at least 100 FTU/serving.

In a further embodiment, a ready-to-use food according to the present invention is used to correct deficiencies of mineral salts in undernourished humans.

The present invention therefore also provides a ready-to-use food containing at least 20 weight- % lipids, at least 0.05 weight-% lecithin, at most 10 weight-% water, and enriched with phytase enzyme for use in therapeutic delivery of active phytase to humans. At least 5 FTU phytase units are delivered per serving, preferably, at least 20 FTU/serving, more preferably at least 50 FTU/serving, most preferably, at least 100 FTU/serving. The present invention also provides a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, at most 10 weight-% water, and enriched with phytase enzyme for use in the prevention and/or treatment of deficiencies of mineral salts in undernourished humans.

In a further embodiment, the use of phytase in a ready-to-use food containing at least 20 weight-% lipids, at least 0.05 weight-% lecithin, and at most 10 weight-% water allows to reduce downtime of ready-to-use food production equipment when compared to a product not containing phytase, and also to reduce cleaning costs of the production equipment when compared to a RUF not containing phytase.

The invention is further illustrated by the following examples.

Examples

Example 1: Preparation of ready-to-use food

Ready-to-use food according to the present invention can be prepared according to a method established in the art and described in US 6,346,284.

Example 2: Composition of ready-to-use food

A composition of the present invention comprises the following elements:

Sucrose 25 %

Peanut butter 26 %

Skim milk powder 25 %

Oil 20 %

Lecithin 3 %

Phytase 20 000 FTU/g 200 FTU per serving

Total oil content 33 %

Estimated phytate 0.4 % Example 3: Stability of Phytase in Ready-to-use food 1- Material and Methods

Active and raw materials

Active: Phytase Euphovida 20000G was from DSM; It is a granulated form with an activity of 20Ό00 FTU/g.

Table 1 : Raw materials used for preparation of ready-to-use-therapeutic-foods (RUTF)

This RUTF containing 33 wt.-% lipids, 3 wt.-% lecithin, 0.4 wt.-% water and 0.05 wt.-% Phytase is later referred to as RUTF-1 .

Composition of a RUTF containing 1 1 % water is shown in Table 3 below: Table 3:

This RUTF containing 27 wt.-% lipids, 3 wt.-% lecithin, 1 1 .3 wt.-% water and 0.05 wt.-% Phytase is later referred to as RUTF-2. RUTF-3 consists in peanut butter as is + 0.05 % Euphovida 20000G

RUTF-4 consists in _© Nutella as is + 0.05 % Euphovida 20000G

Equipment

Heat-jacketed Multipurpose stirring vessel with electronically controlled heating / cooling device, connected to vacuum and Nitrogen

Balance

Packaging material

Laminated alu bags

Preparation of RUTF-1 according to the present invention

Heat-jacket of stirring vessel was heated to 45 °C. Peanut butter, peanut oil and lecithin were weighed in the vessel first, followed by powdered sugar and skim milk powder. The vessel was closed and vacuum was applied, followed by nitrogen flush. Stirring device was started, until visually homogeneous. Euphovida 20000G was added, and stirred for 20 min. the paste was filled immediately in laminated alu bags, followed by heat-sealing.

Preparation of RUTF-2 containing 1 1 % water

Heat-jacket of stirring vessel was heated to 45 °C. Peanut butter, peanut oil and lecithin were weighed in the vessel first, followed by powdered sugar and skim milk powder. The vessel was closed and vacuum was applied, followed by nitrogen flush. Stirring device was started, until visually homogeneous. Euphovida 20000G was added, and stirred for 20 min. Water was added slowly by very gently stirring. The paste was filled immediately in laminated alu bags, followed by heat-sealing.

Fortification of RUTF-3 (peanut butter as is)

Heat-jacket of stirring vessel was heated to 45 °C. Peanut butter was weighed in the vessel. The vessel was closed and vacuum was applied, followed by nitrogen flush. Stirring device was started, until visually homogeneous and smooth. Euphovida 20000G was added, and stirred for 20 min. The paste was filled immediately in laminated alu bags, followed by heat-sealing. Fortification of RUTF-4 (^nutella as is)

Heat-jacket of stirring vessel was heated to 45 °C. _© Nutella was weighed in the vessel. The vessel was closed and vacuum was applied, followed by nitrogen flush. Stirring device was started, until visually homogeneous and smooth. Euphovida 20000G was added, and stirred for 20 min. The paste was filled immediately in laminated alu bags, followed by heat-sealing.

2- Results

Stability trials were performed at three different temperatures: 4 °C as reference temperature, 30 °C and 40 °C as hot weather conditions.

Table 4: Retention of Phytase activity expressed in % of residual activity at different time points, following storage at 4 °C

Table 5: Retention of Phytase activity expressed in % of residual activity at different time points, following storage at 30 °C

Table 6: Retention of Phytase activity expressed in % of residual activity at different time points, following storage at 40°C

Table 4 shows Phytase activity retention in food matrices stored at reference temperature of 4°C. A steady slight decrease of Phytase activity during two months of storage was observed in the range of 7 to 10 % for the different matrices.

Table 5 shows Phytase activity retention in food matrices stored under warm conditions at 30 °C. RUTF-2 (containing 1 1 % water) showed 30 % reduction in Phytase activity compared to non-water containing RUTF-1 with only 10 % reduction, similar to peanut butter as is and ©Nutella as is.

Table 6 shows the Phytase retention in food matrices stored under hot climate conditions at 40 °C. An increase in storage temperature of 10 °C showed strong influence of Phytase activity in the food matrices. RUTF-1 showed 27 % reduction in Phytase activity after two months of storage time, RUFT-2 (containing 1 1 % of water) showed only 37 % retention of Phytase activity. 3- Conclusion

This data demonstrates that in food matrices rich in lipids according to the present invention (RUTF-1 , RUTF-3, and RUTF-4) phytase activity is significantly more stable under warm and hot climate storage conditions stability when compared to RUTF-2.