EA040098B1 - METHOD FOR OBTAINING NUTRIENTAL PRODUCT CONTAINING MILK PROTEINS AND MILK SUGARIDE - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to an improved process for the preparation of nutritional products, such as, for example, infant formula containing milk protein and milk saccharide. The present invention is particularly useful in the preparation of demineralized nutritional products and provides both the final nutritional product and milk saccharide-containing whey protein ingredients useful in the production of such nutritional products.

Background of the invention

Microfiltration (MF) has been found to be an effective means for milk protein fractionation, more specifically the separation of micellar casein and whey protein, which can be recombined to produce nutritional products with an optimized amino acid profile. Examples of such nutritional products are, for example, infant formula (optimized for infants 0-6 months of age), second level formulas (optimized for infants 6-12 months of age) and third level milk formulas (optimized for infants 12+ months of age) or diet food.

WO 2013/068653 discloses a process for fractionating skimmed milk using combinations of 1) skimmed milk MF; 2) ultrafiltration (UF) MF-permeate; 3) nanofiltration (NF) of UF-permeate; and 4) recombining UF retentate (whey protein) and NF retentate (lactose) and other ingredients to form an infant formula product.

WO 2013/137714 discloses a similar method using combinations of 1) skimmed milk MF using a concentration factor by volume of 4-8; 2) ultrafiltration (UF) of the MF permeate using a concentration factor by volume of 3-7; and 3) combining MF-retentate and UF-retentate, thus obtaining a composition with a casein/whey weight ratio of 30/70-50/50.

FR 2809595 discloses a milk derivative containing the following components: total protein 10-80%, minerals 1-5%, sodium 0.02-0.4%, potassium 0.1-1.5%, threonine less than 6 g per 100 g of all amino acids and tryptophan at least 1.8 g per 100 g of amino acids. The milk derivative is produced by a method comprising (a) obtaining a soluble milk phase; (b) selective demineralization of the soluble phase by nanofiltration; and (c) obtaining a milk derivative from the precipitate separated by nanofiltration.

US 2016/044933 A1 relates to a process for treating skimmed milk of animal origin and sweet whey and/or acid whey, comprising the following steps (a), (b) and (c). Step (a) consists in ultrafiltration (UF1) of a first liquid composition containing skimmed milk of animal origin with 70-90% by weight of casein and 10-30% by weight of whey proteins, based on total protein, through a first ultrafiltration membrane with a cut-off by molecular weight of 2.5-25 kDa using a concentration factor by volume of 1.5-6 to obtain retentate (UFR1) and permeate (UFP1). Step (b) consists in ultrafiltration (UF2) of the second liquid composition containing sweet whey and/or acid whey through a second ultrafiltration membrane with a molecular weight cut-off of 2.5-25 kDa using a concentration factor by volume of 2 -15, to obtain retentate (UFR2) and permeate (UFP2). Step (c) consists in mixing the UF retentate obtained in step (a) with the UF retentate obtained in step (b) to obtain a mixture of UF retentates.

Brief description of the invention

The present inventors have observed indications that prior art infant formula bases such as those from WO 2013/068653 and WO 2013/137714 provide unintended fluctuations in the bioavailability of minerals that are provided as di- or trivalent metal ions.

The present inventors have found that the citrate content of the prior art infant formula bases of WO 2013/068653 and WO 2013/137714 is high and furthermore subject to significant fluctuations, for example due to the influence of seasonal changes, general stage of lactation and changes in the type of feed that is supplied to cows.

In addition, the present inventors have realized that changes in citrate content are a problem when formulating final infant formulas that are expected to be nutritionally complete and that provide well-defined and highly controlled amounts of macronutrients (proteins, fats and carbohydrates) to infants. ) and micronutrients (e.g. vitamins and minerals).

The concentration of citrate in infant formula has been shown to affect the bioavailability of important minerals such as, for example, iron, calcium, magnesium, and zinc (see, for example, Glahn et al. and Fairweather-Tait et al.). Altering the amount of citrate in infant formula products will thus lead to inadvertent changes in the bioavailability of minerals that are provided as divalent or trivalent metal ions.

The present inventors have found that the problem can be solved by using electrodialysis (ED) to remove polyvalent ions, and preferably ED is performed

- 1 040098 with the possibility of ensuring the removal of both mono-, di-, and trivalent anions.

Thus, one aspect of the present invention relates to a method for producing a nutritional product, the method comprising the steps

a) providing milk raw materials;

b) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining micellar casein enriched MF retentate and whey protein enriched MF permeate;

c) subjecting the MF permeate to nanofiltration (NF) or nanofiltration/diafiltration using a membrane that allows passage of monovalent ions but retains milk saccharide, thereby obtaining NF retentate and NF permeate;

d) subjecting the NF retentate to electrodialysis, thereby obtaining a demineralized whey protein product containing milk saccharide, which is characterized by a reduced level of calcium, magnesium and phosphorus;

e) adding a casein source and optionally one or more additional ingredients to a demineralized whey protein product containing milk saccharide to form a nutritional product; And

f) optionally converting the nutritional product into a powder.

The present inventors have also found that the above process greatly simplifies the preparation of the milk saccharide-containing whey protein nutritional product and/or demineralized product from step d) while still providing the level of mineral reduction required, for example, for infant formula products. In particular, the present invention does not require the use of ultrafiltration of the whey protein containing streams after step b) and the demineralization is carried out directly in the whey protein containing streams.

Another advantage of the method of the present invention is that it uses a very high level of whey protein and milk saccharide in the milk raw material and a very high proportion of both whey protein and milk saccharide end up in the nutritional product. This is an advantage, for example, in the production of organic nutritional products, such as, for example, organic infant formula, since purified organic ingredients, such as organic lactose, are difficult to find commercially and/or are very expensive. If organic milk raw materials are used, the amount of whey protein and milk saccharide in organic milk raw materials is usually sufficient to provide at least 90% wt./weight. whey protein and at least 90% wt./weight. carbohydrate for some nutritious foods, such as infant formula.

Another aspect of the present invention relates to a method for producing a demineralized whey protein product containing milk saccharide, the method comprising the steps

i) provision of milk raw materials;

ii) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining an MF retentate and an MF permeate;

iii) subjecting the MF permeate to nanofiltration or nanofiltration/diafiltration, thereby obtaining NF retentate and NF permeate;

iv) subjecting the NF retentate to electrodialysis, thereby obtaining a demineralized whey protein product containing milk saccharide;

v) optionally drying the demineralized milk saccharide-containing whey protein product containing milk saccharide.

A further aspect of the present invention relates to a method for preparing a demineralized whey protein product or a whey protein product containing milk saccharide, the method comprising the steps

1) providing a liquid protein source containing whey protein or whey protein;

2) subjecting the liquid protein source to an inorganic multivalent ion reduction procedure, the reduction procedure comprising adjusting the liquid protein source to a pH of at least 6, heating it to a temperature of at least 30° C., and separating the resulting precipitate from the liquid protein source thereby obtaining a demineralized whey protein product or a whey protein product containing milk saccharide;

3) optionally drying the demineralized whey protein product or whey protein product containing milk saccharide.

Another aspect of the present invention relates to a nutritional product, for example, obtained using the method described in this document, containing proteins, milk saccharide, fats and minerals and providing

- 2 040098 total carbohydrates in the range of 40-55% (w/w total solids);

the total amount of proteins in the total in the range of 9-14% (wt./wt. all solids);

total milk saccharide in the range of 40-55% (w/w total solids);

the weight ratio of whey protein and casein in the range of 50:50-70:30, preferably in the range of 55:45-65:45 and even more preferably about 60:40;

the total amount of calcium is not more than 0.7% (wt./wt. of all solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.5% (wt./wt. of all solids);

not more than 0.3% total sodium (w/w total solids);

the total amount of potassium is not more than 0.8% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

In the context of the present invention, the term chlorine refers to elemental chlorine and total chlorine refers to the total amount of elemental chlorine in any form.

In another aspect, this invention relates to a demineralized whey protein product containing milk saccharide, for example, obtained using the method described herein, providing for a total amount of lactose in the range of 65-85% (wt./wt. all solid substances);

a total amount of whey protein and whey protein in the range of 10-25% (w/w total solids);

a weight ratio of whey protein and micellar casein of at least 95:5;

the total amount of calcium is not more than 1.0% (wt./wt. of all solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.8% (wt./wt. of all solids);

not more than 0.4% total sodium (w/w total solids);

the total amount of potassium is not more than 1.3% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

Brief description of graphic materials

In FIG. 1 describes a variant of the method of the present invention for producing a casein-containing nutritional product, such as, for example, infant formula.

In FIG. 2 describes another embodiment of the method of the present invention for producing a casein-free whey protein ingredient suitable for the production of, for example, infant formula.

Detailed description of the invention

Thus, a broad aspect of the present invention relates to a method for producing a nutritional product, the method comprising the steps

a) providing raw milk;

b) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining micellar casein enriched MF retentate and whey protein enriched MF permeate;

c) subjecting the MF permeate to nanofiltration or nanofiltration/diafiltration using a membrane that allows passage of monovalent ions but retains milk saccharide and whey protein, thereby obtaining nanofiltration NF retentate and NF permeate;

d) subjecting the NF retentate to an inorganic multivalent ion reduction procedure, thereby obtaining a demineralized whey protein product containing milk saccharide, which is characterized by reduced levels of calcium, magnesium and phosphorus;

e) adding a casein source and optionally one or more additional ingredients to a demineralized whey protein product containing milk saccharide to form a nutritional product; And

f) optionally converting the nutritional product into a powder.

The inventors of the present invention have found that prior art methods for producing nutritious milk protein containing products with reduced mineral content, such as, for example, infant formulas, based on fractionated milk proteins can be significantly improved by the method of the present invention, wherein the whey protein milk is not separated from milk saccharides after the protein fractionation step, in contrast to e.g.

It is particularly preferred that step d) comprises or even consists of an electrodialysis step.

Thus, a preferred aspect of the present invention relates to a method for producing a nutritional product, the method comprising the steps

a) providing raw milk;

- 3 040098

b) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining micellar casein enriched MF retentate and whey protein enriched MF permeate;

c) subjecting the MF permeate to nanofiltration (NF) or nanofiltration/diafiltration using a membrane that allows passage of monovalent ions but retains milk saccharide, thereby obtaining NF retentate and NF permeate;

d) subjecting the NF retentate to electrodialysis, thereby obtaining a demineralized whey protein product containing milk saccharide, which is characterized by a reduced level of calcium, magnesium and phosphorus;

e) adding a casein source and optionally one or more additional ingredients to a demineralized whey protein product containing milk saccharide to form a nutritional product; And

f) optionally converting the nutritional product into a powder.

In the context of the present invention, the term nutritional product refers to an edible product that contains at least protein and carbohydrate and optionally also lipids. The nutritional product may, for example, be an infant nutritional product such as, for example, infant formula, second level milk formula, or third level milk formula. The nutritional product may be nutritionally complete for the intended consumer, such as a 0-6 month old infant or a 6-12 month old infant, or it may be a nutritional supplement.

The nutritional product may be in the form of a liquid product, a concentrated liquid product, a paste, or a powder.

Another advantage of the present invention is that it reduces the energy required per kg of processed milk raw material or per kg of a whey protein composition containing milk saccharide.

The present invention further provides a lower consumption of milk raw materials per kg of nutritional product or per kg of demineralized product based on milk whey protein containing milk saccharide than in the prior art, especially if lactose crystallization is used in the prior art to purify lactose obtained from MF- or UF permeate.

In some preferred embodiments of the present invention, the nutritional product is a liquid. A liquid nutritional product is often considered more convenient to use than powdered nutritional products and is ready for consumption.

In the case of a liquid nutritional product, the nutritional product contains water in an amount of at least 75% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product is not more than 25% wt./weight. relative to the weight of the nutrient. For example, a liquid nutritional product may contain water in an amount of at least 85% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product can be no more than 15% wt./weight. relative to the weight of the nutrient.

In still other preferred embodiments of the present invention, the nutritional product is a concentrated nutritional product.

In the case of a concentrated nutritional product, the nutritional product contains water in an amount in the range of 30-74% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product is usually in the range of 26-70% wt./weight. relative to the weight of the nutrient. For example, a concentrated nutritional product may contain water in an amount in the range of 40-60% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product may be in the range of 40-60% wt./weight. relative to the weight of the nutrient.

The values for water content and total solids content of the nutritional product are determined according to NMKL 110 2nd Edition, 2005 (Total solids (Water) - Gravimetric determination in milk and milk products). NMKL is short for Nordisk Metodikkomite for Nsringsmidler (Scandinavian Food Analysis Committee).

In still other preferred embodiments of the present invention, the nutritional product is a spreadable nutritional product.

In the case of a pasty nutritional product, the nutritional product contains water in an amount in the range of 7-29% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product is usually in the range of 71-93% wt./weight. relative to the weight of the nutrient. For example, the spreadable nutritional product may contain water in an amount in the range of 15-25% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product may be in the range of 75-85% wt./weight. relative to the weight of the nutrient.

In additional preferred embodiments of the present invention, the nutritional product is a powdered nutritional product.

- 4 040098

In the case of a powdered nutritional product, the nutritional product contains water in an amount in the range of 1-6% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product is usually in the range of 94-99% wt./weight. relative to the weight of the nutrient. For example, the powdered nutritional product may contain water in an amount in the range of 2-4% w/w. relative to the weight of the nutritional product and the solids content of the nutritional product may be in the range of 96-98% wt./weight. relative to the weight of the nutrient.

The method is preferably carried out in the sequence a), b), c), d) and e) or, if the nutritional product is powdered, in the sequence a), b), c), d), e) and f).

A schematic example of steps a) to f) of the method is shown in FIG. 1. In this case, the raw milk is subjected to microfiltration, which provides a permeate (P), containing mainly whey protein, milk saccharide, water and minerals, and a retentate (R), containing mainly casein micelles, water and additionally small amounts of protein whey milk, milk saccharide and minerals. The permeate is subjected to nanofiltration to provide NF-permeate (P) containing monovalent ions and water, and NF-retentate (R) containing whey protein, milk saccharide, water and other minerals. The NF retentate is subjected to a polyvalent inorganic ion reduction procedure, for example by mineral precipitation or electrodialysis, and a mineral-containing precipitate and a milk-saccharide-containing milk whey protein with a reduced amount of minerals are obtained. In this example, the milk saccharide mainly provides for lactose. The milk saccharide-containing, mineral-reduced whey protein is then mixed with a casein source, such as, for example, skim milk, and the mixture is converted into a powdered nutritional product by spray drying.

In some preferred embodiments of the present invention, the nutritional product contains at least 60% of the milk saccharide present in the milk raw material. Preferably, the nutritional product contains at least 70% of the milk saccharide present in the milk raw material. Even more preferably, the nutritional product contains at least 80% of the milk saccharide present in the milk raw material.

In other preferred embodiments of the present invention, the nutritional product contains at least 80% of the milk saccharide present in the milk raw material. Preferably, the nutritional product contains at least 90% of the milk saccharide present in the milk raw material. Even more preferably, the nutritional product contains at least 95% of the milk saccharide present in the milk raw material.

It is particularly preferred that the nutritional product contains at least 97% of the milk saccharide present in the milk raw material.

In the context of the present invention, the term milk saccharide refers to a) lactose, also sometimes referred to as milk sugar, b) glucose and galactose, which are monosaccharides, reaction products obtained from the hydrolysis of lactose, and c) galactooligosaccharides (GOS), which can, for example, obtained by hydrolysis of lactose by some enzymes - beta-galactosidases.

Dairy feedstocks and streams containing milk saccharide also typically contain milk oligosaccharides in addition to milk saccharide, and the advantage of the present process is that less milk oligosaccharides are lost during processing than prior art processes that use lactose crystallization to purify lactose. .

GOS may be present in the raw milk initially, or they may be obtained during the processing of the milk raw material and/or subsequent product streams. GOS is the result of transgalactosylation of lactose, glucose and galactose and usually contains mainly disaccharides, trisaccharides and tetrasaccharides.

Thus, total milk saccharides refers to total galactose, glucose, lactose, and GOS (up to and including GOS tetrasaccharides). The total amount of milk saccharide is measured according to example 1.

The milk raw material that is provided in step a) is a liquid raw material that is subjected to a microfiltration step in which casein micelles and whey protein are separated.

The content of milk saccharides can vary over a wide range depending on, for example, seasonal changes in the milk source and the pre-treatment to which the milk source was subjected to when receiving milk raw materials.

In some preferred embodiments of the present invention, the dairy raw material provides for a total amount of milk saccharides in the range of 0.5-10% wt./weight. Preferably, the dairy raw material provides for a total amount of milk saccharides in the range of 2-7% w/w. Even more preferably, the dairy raw material provides for a total amount of milk saccharides in the range of 2-6%.

In other preferred embodiments of the present invention, the dairy raw material provides for a total amount of milk saccharides in the range of 0.5-7% wt./weight. Preferably mo

- 5 040098 local raw materials provide for the total amount of milk saccharides in the range of 1.0-5% wt./weight. For example, raw milk provides for a total amount of milk saccharides in the range of 1.0-3%.

In additional preferred embodiments of the present invention, the dairy raw material provides for a total amount of milk saccharides in the range of 4-10% wt./weight. Preferably, the dairy raw material provides for a total amount of milk saccharides in the range of 5-9% w/w. For example, raw milk provides for a total amount of milk saccharides in the range of 6-8%.

In a number of preferred embodiments of the present invention, the dairy raw material provides for a total amount of lactose in the range of 0.5-10% wt./weight. Preferably, the dairy raw material provides for a total amount of lactose in the range of 2-7% w/w. Even more preferably, the dairy raw material provides for a total amount of lactose in the range of 2-6%.

In other preferred embodiments of the present invention, the dairy raw material provides for a total amount of lactose in the range of 0.5-7% wt./weight. Preferably, the raw milk contains a total amount of lactose in the range of 1.0-5% w/w. For example, raw milk provides for a total amount of lactose in the range of 1.0-3%.

In additional preferred embodiments of the present invention, the dairy raw material provides for a total amount of lactose in the range of 4-10% wt./weight. Preferably, the raw milk contains a total amount of lactose in the range of 5-9% w/w. For example, dairy raw materials provide for a total amount of lactose in the range of 6-8%.

Although other carbohydrates can be added to the milk raw material, it is preferred that the carbohydrate in the milk raw material comprises at least 95% milk saccharide, and even more preferably consists mainly of milk saccharide.

Milk raw materials contain, in addition to milk saccharide, milk protein, including both casein and whey protein.

Casein in milk raw materials is mainly present in the form of casein micelles, similar or even identical to casein micelles found, for example, in skim milk.

The term whey refers to the liquid phase of milk in which casein micelles and milk fat globules are dispersed.

In the context of the present invention, the terms whey protein or whey protein refers to proteins found in milk whey. Milk whey proteins typically include beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin, immunoglobulin and osteopontin, as well as lactoferrin and lactoperoxidase. Whey protein may additionally contain a significant amount of beta-casein if the milk raw material was stored at a low temperature without subsequent heat treatment before the protein fractionation step by microfiltration.

The term protein refers to polypeptides containing at least 10 amino acids and encompasses both individual polypeptides and aggregates of polypeptides.

In some preferred embodiments of the present invention, the whey protein in the raw milk is in an undenatured, native form, i. the same form as in raw milk that has not been subjected to denaturing heat treatment. Thus, it is also preferred that the milk raw material and the product stream from which the milk raw material has been derived are not subjected to conditions that result in significant protein denaturation, such as, for example, high temperature for a long time. In the context of the present invention, the term significant protein denaturation means that the degree of denaturation of beta-lactoglobulin is at least 10% w/w. The degree of denaturation of beta-lactoglobulin is measured according to the method described in WO 2012/010699.

The term non-protein nitrogen (NPN) refers to nitrogen found in molecules that are not protein. In milk, a significant proportion of NPNs are urea, ammonium salts, and small peptides containing less than 10 amino acids.

The term whey refers to the liquid that remains in the liquid phase when the casein is precipitated from milk through, for example, acidification and/or protein degradation (eg, using rennet in cheese production). Whey obtained by rennet precipitation of casein is commonly referred to as sweet whey, while whey obtained by acid precipitation of casein is commonly referred to as acid whey, acidified whey, or casein whey.

The term whey protein refers to proteins found in whey. Whey proteins typically include beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin and immunoglobulin, lactoferrin, lactoperoxidase, and globular membrane protein from milk fat. In addition, the whey protein found in sweet whey usually also contains a casein macropeptide. The term whey protein includes, of course, also whey protein.

In some embodiments, the implementation of the present invention, the raw milk provides a total amount of proteins in the range of 1-12% wt./weight. Preferably, the raw milk provides

- 6 040098 the total amount of proteins in the range of 2-10% wt./weight. Even more preferably, the raw milk contains a total protein content in the range of 3-8%.

The milk raw material is preferably characterized by a weight ratio of casein to whey protein in the range of 70:30-90:10, such as, for example, in the range of 75:25-85:15 and usually in the range of 77:23-83:17.

While the fat content of milk may vary, it is often preferred that it be less than about 6%. In some embodiments, the implementation of the present invention, the raw milk provides a total amount of fat in the range of 0.01-5% wt./weight. Preferably, the raw milk provides for a total fat content in the range of 0.01-2% w/w. Even more preferably, the raw milk provides for a total fat content in the range of 0.01-0.5% w/w.

The solids content of raw milk may vary depending on the raw material used, but is usually in the range of 1-30% wt./weight. Preferably, the solids content of the milk raw material is in the range of 4-25% w/w. Even more preferably, the solids content of the milk raw material is in the range of 5-15% w/w.

A number of different types of raw materials can be used in this invention. For example, the dairy raw material may, for example, contain whole milk, skim milk, fat-free milk, low-fat milk, full-fat milk, and concentrated milk, or even be composed of them.

The term concentrated milk refers to milk that has been concentrated by evaporation or ultrafiltration, nanofiltration and/or reverse osmosis. It is particularly preferred that the condensed milk is concentrated non-evaporated milk, i. e. milk, which was concentrated by filtration.

The pH of the milk raw material is preferably at least 5.0, and usually the pH of the milk raw material is similar to that of skimmed milk, i. e. in the range of 6.1-6.8 when measured at 25°C.

Dairy raw materials are usually obtained from the milk of ruminants, such as, for example, cows, sheep, goats, buffaloes, camels, reindeer and/or llamas. According to the present invention, dairy raw materials obtained from cows are the most preferred.

Dairy raw materials usually contain at least some amount of citrate. Dairy raw materials may, for example, contain citrate in an amount in the range of 0.01-1.0% wt./weight. relative to the weight of the raw milk, preferably in the range of 0.05-0.5% wt./weight. and even more preferably in the range

0.1-0.4% w/w relative to the weight of raw milk.

The present invention is particularly suitable for processing organic milk into organic nutritional products. In some embodiments of the present invention, the dairy raw material is thus an organic dairy raw material derived from an organic milk source. In some particularly preferred embodiments of the present invention, the dairy raw material is organic skimmed milk or organic concentrated skimmed milk.

In the context of the present invention, the term organic milk refers to the milk produced by cattle reared according to the following. Cattle must have free access to certified organic pastures during the entire grazing period. This period is specific to the geographic climate of the farm, but should be at least 120 days per year and preferably at least 150 days. Due to the weather, season or climate, the grazing period may or may not be continuous. Organic cattle diets must contain at least 30% dry matter (on average) from certified organic pasture. Dry matter intake (DMI) is the amount of feed that an animal consumes per day, excluding moisture. The rest of the diet must also be certified organic, including hay, grains, and other agricultural products. Livestock should be raised free of antibiotics, added growth hormones, mammalian or avian by-products, or other prohibited feed components (eg, urea or arsenic compounds).

In some preferred embodiments of the present invention, providing the milk raw material in step a) includes an ultrafiltration (UF) and optional UF/diafiltration step of the milk source, thereby providing

UF milk retentate;

UF-permeate of milk, while at least part of the UF-retentate of milk is used as a dairy raw material.

In some embodiments of the present invention, a portion of the milk UF retentate is used as a raw milk material and at least a portion of the remaining milk UF retentate is used as a casein source in step e). For example, part of the milk UF-retentate can be used as a raw milk material, and all of the remaining milk UF-retentate can be used as a casein source in step e).

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The milk UF retentate can be used as a dairy raw material as such, or it can be diluted with water, NF permeate and/or RO permeate before being used as a dairy raw material.

The concentration ratio by volume is preferably kept fairly low and is typically at most 3, preferably at most 2.5, and even more preferably at most 2.0. In some preferred embodiments of the present invention, the UF treatment of the milk source does not involve diafiltration.

Suitable UF membranes allow passage of lactose and small peptides but retain whey protein.

The lactose in the UF milk permeate can, for example, be purified to a concentration of at least 90% w/w. lactose relative to dry matter, preferably at least 95% wt./weight. lactose.

Techniques for purifying lactose from protein-free UF-permeate milk/whey streams are well known in the art and may, for example, include lactose crystallization and demineralization (see, for example, Membrane filtration and related molecular separation technologies published by APV Systems, 2o0o , ISBN 87-88016757).

In step b), the raw milk is microfiltered (MF) or microfiltered/diafiltered (MF/DIA) using a type of membrane that permits passage of whey proteins, milk saccharides and minerals but retains casein micelles. Thus step b) provides an MF retentate enriched with micellar casein and an MF permeate enriched with whey protein, lactose and minerals.

The term microfiltration/diafiltration includes the dilution of the milk raw material and/or one or more intermediate retentate streams during the microfiltration process to increase the degree of removal of whey protein and milk saccharide from the retentate stream.

Suitable, but non-limiting, examples of diluents that can be used for MF/DIA dilution are tap water, demineralized water, reverse osmosis (RO) permeate, NF-permeate from step c). The RO-permeate may, for example, be the RO-permeate obtained by RO-treating the NF-permeate from step c).

As will be apparent to those skilled in the art, the MF or MF/DIA in step b) can be carried out using a single MF membrane or using multiple MF membranes, for example in series. In the case of MF/DIA in step b), the same MF membrane can be used several times.

The terms MF-retentate and MF-permeate refer to the final retentate and combined permeates obtained from MF or MF/DIA.

The MF or MF/DIA in step b) can be performed over a wide range of VCFs. In some embodiments, implementation of the present invention, the concentration factor by volume (VCF) MF or MF/DIA in stage b) is in the range of 0.3-5. Preferably the VCF for MF or MF/DIA in step b) is in the range 0.5-4. Even more preferably, the VCF for MF or MF/DIA in step b) is in the range of 0.5-3.

VCF is calculated by dividing the volume of raw material by the volume of retentate.

MF or MF/DIA is preferably carried out to obtain a total protein concentration in the MF retentate of not more than 15% wt./weight. relative to the total weight of the MF-retentate, preferably not more than 12% wt./weight. and even more preferably not more than 10% wt./weight. The total protein concentration in the MF retentate is typically in the range of 1-15% w/w. relative to the total weight of the MF-retentate, preferably in the range of 3-12% wt./weight. and even more preferably in the range of 5-10% wt./weight. relative to the total weight of the MF retentate.

In some embodiments of the present invention, the temperature of the milk raw material during MF or MF/DIA in step b) is in the range of 1-66°C. Preferably, the temperature of the milk raw material during MF or MF/DIA in step b) is in the range of 45-66°C. Even more preferably, the temperature of the milk raw material during MF or MF/DIA in step b) is in the range of 55-66°C.

In some preferred embodiments of the present invention, the temperature of the milk raw material during MF or MF/DIA in step b) is in the range of 45-55°C.

In some preferred embodiments of the present invention, the temperature of the milk raw material during MF or MF/DIA in step b) is in the range of 1-20°C and even more preferably in the range of 4-15°C, such as, for example, 5-10° C. The low temperature at MF or MF/DIA is particularly useful when the milk raw material is stored at no more than 10° C. for at least 0.5 hour immediately prior to MF or MF/DIA. Preferably, the raw milk is stored at no more than 5° C. for at least 1 hour immediately prior to MF or MF/DIA. The cooled milk raw material is preferably transferred to the MF or MF/DIA without heating it to a temperature above 30°C, preferably without heating it to a temperature above 20°C and even more preferably without heating it to a temperature above 15°C.

Beta-casein dissociates from casein micelles at low temperatures and associates into casein micelles.

- 8 040098 zein at a higher temperature. When raw milk is stored at a low temperature, therefore, more beta-casein is released from the casein micelles into the milk whey. When the chilled milk raw material is fractionated with MF or MF/DIA at low temperature, the dissociated beta-casein is transferred to the MF permeate. Beta-casein is believed to be an attractive alternative to casein micelles in infant formulas, as human breast milk is composed primarily of beta-casein and whey protein. The use of an increased amount of beta-casein in infant formula relative to total casein thus makes infant formula products closer to human milk.

The transmembrane pressure (TMP) used for MF or MF/DIA is typically in the range of 0.1-5 bar, preferably 0.2-2 bar and even more preferably in the range of 0.3-1, such as for example 0 .3-0.8 bar.

As stated above, the MF membrane(s) used for MF or MF/DIA must be capable of retaining casein micelles while allowing whey protein, lactose and minerals to pass through it(s).

The pore size of MF membrane(s) is typically in the range of 0.01-1.0 microns. Preferably, the pore size of the MF membrane(s) is in the range of 0.05-0.8 microns. Even more preferably, the pore size of the MF membrane(s) is in the range of 0.1-0.5 microns.

If the filtration performance of the MF membrane(s) is provided relative to a molecular weight cutoff, the MF membrane(s) typically has(are) characterized by a molecular weight cutoff in the range of 200-2000 kDa. Preferably, the MF membrane(s) is/are characterized by a molecular weight cut-off in the range of 300-1500 kDa. Even more preferably, the MF membrane(s) is/are characterized by a molecular weight cut-off in the range of 400-1000 kDa.

The MF membrane(s) may, for example, be polymeric membranes or ceramic membranes.

Non-limiting examples of suitable membranes are, for example, ceramic membranes with a pore size of about 0.14 microns (Inside Ceram™, Tami Industries, Nyon, France) or polymeric FR membranes with a molecular weight cut-off of about 800 kDa (PVDF 800 kDa; from Synder Filtration, USA).

It is particularly preferred that a significant amount of the milk saccharide in the milk raw material is transferred to the MF permeate.

In some preferred embodiments of the present invention, MF or MF/DIA is used sufficiently to transfer at least 80% wt./weight. from the total amount of milk saccharide in raw milk into MF-permeate. Preferably MF or MF/DIA is used sufficiently to transfer at least 90% wt./weight. from the total amount of milk saccharide in raw milk into MF-permeate. Even more preferably, MF or MF/DIA is used sufficiently to transfer at least 95% wt./weight. from the total amount of milk saccharide in raw milk into MF-permeate. Most preferably, MF or MF/DIA is used sufficiently to transfer at least 98% wt./weight. from the total amount of milk saccharide in the raw milk in the MF-permeate, for example, about 100% wt./weight.

In some preferred embodiments of the present invention, MF or MF/DIA is used sufficiently to transfer at least 80% wt./weight. from the total amount of lactose in raw milk into MF-permeate. Preferably MF or MF/DIA is used sufficiently to transfer at least 90% wt./weight. from the total amount of lactose in raw milk into MF-permeate. Even more preferably, MF or MF/DIA is used sufficiently to transfer at least 95% wt./weight. from the total amount of lactose in raw milk into MF-permeate. Most preferably, MF or MF/DIA is used sufficiently to transfer at least 98% wt./weight. from the total amount of lactose in raw milk in MF-permeate, for example, about 100% wt./weight.

In addition, it is preferable that a significant amount of whey protein in the milk raw material is transferred to the MF-permeate.

In some preferred embodiments of the present invention, MF or MF/DIA is used sufficiently to transfer at least 50% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate. Preferably MF or MF/DIA is used sufficiently to transfer at least 60% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate. Even more preferably, MF or MF/DIA is used sufficiently to transfer at least 70% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate. For example, MF or MF/DIA can be used sufficiently to transfer at least 80% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate. Alternatively, MF or MF/DIA can be used sufficiently to transfer at least 90% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate. In some preferred embodiments of the present invention

- 9 040098

MF or MF/DIA is used sufficiently to transfer at least 95% wt./weight. from the total amount of whey protein in milk raw materials in MF-permeate.

More information regarding the implementation of MF or MF/DIA can be found in Dairy processing Handbook, 2015, (ISBN 978-9176111321) and Membrane filtration and related molecular separation technologies, Werner Kofod Nielsen, APV Systems, 2000, ISBN 87-88016757, which are included herein by reference for all purposes.

Step c) involves subjecting the MF permeate to nanofiltration (NF) or nanofiltration/diafiltration (NF/DIA) using a membrane that allows the passage of monovalent ions but retains at least lactose and whey protein, thereby obtaining a nanofiltration (NF) retentate and NF-permeate. Thus, NF or NF/DIA provide an efficient way to both remove monovalent ions and small NPN molecules such as urea, for example, from MF permeate and optionally also concentrate the MF permeate.

The term nanofiltration/diafiltration refers to the dilution of the MF permeate and/or one or more intermediate retentate streams during the nanofiltration process to increase the degree of removal of monovalent ions from the retentate stream.

Suitable, but non-limiting, examples of diluents that can be used to dilute at NF/DIA are tap water, demineralized water, reverse osmosis (RO) permeate. The RO-permeate may, for example, be the RO-permeate obtained by RO-treating the NF-permeate from step c).

As will be apparent to those skilled in the art, the NF or NF/DIA in step c) can be carried out using a single NF membrane or using multiple NF membranes, for example in series. In the case of NF/DIA in step c), the same NF membrane can be used several times.

The terms NF-retentate and NF-permeate refer to the final retentate and combined permeates obtained from NF or NF/DIA.

The MF permeate may be subjected immediately to NF or NF/DIA, or it may be subjected to treatment steps to NF or NF/DIA that do not significantly alter the solids composition of the MF permeate. Such processing steps may, for example, be pasteurization, antibacterial microfiltration, bactofugation, dilution or concentration.

Similar to microfiltration, one skilled in the art can find guidance for performing nanofiltration or nanofiltration/diafiltration in Dairy processing Handbook, 2015 (ISBN 978-9176111321) and Membrane filtration and related molecular separation technologies, Werner Kofod Nielsen, APV Systems, 2000, ISBN 87-88016757, which are incorporated herein by reference for all purposes.

VCF for NF or NF/DIA depends on the actual implementation of the method. The VCF for NF or NF/DIA is often, but not necessarily always, in the range of 0.1-30, such as, for example, in the range of 0.5-20 or in the range of 1-15.

The temperature of the milk raw material during NF/DIA is usually in the range of 1-20°C, preferably in the range of 2-18°C and even more preferably in the range of 5-15°C.

The transmembrane pressure used for NF or NF/DIA depends on the actual implementation of the process and the membrane(s) used. The transmembrane pressure is often in the range of 5-35 bar and preferably in the range of 15-22 bar.

The NF membrane used for NF or NF/DIA may be any type of NF membrane that is suitable for separating lactose and optionally also glucose and galactose from monovalent salts. Suitable examples of such membranes are NF245 membrane (DOW Filmtec, USA) or DL membrane (GE Water, USA).

The NF membrane(s) used are typically characterized by a lactose retention of at least 80% and preferably at least 90% and a Na, K and Cl retention of no more than about 50%. Suitable NF membranes often have a molecular weight cut-off in the range of 150-500 Daltons and preferably in the range of 150-300 Daltons.

In some embodiments of the present invention, at least some of the NF-permeate is used as a diafiltration diluent during MF/DIA in step b).

In some preferred embodiments of the present invention, NF or NF/DIA is used sufficiently to remove at least 50% mol./mol. each of sodium, potassium and chlorine from MF-permeate. Preferably NF or NF/DIA is used sufficiently to remove at least 60% mol./mol. each of sodium, potassium and chlorine from MF-permeate. Even more preferably, NF or NF/DIA is used sufficiently to remove at least 70% mol./mol. each of sodium, potassium and chlorine from MF-permeate.

For example, NF or NF/DIA can be used sufficiently to remove at least 80% mol./mol. each of sodium, potassium and chlorine from MF-permeate. NF or NF/DIA can, for example, be used sufficiently to remove at least 85% mol./mol. each of sodium, potassium and chlorine from MF-permeate. Alternatively, NF or NF/DIA can be applied sufficiently

- 10 040098 to remove at least 90% mol./mol. each of sodium, potassium and chlorine from MF-permeate.

Na, K, Ca, Mg, Cl and P concentrations are measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES).

In some preferred embodiments, the implementation of the present invention NF-retentate provides for a total amount of sodium of not more than 0.4% (wt./wt. all solids);

the total amount of potassium is not more than 1.3% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

Preferably the NF-retentate has a total sodium content of not more than 0.4% (w/w total solids);

the total amount of potassium is not more than 1.1% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.7% (wt./wt. of all solids).

For example, the NF retentate may have a total sodium of no more than 0.2% (w/w total solids);

the total amount of potassium is not more than 0.5% (wt./wt. all solids); and the total amount of chlorine is not more than 0.4% (wt./wt. of all solids).

In some preferred embodiments, the implementation of the present invention NF-retentate provides a total amount of sodium in the range of 0.01-0.4% (wt./wt. all solids);

total potassium in the range of 0.01-1.3% (w/w total solids); and total chlorine in the range of 0.01-0.8% (w/w total solids).

Preferably the NF-retentate contains a total amount of sodium in the range of 0.05-0.4% (w/w total solids);

total potassium in the range of 0.05-1.1% (w/w total solids); and total chlorine in the range of 0.02-0.7% (w/w total solids).

For example, the NF retentate provides for a total sodium in the range of 0.05-0.2% (w/w total solids);

total potassium in the range of 0.1-0.5% (w/w total solids); and total chlorine in the range of 0.02-0.4% (w/w total solids).

In step d), the NF-retentate is subjected to a procedure for reducing the amount of inorganic multivalent ions, thereby obtaining a demineralized whey protein product containing milk saccharide, which is characterized by a reduced level of calcium, magnesium and phosphorus relative to the NF-retentate.

In the context of the present invention, the term polyvalent inorganic ion reduction procedure refers to a procedure for reducing the amount of divalent cations Ca ²⁺ , Mg ²⁺ and inorganic molecules containing phosphorus.

The NF-retentate may be immediately subjected to the polyvalent inorganic ion reduction procedure, or it may be subjected to processing steps prior to the reduction procedure that do not significantly alter the solids composition of the NF-retentate. Such processing steps may, for example, be pasteurization, antibacterial microfiltration, bactofugation, dilution and/or concentration.

In some preferred embodiments of the present invention, whey protein-containing streams are not ultrafiltered after step b). It is especially preferred that the whey protein-containing streams after step b) are not subjected to ultrafiltration, which separates the whey protein from the milk saccharide.

The present inventors have found that this is advantageous because separate processing of milk saccharide streams and whey protein streams is avoided.

The polyvalent inorganic ion reduction procedure preferably does not remove significant amounts of milk saccharide or whey protein from the NF retentate.

For example, the procedure for reducing the number of polyvalent inorganic ions of stage d) may include electrodialysis, precipitation of minerals and/or ion exchange.

In some embodiments, implementation of the present invention, the procedure for reducing the number of polyvalent inorganic ions in stage d) involves or even consists of electrodialysis (ED). Electrodialysis is well known to those skilled in the art and is, for example, described in Membrane filtration and related molecular separation technologies, published by APV Systems, 2000, ISBN 87-88016757; in Ion exchange membranes Fundamentals and Applications, Yoshinobu Tanaka, 2nd edition, Elsevier, 2015, ISBN: 978-0-444-63319-4; and Ion exchange membranes Preparation, characterization, modification and application, Toshikatsu Sata, The Royal Society of Chemistry, 2004, ISBN 0-85404-5902, which are incorporated herein for all purposes.

Electrodialysis can be performed in a number of different configurations and it is important to the present invention that the ED be configured to remove not only small monovalent metal cations and anions, but also polyvalent ions such as, for example, Ca ²⁺ , Mg ²⁺ , and phosphates.

- 11 040098

As described herein, it is further preferred that the ED be configured to remove citrate.

Briefly, ED typically uses the transfer of ions from a feed solution through ion exchange membranes to one or more adjacent solutions under the action of an applied DC electric field. This is done in a configuration called an electrodialysis cell. The cell typically contains a feed chamber (often referred to as a diluate chamber) defined by an anion exchange membrane and a cation exchange membrane and is located between two concentrating chambers (often referred to as brine chambers). The direct current electric field attracts cations from the feedstock to the negative electrode and anions to the positive electrode, and at least smaller cations and anions are able to permeate through the cation exchange membrane and the anion exchange membrane, respectively. Thus, charged molecular particles are removed from the raw material.

In virtually all electrodialysis methods, multiple electrodialysis cells are arranged in a configuration called an electrodialysis stack, with alternating anion and cation exchange membranes forming multiple electrodialysis cells.

In some preferred embodiments of the present invention, the electrodialysis equipment comprises at least one feed chamber and optionally also a concentrating chamber that contains particulate ion exchange materials such as, for example, an ion exchange resin. The particulate ion exchange material performs the function of retaining ions, allowing them to be transported through ion exchange membranes, and is particularly suitable for liquids with low conductivity. This variant of electrodialysis is sometimes referred to as electrodeionization.

The present inventors have found that it is particularly advantageous to use anion exchange membranes that provide citrate transfer.

In some preferred embodiments of the present invention, the anion exchange membrane has a citrate selective permeability coefficient of at least 0.01, preferably at least 0.05, more preferably at least 0.1, and even more preferably at least 0, 2. For example, the anion exchange membrane has a citrate permeability selectivity of at least 0.3, preferably at least 0.4, more preferably at least 0.5, and even more preferably at least 0.6.

The citrate selectivity of the anion exchange membrane is measured according to Example 1.2. In determining the citrate selectivity of a membrane, the chloride anion is used as the reference anion, and the term citrate selectivity may thus be referred to as the citrate-to-chloride selectivity.

Ion exchange membranes can also be characterized in terms of their selective permeability, i.e. their selectivity for counterion permeability (eg, anion permeability across an anion exchange membrane) relative to their coion permeability (eg, cation permeability across an anion exchange membrane).

In some preferred embodiments of the present invention, the anion exchange membrane used for electrodialysis has a selective permeability of at least 0.4, preferably at least 0.5, more preferably at least 0.6, and even more preferably at least 0. .7. The anion exchange membrane used for electrodialysis preferably has a selective permeability of at least 0.8, more preferably at least 0.9, and even more preferably at least 0.95.

It is equally desirable that the cation exchange membrane has a relatively high selective permeability. Thus, in some preferred embodiments of the present invention, the cation exchange membrane used for electrodialysis has a selective permeability of at least 0.4, preferably at least 0.5, more preferably at least 0.6, and even more preferably at least at least 0.7. The cation exchange membrane used for electrodialysis preferably has a selective permeability of at least 0.8, more preferably at least 0.9, and even more preferably at least 0.95.

Non-limiting examples of suitable membranes are, for example, Ralex CM(H)-PES cation exchange membranes and Ralex AM(H)-PES anion exchange membranes from MEGA (Czech Republic). Other examples of membranes can be found in Tanaka 2015.

The pH of the NF permeate when initially subjected to electrodialysis is usually at least 5.5 and preferably at least 6.0. In some embodiments of the present invention, the pH of the NF permeate, when initially subjected to the electrodialysis used for precipitation, is in the range of 5.5-7.0, in the range of 5.7-6.8, and more preferably in the range of 6.0- 6.5.

In some preferred embodiments of the present invention, the ED concentrate stream has a pH of at most 6.0, preferably at most 5.6, more

- 12 040098 more preferably not more than 5.2 and most preferably not more than 5.0. The relatively low pH of the concentrate stream counteracts the precipitation of calcium phosphate in the concentrate stream.

The temperature of the liquid feedstock and concentrate in electrodialysis is usually in the range of 0-70°C. Preferably, the temperature of the liquid feedstock and concentrate during electrodialysis is in the range of 2-40°C. Even more preferably, the temperature of the liquid feedstock and electrodialysis concentrate is in the range of 4-15°C, such as, for example, preferably in the range of 5-10°C.

The ED voltage depends on the actual setting of the ED system and may, for example, be in the range 1-500 V, such as in the range 50-400 V, such as, for example, the range 100-300 V.

In some embodiments of the present invention, the voltage during ED is kept constant during the ED electrodialysis step.

In some preferred embodiments of the present invention, ED in step d) is carried out until the conductivity of the NF retentate has decreased by at least 40%, preferably by at least 50%, more preferably by at least 60%, even more preferably at least 70% and most preferably at least 80%.

In some preferred embodiments of the present invention, ED in step d) is carried out until the conductivity of the NF retentate has decreased by at least 40%, preferably by at least 50%, more preferably by at least 60%, even more preferably at least 70% and most preferably at least 80%.

It is also possible to control the ED process with the level of citrate reduction. Thus, in some embodiments of the present invention, ED in step d) is carried out until the amount of citrate in the original NF retentate is reduced by at least 30%, preferably by at least 50%, more preferably by at least 70%, even more preferably at least 80%, and most preferably at least 90%.

In some embodiments of the present invention, it is further preferred that only a limited amount of sialyllactose is removed in the NF retentate provided in step c). This can be done by selecting an anion exchange membrane that is impermeable to sialyllactose and/or stopping the ED process before removing the sialyllactose. Preferably, the ED process in step d) removes no more than 50% w/w. sialyllactose in NF-retentate, preferably not more than 40% w/w, more preferably not more than 30% w/w, even more preferably not more than 20% w/w. and most preferably not more than 10% wt./weight. Preferably, the ED process in step d) does not remove sialyllactose.

In other embodiments, implementation of the present invention, the procedure for reducing the number of polyvalent inorganic ions of stage d) involves ion exchange or even consists of it. Ion exchange is also a technique well known to those skilled in the art, which is, for example, described in Protein Purification: Principles and Practice, Robert K. Scopes, 3rd edition, Springer Verlag New York, Inc., ISBN 0-387-94072- 3; or in Membrane filtration and related molecular separation technologies published by APV Systems, 2000, ISBN 87-88016757, which are incorporated herein for all purposes, which are incorporated herein for all purposes.

However, in some preferred embodiments of the present invention, the procedure for reducing the number of polyvalent inorganic ions of step d) does not involve electrodialysis. In some preferred embodiments of the present invention, the procedure for reducing the number of polyvalent inorganic ions of step d) does not involve ion exchange.

In some preferred embodiments of the present invention, the procedure for reducing the amount of polyvalent inorganic ions of step d) does not involve either electrodialysis, ion exchange or ultrafiltration, which separates the milk saccharide from whey proteins.

In some preferred embodiments of the present invention, the procedure for reducing the number of polyvalent inorganic ions of step d) involves or even consists of precipitation of minerals, which includes the formation of a mineral-containing precipitate using at least one of the following:

adjusting the pH of the NF retentate to at least 6.0;

heating the NF-retentate to a temperature of at least 30°C; and concentrating the NF-retentate; and isolating the mineral precipitate from the NF-retentate, thereby obtaining a demineralized whey protein product containing milk saccharide and sediment minerals.

The pH used for precipitation is at least 6.0 and preferably at least 6.3. In some embodiments of the present invention, the pH used for precipitation is at least 6.5. Preferably, the pH used for precipitation is at least 7.0. Even more preferably, the pH used for precipitation is at least 8.0.

The pH values that are presented in this document, measured at 25°C, unless otherwise indicated.

- 13 040098

In some preferred embodiments of the present invention, the pH used for precipitation is the same as the pH normally found in milk. This is particularly advantageous since the need for pH adjustment is either reduced or eliminated. The addition of an alkalizing agent, for example in the form of NaOH or KOH, also contributes to more mineral cations, which can then be removed.

Thus, in some preferred embodiments of the present invention, the pH used for precipitation is in the range of 6.0-7.0. Preferably, the pH used for precipitation is in the range of 6.2-6.9. Even more preferably, the pH used for precipitation is in the range of 6.3-6.8.

In other preferred embodiments of the present invention, the pH used for precipitation is higher than the pH normally found in milk.

Thus, in other preferred embodiments of the present invention, the pH used for precipitation is in the range of 6.9-9. Preferably, the pH used for precipitation is in the range of 7.2-8.5. Even more preferably, the pH used for precipitation is in the range of 7.5-8.0. An increased pH results in more efficient precipitation and/or allows the precipitation step to be carried out at a lower temperature than if a lower pH were used.

In additional preferred embodiments of the present invention, the pH used for precipitation is in the range of 6.0-9, preferably 6.2-8.0 and even more preferably in the range of 6.5-7.5.

The temperature used for precipitation is at least 30°C. Preferably the temperature used for precipitation is at least 40°C. Even more preferably, the temperature used for deposition is at least 50°C.

In some preferred embodiments of the present invention, the temperature used for deposition is in the range of 30-75°C. Preferably the temperature used for deposition is in the range of 45-65°C. Even more preferably, the temperature used for the deposition is in the range of 55-65°C

In some preferred embodiments of the present invention, the precipitation pH is in the range of 6.1-6.8, preferably 6.3-6.7, and the temperature is maintained in the range of 55-75°C. For example, the pH for precipitation may be in the range of 6.3-6.7 and the temperature is maintained in the range of 60-70°C.

In other preferred embodiments of the present invention, the precipitation pH is in the range of 6.9-9, preferably 7.0-8.0, and the temperature is maintained in the range of 55-75°C. For example, the precipitation pH may be in the range of 7.0-8.0 and the temperature is maintained in the range of 60-70°C.

Precipitation conditions will preferably be maintained for a time sufficient to precipitate significant amounts of calcium, magnesium and phosphorus. For example, the conditions for precipitate formation are typically maintained for at least 1 minute, and preferably even longer, such as, for example, at least 10 minutes or even at least 15 minutes.

In some preferred embodiments of the present invention, sedimentation conditions are maintained for at least 20 minutes. Preferably, sedimentation conditions are maintained for at least 30 minutes. For example, sedimentation conditions are maintained for at least 1 hour.

In some embodiments of the present invention, sedimentation conditions are maintained for 1 minute to 48 hours. For example, sedimentation conditions are maintained for 5 minutes to 5 hours. Alternatively, sedimentation conditions are maintained for 10 minutes to 2 hours. In some preferred embodiments of the present invention, sedimentation conditions are maintained for 15 minutes to 1 hour.

In some preferred embodiments of the present invention, the conditions for the formation of sediment support for a time sufficient to precipitate at least 30% wt./weight. calcium in NF-retentate, preferably at least 35% wt./weight. and even more preferably at least 40% wt./weight.

You can precipitate more calcium, and in some preferred embodiments of the present invention, the conditions for the formation of the precipitate support for a time sufficient to precipitate at least 50% wt./weight. calcium in NF-retentate, preferably at least 60% wt./weight. and even more preferably at least 70% wt./weight.

In some preferred embodiments of the present invention, the conditions for the formation of sediment support for a time sufficient to precipitate at least 30% wt./weight. phosphorus in the NF-retentate, preferably at least 35% wt./weight. and even more preferably at least 40% wt./weight.

More phosphorus can be precipitated, and in some preferred embodiments of the present invention, the conditions for precipitate formation are maintained for a period of time

- 14 040098 accurate for deposition of at least 50% wt./weight. phosphorus from NF-retentate, preferably at least 60% wt./weight. and even more preferably at least 70% wt./weight.

The mineral precipitate can be separated from the rest of the NF-retentate by conventional separation techniques such as, for example, centrifugation, microfiltration or decanting.

In the context of the present invention, the terms demineralized and demineralized are used interchangeably and mean that at least some of the minerals have been removed from the feed to produce the composition in question. The demineralized and demineralised product or composition preferably contains no more than 0.6% (w/w TS) of calcium, preferably no more than 0.4% w/w. calcium and even more preferably not more than 0.2% wt./weight. calcium, such as, for example, preferably not more than 0.1% wt./weight. calcium.

In some preferred embodiments of the present invention, the demineralized whey protein product containing milk saccharide obtained in step d) provides a total amount of calcium of not more than 1.0% (w/w total solids);

the total amount of magnesium is not more than 0.1 (wt./wt. all solids); and the total amount of phosphorus is not more than 0.8% (wt./wt. of all solids).

For example, the demineralized whey protein product containing milk saccharide obtained in step d) may contain a total amount of calcium of not more than 0.6% (w/w total solids);

the total amount of magnesium is not more than 0.1 (wt./wt. all solids); and the total amount of phosphorus is not more than 0.4% (wt./wt. of all solids).

The demineralized whey protein product containing milk saccharide which is obtained in step d) may, for example, contain a total amount of calcium in the range of 0.01-1.0% (w/w total solids);

total magnesium in the range of 0.001-0.1 (w/w total solids); and total phosphorus in the range of 0.01-0.6% (w/w total solids).

In some preferred embodiments of the present invention, the demineralized whey protein product containing milk saccharide obtained in step d) provides a total amount of calcium in the range of 0.1-0.6% (w/w total solids) ;

total magnesium in the range of 0.01-0.1 (w/w total solids); and total phosphorus in the range of 0.05-0.4% (w/w total solids).

In step e) a casein source and optionally one or more additional ingredients are added to a demineralized whey protein product containing milk saccharide to form a nutritional product. Step e) may further include processing steps such as mixing, homogenization, evaporation and/or heat treatment.

Various sources of casein can be used. In some embodiments, the casein source provides one or more of milk, concentrated milk, milk powder, milk UF retentate, milk protein concentrate, betacasein isolate, micellar casein isolate, caseinate, or a combination thereof. Preferably, the casein from the casein source is micellar casein, such as that present in skim milk, and/or beta-casein.

Skimmed milk in the form of skimmed milk liquid or skimmed milk powder is particularly preferred.

In addition, skimmed milk UF retentate is a particularly preferred source of casein.

In some preferred embodiments of the present invention, both the casein source in step e) and the milk raw material in step a) are UF retentates of milk and, for example, from the same batch of milk or from the same category of milk, such as organic milk.

One or more additional ingredients that may be included in the nutritional product may preferably be selected from those commonly used in children's products.

For example, a nutritional product, such as an infant formula, may include at least one of human milk oligosaccharides (HMOs), such as, for example, 2'-FL and LNnT. Research has shown multiple roles for NMOs in improving central nervous system (CNS) function. In addition to including at least one of the 2'-FL and LNnT described above, in some aspects, the nutritional product contains additional sialylated or fucosylated human milk oligosaccharides (HMOs).

Any or all of the HMOs used in a nutritional product may be isolated or fortified from milk excreted by mammals, including without limitation human, cow, sheep, pig or goat. HMOs can also be produced through microbial fermentation, enzymatic methods,

- 15 040098 chemical synthesis or combinations thereof.

Suitable sialylated HMOs for inclusion in infant formula may, for example, include at least one sialic acid residue in the backbone of the oligosaccharide. In some aspects, the sialylated HMO contains two or more sialic acid residues.

Alternatively or additionally, the nutritional product may also contain other types of oligosaccharides such as, for example, trans-galactooligosaccharides (GOS), fructooligosaccharides (FOS) and/or polydextrose.

The nutritional product, for example in the form of an infant formula, may further comprise one or more polyunsaturated fatty acids (PUFA), such as, for example, docosahexaenoic acid (DHA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) , linoleic acid, linolenic acid (alpha-linolenic acid) and gamma-linolenic acid.

Research has shown multiple roles for PUFA in supporting brain function and vision development in infants. Applicant believes that the inclusion of DHA and AA in infant formula may improve neurological functions such as cognition, learning and memory associated with the CNS.

In certain aspects, the PUFAs are provided as free fatty acids, as a triglyceride, as a diglyceride, as a monoglyceride, as a phospholipid, or as a mixture of one or more of the foregoing, preferably as a triglyceride. PUFA can be obtained from oil sources such as vegetable oils, marine plankton, mushroom oils, and fish oils. In certain aspects, PUFAs are derived from fish oils such as menhaden, salmon, anchovy, cod, halibut, tuna, or herring.

The nutritional product, for example in the form of an infant formula, may further comprise one or more nucleotides, including, for example, the nucleotides: inosine monophosphate, cytidine-5'-monophosphate, uridine-5'-monophosphate, adenosine-5'-monophosphate, guanosine-5 '-1-monophosphate, more preferably cytidine-5'-monophosphate, uridine-5'-monophosphate, adenosine-5'-monophosphate and guanosine-5'-monophosphate.

The concentration of carbohydrates in a nutritional product, such as an infant formula, may, for example, range from about 5% to about 40% w/w, including from about 7% to about 30%, including from about 10%. up to about 25% by weight of the nutritional product. If present, fat concentrations most typically range from about 1% to about 30%, including from about 2% to about 15%, and also including from about 3% to about 10%, by weight of infant formula. If present, protein concentrations most typically range from about 0.5% to about 30%, including from about 1% to about 15%, and also including from about 2% to about 10% by weight of the nutritional product.

In some embodiments, the nutritional product, such as infant formula, includes a fat source or sources other than the PUFAs described above. Suitable fat sources for use herein include any fat or fat source that is suitable for use in oral formulas for infants and that is compatible with the essential elements and features of such a formula.

Additional non-limiting examples of suitable fats or sources thereof for use in the nutritional product described herein include coconut oil, fractionated coconut oil, soybean oil, corn oil, olive oil, safflower oil, high oleic safflower oil, oleic acids ( EMERSOL 6313 OLEIC ACID, Cognis Oleochemicals, Malaysia), MCT Oil (Medium Chain Triglycerides), Sunflower Oil, High Oleic Sunflower Oil, Palm Oil or Palm Kernel Oil, Palm Olein, Canola Oil, Seafood Oils, Fish Oil, Mushroom oils, algae oils, cottonseed oil, and combinations thereof.

The nutritional product may, in addition to whey protein and casein, also contain other types of protein. Non-limiting examples of suitable proteins or protein sources for use in a nutritional product, such as infant formula, include hydrolysed, partially hydrolysed, or non-hydrolysed proteins or protein sources that can be obtained from any known or other suitable source such as animals (e.g., meat , fish), cereal (eg rice, corn), vegetable (eg soy), or combinations thereof. Non-limiting examples of such proteins include highly hydrolysed casein, soy protein isolates, and soy protein concentrates.

The nutritional product may, for example, contain a hydrolysed protein, ie. protein hydrolyzate. As used herein, the terms hydrolysed protein or protein hydrolysates are used interchangeably herein and include highly hydrolyzed proteins, where the degree of hydrolysis is most often at least about 20%, including from about 20% to about 80%, and also including from about 30% up to about 80%, even more preferably from about 40% to about 60%. The degree of hydrolysis is the degree to which peptide bonds are broken during hydrolysis. The degree of protein hydrolysis for the purposes of

The characteristics of the highly hydrolysed protein component of these embodiments are easily determined by one skilled in the art of formulation by quantifying the ratio of amine nitrogen to total nitrogen (AN/TN) of the protein component of the selected liquid formulation. The amine nitrogen providing component is quantified using USP titration methods for the determination of amine nitrogen content, while the total nitrogen providing component is determined using the Kjeldahl method using Tecator, all of which are methods well known to those skilled in the art of analytical chemistry.

Suitable hydrolysed proteins include soy protein hydrolysate, casein protein hydrolysate, whey protein hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg protein hydrolysate, gelatin protein hydrolysate, combinations of animal and vegetable protein hydrolysates, and their combinations. Particularly preferred protein hydrolysates include whey protein hydrolyzate and hydrolysed sodium caseinate.

The nutritional product may, in addition to the milk saccharide, contain an additional carbohydrate. Non-limiting examples of suitable carbohydrates or sources thereof include maltodextrin, hydrolysed or modified starch or corn starch, glucose polymers, corn syrup, dry corn syrup, rice derived carbohydrates, pea derived carbohydrates, potato derived carbohydrates, tapioca, sucrose, fructose, lactose. , high fructose corn syrup, honey, sugar alcohols (eg, maltitol, erythritol, sorbitol), artificial sweeteners (eg, sucralose, acesulfame potassium, stevia), and combinations thereof. A particularly desirable carbohydrate is low dextrose equivalent (DE) maltodextrin.

The casein source is usually added to the demineralized whey product containing milk saccharide in an amount sufficient to obtain the desired weight ratio between casein and whey protein in the nutritional product. In some preferred embodiments of the present invention, a whey product containing milk saccharide and a source of casein are mixed, thereby achieving a weight ratio of whey protein and casein in the range of 1-9, preferably 1-3, and even more preferably 1.2- 1.9, such as about 1.5.

In the context of the present invention, the weight ratio between the two components A and B is defined as the weight of component A divided by the weight of component B. Thus, if the composition contains 9% wt./weight. A and 6% wt./wt. B, the weight ratio will be 9%/6%=1.5.

In some embodiments of the present invention, at least some of the purified milk saccharide (eg, lactose) from the UF milk permeate obtained in step a) is added as an ingredient during step e).

In some embodiments of the present invention, the milk saccharide (eg, lactose) of the nutritional product is obtained from the same milk source, i. batches of milk as raw milk.

In some embodiments of the present invention, the nutritional protein is derived from the same milk source as the raw milk.

In some preferred embodiments of the present invention, the protein and milk saccharide of the nutritional product are provided with organic ingredients. Preferably the nutritional product is an organic product.

If the nutritional product is intended for sale or use as a liquid product, it may be preferable to heat the nutritional product with an F ₀ value equivalent to at least 72°C for 15 seconds, or even better, with an F ₀ value equivalent to at least 142°C for 4 s. The heat treatment may, for example, be a UHT treatment that sterilizes the liquid nutritional product.

The pH of the nutritional product is preferably in the range of 6-7 and even more preferably in the range of 6.0-7.0, such as, for example, in the range of 6.2-7.0.

The pH of the nutritional product is measured by standardizing the product to a solids content corresponding to about 10 g of solids in 90 g of demineralized water and measuring the pH at 25°C.

In some preferred embodiments of the present invention, the method further comprises step f) converting the nutritional product obtained in step e) from liquid form to powder form. You can use any suitable method of converting to powder, such as spray drying or freeze drying. Suitable methods and implementation details can be found, for example, in Westergaard, Milk Powder Technology - evaporation and spray drying, 5th edition, 2010, Gea Niro, Copenhagen.

Further, it is preferred that the nutritional product either in liquid or concentrated or powder form be packaged. The packaging may, for example, be carried out under aseptic or sterile conditions, and may, for example, involve filling and sealing the nutritional product in sterile containers.

- 17 040098

The present inventors have also found that it may be preferable to stop the process using steps a) to d) above to obtain a whey protein product containing milk saccharide with a reduced mineral content. Such a whey protein product is an interesting ingredient for making, for example, infant formula products and can be produced in an efficient manner with the present invention.

Thus, another aspect of the present invention relates to a method for obtaining a demineralized whey protein product containing a milk saccharide, the method comprising the steps

i) provision of milk raw materials;

ii) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining an MF retentate and an MF permeate;

iii) subjecting the MF permeate to nanofiltration (NF) or nanofiltration/diafiltration (NF/DIA) to obtain NF retentate and NF permeate;

iv) subjecting the NF retentate to an inorganic polyvalent ion reduction procedure, thereby obtaining a demineralized whey protein product containing milk saccharide; And

v) optionally drying the demineralized whey protein product containing milk saccharide.

Step i) is identical to step a) and all features mentioned in the context of step a) also apply to step i).

Step ii) is identical to step b) and all features indicated in the context of step b) also apply to step ii).

Step iii) is identical to step c) and all features indicated in the context of step c) also apply to step iii).

Step iv) is identical to step d) and all features indicated in the context of step d) also apply to step iv).

A schematic example of steps i)-iv) of the method is shown in FIG. 2. In this case, the raw milk is subjected to microfiltration, which provides a permeate (P), containing mainly whey protein, milk saccharide, water and minerals, and a retentate (R), containing mainly casein micelles, water and additionally small amounts of protein whey milk, milk saccharide and minerals. The permeate is subjected to nanofiltration to provide NF-permeate (P) containing monovalent ions and water, and NF-retentate (R) containing whey protein, milk saccharide, water and other minerals. The NF retentate is subjected to a polyvalent inorganic ion reduction procedure, for example by mineral precipitation, and a mineral-containing precipitate and a milk saccharide-containing whey protein with a reduced amount of minerals are obtained. In this example, the milk saccharide mainly provides for lactose. The milk saccharide-containing reduced mineral whey protein may be used as a liquid ingredient as such or may be formulated into a powder, for example by spray drying.

Thus, the method may further comprise step v) of drying the demineralized whey protein product containing milk saccharide and thereby converting it into a powder. You can use any suitable method of converting to powder, such as spray drying or freeze drying. Suitable methods and implementation details can be found, for example, in Westergaard, Milk Powder Technology - evaporation and spray drying, 5th edition, 2010, Gea Niro, Copenhagen.

It is further preferred that a demineralized whey protein product containing milk saccharide, usually in powder form, be packaged. The packaging may, for example, be carried out under aseptic or sterile conditions, and may, for example, involve filling and sealing the nutritional product in sterile containers.

The present inventors have found that the mineral precipitation described above can also be applied to other types of milk protein solutions than those provided in steps a) to c) above, especially to other types of whey protein solutions or whey protein solutions.

A further aspect of the present invention relates to a method for producing a demineralized whey protein product or a whey protein product, the method comprising the steps

1) providing a liquid protein source containing whey protein or whey protein and preferably also milk saccharide;

2) subjecting the liquid protein source to an inorganic multivalent ion reduction procedure, the reduction procedure comprising adjusting the pH of the liquid protein source to at least 6 and heating it to a temperature of at least

- 18 040098 re 30°C, and separating the resulting precipitate from the NF-retentate, thereby obtaining a product based on whey protein or a product based on whey protein;

3) optionally drying the whey protein based product or the whey protein based product.

Step 2) is identical to step d), and all the features indicated in the context of step d) also apply to step 2), the only difference being that the liquid protein source is subjected to the inorganic polyvalent ion reduction procedure, and not the NF-retentate.

In some embodiments, the implementation of the present invention, the liquid protein source provides a total amount of whey protein and whey protein in the range of 1-15% wt./weight. Preferably, the liquid protein source provides for a total amount of whey protein and whey protein in the range of 2-10% w/w. Even more preferably, the liquid protein source provides for a total amount of whey protein and whey protein in the range of 3-8%.

The liquid protein source preferably contains less than 5% (w/w total protein) casein, and preferably is substantially free of casein, in which case it contains no more than 1% casein (w/w total protein).

An additional aspect of the present invention relates to a nutritional product, for example, obtained using the method described in this document, containing

20-90% (w/w TS) carbohydrates;

5-40% (w/w TS) proteins;

0-40% (w/w TS) lipids;

at least 15% wt./weight. whey protein relative to total protein; and not more than 1% (w/w TS) citrate.

In some preferred embodiments of the present invention, the nutritional product is suitable for infant formula.

The nutritional product preferably contains at least one ruminant milk protein. Preferably, at least the whey protein of the nutritional product is a ruminant whey protein, and preferably a bovine whey protein. In some preferred embodiments of the present invention, the protein of the nutritional product is obtained from a cow, and is preferably obtained from cow's milk.

In some preferred embodiments of the present invention, the nutritional product is a nutritional product such as, for example, infant formula, second level milk formula, or third level milk formula.

In some preferred embodiments of the present invention, the nutritional product is an infant formula.

Preferably, the nutritional product is an infant formula containing

35-70% (w/w TS) carbohydrates;

5-15% (w/w TS) proteins;

20-40% lipids (w/w TS);

30-70% w/w whey protein relative to total protein; And

30-70% w/w casein relative to total protein.

In the context of the present invention, the term infant formula refers to nutritionally complete foods for infants aged 0-6 months, which foods comply with the US Code of Federal Regulations, title 21, chapter I, subpart B, part 107 (infant formula) , subsection D (nutrient requirements); section 107.100 Nutrient Guidelines, as amended April 1, 2015

In some preferred embodiments of the present invention, the nutritional product, for example in the form of infant formula, contains proteins, milk saccharide, fats and minerals, and provides a total carbohydrate content in the range of 40-55% (w/w total solids);

total proteins in the range of 9-14% % (w/w total solids);

total milk saccharide in the range 40-55% (w/w total solids), weight ratio of whey protein and casein in the range 50:50-70:30, preferably in the range 55:45-65:45 and more more preferably about 60:40;

the total amount of calcium is not more than 0.7% (wt./wt. of all solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.5% (wt./wt. of all solids);

not more than 0.3% total sodium (w/w total solids);

the total amount of potassium is not more than 0.8% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

In other preferred embodiments of the present invention, the nutritional

- 19 040098 A duct is an infant formula ingredient, also referred to as an infant formula base, that typically lacks some of the ingredients needed to provide a nutritionally complete infant formula.

In some preferred embodiments of the present invention, the nutritional product is an infant formula base product comprising

30-70% w/w whey protein relative to total protein; And

30-70% w/w casein relative to total protein.

Preferably the infant formula base comprises

35-70% (w/w TS) milk saccharide;

5-15% (w/w TS) proteins;

20-40% lipids (w/w TS);

30-70% w/w whey protein relative to total protein; And

30-70% w/w casein relative to total protein.

Even more preferably, the infant formula base comprises

35-70% (w/w TS) milk saccharide;

5-15% (w/w TS) proteins;

20-40% lipids (w/w TS);

50-70% w/w whey protein relative to total protein; And

30-50% w/w casein relative to total protein.

In the context of the present invention, the term infant formula base refers to an ingredient that contains at least the proteins and carbohydrates required for infant formula, and optionally also lipids, but which is not nutritionally complete, meaning that it lacks at least the amount of certain micronutrients required under 21 U.S. Code of Federal Regulations, Chapter I, Subpart B, Part 107 (Infant Formula), Subpart D (Nutritional Requirements); section 107.100 Nutrient Guidelines, as amended April 1, 2015

Preferably, the infant formula base contains only milk solids, i. only solids derived from milk.

In some embodiments of the present invention, the nutritional product is a second level milk formula or a third level milk formula.

In some preferred embodiments of the present invention, the nutritional product is a demineralized whey protein product containing a milk saccharide containing

20-90% (w/w TS) milk saccharide;

5-40% (w/w TS) proteins;

0-10% lipids (w/w TS);

at least 60% wt./weight. whey protein relative to total protein and not more than 40% casein, preferably from which at least 50% wt./weight. are betacasein, relative to total protein.

In some preferred embodiments of the present invention, the demineralized whey protein product containing milk saccharide contains

20-90% (w/w TS) milk saccharide;

5-40% (w/w TS) proteins;

0-10% lipids (w/w TS);

at least 70% wt./weight. whey protein relative to total protein; and no more than 30% casein relative to total protein.

Alternatively, but also preferably, the demineralized whey protein product containing milk saccharide may contain

20-90% (w/w TS) milk saccharide;

5-40% (w/w TS) proteins;

0-10% lipids (w/w TS);

60-80% w/w whey protein relative to total protein; And

20-40% w/w casein, of which at least 50% wt./weight. are betacasein, relative to total protein.

A demineralized whey protein product containing milk saccharide may, for example, contain

20-80% (w/w TS) milk saccharide;

10-30% (w/w TS) proteins;

0-10% lipids (w/w TS);

at least 90% wt./weight. whey protein relative to total protein; and no more than 10% casein relative to total protein.

For example, the nutritional product may be a demineralized whey protein product containing milk saccharide, for example prepared by the process defined herein, providing for a total lactose in the range of 65-85%;

the total amount of whey protein and whey protein in the range of 10-25%, the weight ratio of whey protein and micellar casein is at least 95:5;

the total amount of calcium is not more than 1.0% (wt./wt. of all solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.8% (wt./wt. of all solids);

not more than 0.4% total sodium (w/w total solids);

the total amount of potassium is not more than 1.3% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

In the context of the present invention, the percentages of the components are the weight percentages of the component relative to the total weight of the composition in question, unless otherwise indicated.

The carbohydrate in the nutritional composition may be selected from any edible carbohydrate and may include both monosaccharides, disaccharides, oligosaccharides and/or polysaccharides.

In some particularly preferred embodiments of the present invention, the carbohydrate in the nutritional composition, in particular the infant formula base and/or the demineralized whey protein product containing milk saccharide, contains at least 80% w/w. milk saccharide relative to the total amount of carbohydrates, preferably at least 90% wt./weight. milk saccharide relative to the total amount of carbohydrates, and even more preferably at least 95% wt./weight. milk saccharide relative to total carbohydrates.

In some preferred embodiments of the present invention, the carbohydrate consists of milk saccharide and sialyllactose and contains only trace amounts of other bovine milk oligosaccharides.

Milk saccharide usually contains a significant amount of digestible milk saccharide, i. sum of lactose, glucose and galactose. In some particularly preferred embodiments of the present invention, the carbohydrate in the nutritional composition, in particular the infant formula base and/or the demineralized whey protein product containing milk saccharide, contains at least 80% w/w. digestible milk saccharide relative to the total amount of carbohydrates, preferably at least 90% wt./weight. digestible milk saccharide relative to the total amount of carbohydrates, and even more preferably at least 95% wt./weight. digestible milk saccharide relative to total carbohydrates.

Lactose is a particularly preferred type of milk saccharide, and in some embodiments of the present invention, the carbohydrate in a nutritional composition, in particular an infant formula base and/or a demineralized whey protein product containing milk saccharide, contains at least 50 wt%. weight. lactose relative to the total amount of carbohydrates, preferably at least 80% wt./weight. lactose relative to the total amount of carbohydrates, and even more preferably at least 95% wt./weight. lactose relative to total carbohydrates.

In some preferred embodiments of the present invention, the nutritional composition contains sialyllactose in an amount of at least 0.01% w/w. relative to total carbohydrates. Preferably, the nutritional product contains sialyllactose in an amount of at least 0.02% w/w, more preferably at least 0.03% w/w. and even more preferably at least 0.06% wt./weight. relative to total carbohydrates.

The amount of sialyllactose is defined as the sum of 3'-sialyllactose and 6'-sialyllactose and is determined according to Lee at al., J. Dairy Sci., 2015 November, 98(11): 7644-7649. If the test sample contains proteins and/or fats, these can be removed by filtering the sample (or its solution) in a filter centrifuge with a nominal molecular weight cutoff of approximately 3 kDa (e.g. Amicon Ultra-0.5 filter centrifuge 3K; Merck KGaA) and subjecting permeate analysis.

Large amounts of sialyllactose are even more preferred. In some preferred embodiments of the present invention, the nutritional composition contains sialyllactose in an amount of at least 0.10% w/w. relative to total carbohydrates. Preferably, the nutritional product contains sialyllactose in an amount of at least 0.15% w/w, more preferably at least 0.2% w/w. and even more preferably at least 0.3 wt./weight. relative to total carbohydrates.

In some preferred embodiments of the present invention, for example, where the nutritional composition has not been supplemented with sialyllactose, the nutritional composition contains sialyllactose in an amount in the range of 0.01-0.5% w/w. relative to total carbohydrates. Prev

- 21 040098 respectfully nutritional product contains sialyllactose in an amount in the range of 0.02-0.4% w/w, more preferably in the range of 0.03-0.3% w/w. and even more preferably in the range of 0.05-0.3% wt./weight. relative to total carbohydrates.

A particular advantage is achieved if the nutritional product is an infant formula base or whey protein, since the use of natural sialyllactose content of cow's milk reduces the amount of additional sialyllactose that must be added to infant formula to obtain a concentration of sialyllactose in human breast milk.

In some preferred embodiments of the present invention, at least 50% wt./weight. casein of the nutritional product, e.g. infant formula, infant formula base or demineralized whey protein product containing milk saccharide, is beta-casein, preferably at least 60% w/w, more preferably at least 70% wt./weight., even more preferably at least 80% wt./weight. and most preferably at least 90% wt./weight. caseins are beta-casein. This embodiment is particularly preferred because the higher content of beta-casein makes the protein composition of the nutritional product closer to that of human milk.

In some preferred embodiments of the present invention, the nutritional product comprises one or more of the following:

the total amount of calcium is not more than 0.7% (wt./wt. of all solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.5% (wt./wt. of all solids);

not more than 0.3% total sodium (w/w total solids);

the total amount of potassium is not more than 0.8% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

Preferably, the nutritional product provides for a total amount of calcium of not more than 0.7% (w/w total solids);

the total amount of magnesium is not more than 0.1% (wt./wt. all solids);

the total amount of phosphorus is not more than 0.5% (wt./wt. of all solids);

not more than 0.3% total sodium (w/w total solids);

the total amount of potassium is not more than 0.8% (wt./wt. of all solids); and the total amount of chlorine is not more than 0.8% (wt./wt. of all solids).

In some preferred embodiments of the present invention, the nutritional product, such as an infant formula base or a demineralized whey protein product containing milk saccharide, contains citrate in an amount of not more than 0.8% (wt./wt. TS), preferably not more than 0.6% (w/w TS) and even more preferably not more than 0.4% (w/w TS). Even lower citrate content may be preferred. Thus, in some preferred embodiments of the present invention, the nutritional product provides an amount of citrate that is not more than 0.3% (wt./wt. TS), preferably not more than 0.2% (wt./wt. TS) and even more preferably not more than 0.1% (w/w TS).

The nutritional product may, for example, have a citrate to total protein weight ratio of at most 0.06, preferably at most 0.04, more preferably at most 0.02, and even more preferably at most 0.01.

The present inventors have observed indications that the precipitate formed from the mineral precipitation that may occur in step d) can be used as a source of milk minerals and, for example, a source of organic milk minerals if the milk raw material is organic.

Thus, another aspect of the present invention relates to containing milk minerals product, which is obtained by the method described in this document. More specifically, the milk mineral-containing product contains or even consists of the dry matter of the mineral precipitate and may be in the form of a powder containing not more than 10% w/w. water, or in the form of a wet sediment containing at least 11% wt./weight. water.

Another aspect relates to a method for preparing a nutritional product, such as, for example, infant formula, the method comprising providing a base for infant formula and/or a demineralized whey protein product containing a milk saccharide as defined herein;

combining an infant formula base and/or a demineralized whey protein product containing a milk saccharide with one or more additional ingredients;

processing a combination of an infant formula base and/or a demineralized whey protein product containing a milk saccharide with one or more additional ingredients to form a nutritional product, such as an infant formula.

The additional ingredient(s) used to make a nutritional product, such as, for example, infant formula, may, for example, be one or more of

- 22 040098 nutrients specified in 21 CFR Title I Subpart B Part 107 (Infant Formula) Subpart D (Nutrient Requirements); section 107.100 Nutrient Guidelines, as amended April 1, 2015. For example, additional ingredients can be selected from the nutrients listed in Table 1. 8 for example 9.

The combination processing step typically includes one or more of the following steps: mixing, homogenization, heating, drying, and/or packaging.

Another aspect of the present invention relates to the use of electrodialysis for the demineralization of milk whey, wherein the whey has optionally been concentrated by nanofiltration and wherein the electrodialysis uses an anion exchange membrane with a citrate selectivity of at least 0.01.

The present invention has been described above with reference to specific embodiments. However, within the scope of the present invention, embodiments other than those described above are equally possible. Various features and steps of various embodiments and aspects of the present invention may be combined in other ways than those described herein, unless otherwise indicated.

Examples

Example 1.1. Quantitative determination of milk saccharides.

The following method was used to quantify milk saccharides in a nutritional product.

g Sample nutritional product to be analyzed, regulated by the content of solids to about 10% wt./weight. by adding demineralized water (or by evaporation at low pressure) and taking a 2 g aliquot of the adjusted sample.

Carrez reagents 1 and 2 were added to the aliquot in sufficient amounts to cause all solids to coagulate. The resulting biphasic mixture was filtered using standard filter paper and again using a disposable PTFE syringe microfilter (0.45 micron pore size). At this point, the clear solution was heat-treated (90° C. for 10 min) to denature any remaining protein and the solution filtered with a disposable PFVD syringe microfilter (0.1 micron pore size). The resulting clear saccharide solutions were then placed into HPLC vials and analyzed.

The HPLC method used was as follows:

system: Agilent;

column: Agilent HiPlex Na polymer ion exchange column;

eluent: MilliQ water;

column: temperature: 85°C;

flow rate: 0.2 ml/min;

pressure: 21 bar (max. 25 for this column);

detector: RID at 35°C.

Quantification of glucose, galactose, disaccharides (including lactose and DP2 galactooligosaccharides), trisaccharides (DP3 galactooligosaccharides) and tetrasaccharides (DP4 galactooligosaccharides) is based on peak area. Response factors were also calculated for all saccharides. The retention times and corresponding response factors of the above saccharides were determined using analytical standards glucose, galactose, lactose (DP2), GOS trisaccharides (4-galactosyl lactose) and GOS tetrasaccharides (maltotetraose - used as a model for DP4 GOS). These standards can, for example, be obtained from Carbosynth (UK) or Dextra Laboratories Ltd (UK).

The amount of lactose was measured according to COULIER et al., J. Agric. Food Chem., 2009, 57, 8488-8495.

The results obtained from the analyzes correlated with the analyzed weight of the nutrient sample, and the concentrations of glucose, galactose, lactose, disaccharides (including lactose), trisaccharides and tetrasaccharides are presented as weight percent of the type of saccharide relative to the total weight of the nutritional product.

Example 1.2. Membrane characterization: determination of the selective permeability coefficient for citrate.

The citrate selective permeability coefficient for an anion exchange membrane was determined according to Tanaka 2015 (Ion exchange membranes Fundamentals and Applications, 2nd edition, Elsevier, 2015, ISBN: 978-0-444-63319-4, p. 41-43).

The reference anion for determination was chloride, and the electrolyte solution used for the test was an aqueous solution of 0.5 M sodium citrate and 0.5 M sodium chloride obtained by dissolving salts in demineralized water.

The temperature of the liquids during the method was set to 25°C.

Citrate permeability coefficient for an anion exchange membrane

- 23 040098 defined as

Im citrate ,_ (^citrate/^chloride) ⁷ chloride ^- 777 777} 7 citrate/ '-' chloride/ where C _citrate /Cx _L o _ride is the ratio of citrate concentration to chloride concentration in the storage tank electrolyte described in Tanaka 2015 , And

C citrate/C _chloride is the ratio of the concentration of citrate to the concentration of chloride in the concentrate solution, once the electrolyte solution of the concentrated solution becomes constant.

Example 1.3. Determination of citrate concentration.

Citrate concentration is measured using the Enzyplus EZA 785+ test kit, citric acid (Biocontrol, Italy), which contains the following kit components.

R1: (lyophilized) glycyl-glycine buffer, ZnCl ₂ , NADH, L-MDH, L-LDH, sodium azide (0.1%) as preservative; should be re-bred.

R2: (powder) citrate lyase (13 units); should be re-bred.

R3: (1 ml) citric acid standard solution (0.30 g/l); ready to use.

The method uses UV absorption to determine citrate.

The amount of citrate is presented in weight percent relative to the total weight of the original sample.

The principle of analysis.

Citrate is converted to oxaloacetate and acetate according to the following reaction catalyzed by the enzyme citrate lyase (CL), see reaction (1).

(1) Citrate + CL => oxaloacetate + acetate + CL

The enzymes, L-malate dehydrogenase (L-MDH) and L-acetate dehydrogenase (L-LDH), reduce oxaloacetate and its decarboxylated derivative, pyruvate, to L-malate and L-lactate by reduction of nicotinamide adenine dinucleotide (NADH), see reactions (2, 3).

(2) Oxaloacetate + NADH + H ⁺ + L-LDH -> ί-malate + NAD ⁺ (3) Pyruvate + NADH + H ⁺ L-MDH -> L-lactate + NAD ⁺

The amounts of oxidized NADH from reactions (2) or (3) correspond stoichiometrically to the amount of citrate in the original sample. The concentration of NADH is determined by its absorption at a wavelength of 340 nm.

Sample pretreatment.

g of the Powder to be tested was transferred to a 150 ml beaker. The exact weight (m _{initial sample} ) of the powder sample in grams was recorded with 4 digits. If the sample was a liquid sample, the sample volume corresponding to 1 g of all solids was used and the exact amount of all solids was recorded as m _{initial sample} .

20 ml of 1M perchloric acid was added and the sample and mixture were placed under moderate stirring conditions for 5 minutes.

40 ml ultrapure water was added and the pH was adjusted to 10.0 with 2 M KOH. The mixture was then transferred to a 100 ml volumetric flask, washing the beaker with ultrapure water, and ultrapure water was added to the volumetric flask to make a liquid volume of 100 ml. If a fat layer forms, the fat layer should be above the 100 ml mark on the volumetric flask. The volumetric flask was closed and shaken to thoroughly mix its contents.

The volumetric flask was placed in a refrigerator for 20 minutes to ensure separation of the fat phase from the rest of the liquid and then the mixture was subjected to filtration. The original ml of liquid that passed through the filter was discarded, but the rest of the permeate was collected for further analysis and was called the diluted sample.

Enzymatic reaction and absorbance measurements.

The enzymatic reaction and absorbance measurements were performed according to the following table. It should be noted that the term sample in the table refers to the diluted sample.

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Measure into a cuvette Idle arr. Sample Distilled water R1 (buffer solution, east) Sample (or control) 2,000 ml 1,000 ml 1,800 ml 1,000 ml 0.200 ml Stir ¹ , read the absorption values of the solutions (AJ after about 3 min and then add: R2(CL) | 0.020 ml | 0.020 ml Stir ¹ , read the absorption values of the solutions (A ₂ ) after about 10 minutes. If the reaction has not stopped after 15 min, continue reading absorbance values at 2 min intervals until the values remain unchanged for 2 min ² .

¹ For example, with a plastic spatula or by gently inverting the cuvette closed with Parafilm®.

² The reaction has stopped when the absorbance value is constant, continue reading the absorbance values until the values increase continuously over 2 min. If the absorbance continues to increase, this may be due to the influence of colored compounds or enzymes in the sample. These interfering substances can be removed during sample preparation.

Calculation.

Let us determine the absorbance difference (A1-A2) for the blank sample and the sample. Subtract the absorbance difference of the blank sample from the absorbance difference of the sample, thereby obtaining ΔA _{citric acid} .

DAcitric acid = (A1 ^_ A2) sample or standard ^- (A1 ^_ A2) blank

The ΔA value _{of citric acid} should generally be at least 0.100 absorbance units to provide reasonably accurate results.

The concentration of citrate in a diluted sample, C _{diluted sample} , can be calculated as follows:

C diluted sample ⁼ ((V * MW)/(E * d * U * 1000)) * DAcitric acid where V=final volume (ml) [3.02 ml], v=volume of diluted sample (ml) [0, 02 ml],

MW=molecular weight of citric acid [192.10 g/mol], ε=NADPH extinction coefficient at 340 nm=6.3 [1mmol' ¹ xcm' ¹ ], d=light path (cm) [ 1 cm].

Hence it follows that

Diluted sample (3.02x192.10)/(6.3x1x0.2x1000)) x ΔΑcitric acid [g/l] = 0.4604 x ΔΑcitric acid [g/l]

The concentration (% w/w) of citrate in the original sample, C _{original sample} , is determined using the following formula:

Original sample [% VOC/VOC] = (0.46 X DAcitric acid)/GPi original sample)

The determination of citrate was always performed in duplicate.

Example 1.4. Determination of the total amount of proteins.

The total protein content (actual protein) in the sample was determined as described below.

1) Total nitrogen was determined in the sample according to ISO 8968-1/2|IDF 020-1/2 Milk. Determination of nitrogen content. Part 1/2. Determination of nitrogen content using the Kjeldahl method.

2) Non-protein nitrogen was determined in the sample according to ISO 8968-4|IDF 020-4 Milk. Determination of nitrogen content. Part 4. Determination of the content of non-protein nitrogen.

3) Calculate the total amount of protein as ( _total a ₃ from -m non-protein nitrogen) ^x 6.38.

Example 1.5. Determination of the amount of casein.

The amount of casein was determined according to ISO 17997-1:2004, Milk. Determination of nitrogen content in casein. Part 1. Indirect method (reference method).

Example 1.6. Determination of the amount of whey protein in milk.

The amount of whey protein (or whey protein) in the sample was calculated as the amount of total protein minus the amount of casein.

Example 2 Processing of Organic Skimmed Milk.

Dairy raw materials.

1200 kg of pasteurized (73°C/15 s) organic skimmed milk (raw milk) was preheated to 52°C. The pH of the milk raw material was 6.7.

Microfiltration.

The preheated milk raw material was subjected to microfiltration (MF) in a periodic re

- 25 040098 press on a polymeric FR membrane from Synder Filtration (USA), which is characterized by a pore size corresponding to 800 kDa at 50°C and with a transmembrane pressure (TMP) of 0.45 bar. After 720 L of permeate had been collected, diafiltration was started by adding reverse osmosis (RO) filtered tap water to the retentate at the same flow rate as the MF permeate. After adding 3,000 L of RO-filtered tap water, the filtration was completed. Collected 480 kg of MF-retentate and 3720 kg of MF-permeate. 98% lactose and 79% milk whey protein from milk raw materials were collected in MF-permeate, and 97% micellar casein from milk raw materials was collected in MF-retentate.

Nanofiltration.

The MF permeate was concentrated by nanofiltration (NF) and subjected to NF/diafiltration on a batch basis at 10°C on an NF245 membrane from DOW Chemical using a TMP of 19 bar. After collecting 3,000 L of NF-permeate, diafiltration was started by adding RO-filtered tap water to the NF-retentate at the same flow rate as the NF-permeate flow rate. After adding 1.420 L of RO-filtered tap water, the diafiltration was terminated. The diafiltered NF-retentate was finally concentrated to provide 360 kg of NF-retentate. The concentrated NF-retentate contained 97% lactose and 66% whey protein from milk raw materials. During the NF/DIA process, approximately 86% of the monovalent ions (Na, K and Cl) and 13% of the polyvalent ions (Ca, Mg and P) of the MF permeate were transferred to the NF permeate. The pH of the NF retentate was about 6.7.

Procedure for reducing the amount of inorganic polyvalent ions.

The NF-retentate was then heated to 65°C and kept at this temperature for 40 min, after which it was cooled to 10°C. This heat treatment caused calcium and magnesium salts to precipitate and the precipitated salts were removed by batch MF filtration at 10° C. on a Tami 1.4 μm ceramic membrane with a total TMP gradient. When 330 L of permeate was collected, the retentate was diafiltered with 30 L of RO-filtered tap water, which was added at the same flow rate as the permeate flow rate. In total, 360 kg of MF-permeate (referred to as demineralized NF-retentate) and 30 kg of MF-retentate were collected. Collected 97% lactose and 66% whey protein from milk raw materials in demineralized NF-retentate. During MF filtration, approximately 44% of the polyvalent ions (Ca, Mg and P) were transferred to the MF retentate.

Obtaining the product as an organic infant formula.

360 kg of Demineralized NF-retentate was mixed with 202 kg of organic skimmed milk and 36.3 kg of organic vegetable fat mixture. This mixture was pasteurized, evaporated and spray dried to give 128 kg of final organic infant formula powder with a whey protein/casein ratio of 62/38 and an energy value of 2130 kJ per 100 g of powder. The compositions of milk raw materials, purified NF-retentate and infant formula are shown in table. 1.

Table 1

Compositions of dairy raw materials, . demineralized NF-retentate and . infant formula

Component Unit Dairy raw materials Demineralized NF-retentate Food fusion for kids Protein % 3.4 2.0 11.3 Protein representing casein % 2.6 0.1 4.3 whey protein % 0.9 1.9 7.0 NPN*6.25 % 0.2 0.2 0.8 Lactose % 4.8 15.6 51.4 Fat % <0.1 <0.1 28.3 Ash substances % 0.7 0.5 2.6 Dry matter % 9.3 18.6 97.0 Calcium % 0.12 0.08 0.42 Magnesium % 0.01 0.01 0.05 Phosphorus % 0.09 0.08 0.38 Sodium % 0.05 0.02 0.13 Potassium % 0.16 0.06 0.43 Chlorine % 0.10 0.04 0.28

Typically, additional organic functional ingredients such as, for example, vitamins, nucleotides, oligosaccharides and polyunsaturated fatty acids (PUFAs), etc., are added to the infant formula.

- 26 040098

Obtaining a product based on organic milk containing calcium phosphate.

kg of the MF retentate obtained from the inorganic polyvalent ion reduction step was evaporated and spray dried to obtain 1.1 kg of a mineral-containing organic milk product containing a significant amount of calcium, magnesium and phosphorus. This product can be used to enrich all types of organic food with milk minerals and especially calcium, magnesium and phosphorus.

Example 3 Processing of Concentrated Organic Skimmed Milk.

Dairy raw materials.

500 kg Pasteurized (73°C/15 s) organic concentrated skimmed milk containing 6.4% wt./weight. proteins, 4.7% wt./wt. lactose, 0.1% w/w fats and 12.5% wt./weight. all solids (raw milk), preheated to 52°C. The pH of the raw milk was 6.7.

Microfiltration.

The preheated milk raw material was subjected to batch microfiltration (MF) on a polymeric FR membrane from Synder Filtration (USA), which has a pore size corresponding to 800 kDa at 50°C and a transmembrane pressure (TMP) of 0.45 bar. After collecting 100 L of permeate, diafiltration was started by adding reverse osmosis (RO) filtered tap water to the retentate at the same flow rate as that of the MF permeate. After adding 3,000 L of RO-filtered tap water, the filtration was completed. Collected 400 kg of MF-retentate and 3,100 kg of MF-permeate. 96% lactose and 78% whey protein from milk raw materials were collected in MF-permeate and 98% micellar casein from milk was collected in MF-retentate.

Nanofiltration.

The MF permeate was concentrated by nanofiltration (NF) and subjected to NF/diafiltration on a batch basis at 10°C on an NF245 membrane from DOW Chemical using a TMP of 19 bar. After collecting 2.790 L of NF-permeate, diafiltration was started by adding RO-filtered tap water to the NF-retentate at the same flow rate as the NF-permeate flow rate. After adding 775 L of RO-filtered tap water, the diafiltration was terminated. The diafiltered NF-retentate was finally concentrated to provide 155 kg of NF-retentate. The concentrated NF-retentate contained 95% lactose and 72% whey protein from milk raw materials. During the NF/DIA process, approximately 78% of the monovalent ions (Na, K and Cl) and approximately 11% of the polyvalent ions (Ca, Mg and P) of the MF permeate were transferred to the NF permeate. The pH of the NF retentate was about 6.7.

Procedure for reducing the amount of inorganic polyvalent ions.

The concentrated NF-retentate was then heated to 65°C and kept at this temperature for 40 min, after which it was cooled to 10°C. This heat treatment caused calcium and magnesium salts to precipitate and the precipitated salts were removed by batch MF filtration at 10° C. on a Tami 1.4 μm ceramic membrane with a total TMP gradient. When 140 L of permeate was collected, the retentate was diafiltered with 15 L of RO-filtered tap water, which was added at the same flow rate as the permeate flow rate. In total, 155 kg of MF-permeate (referred to as demineralized NF-retentate) and 15 kg of MF-retentate were collected. Collected 94% lactose and 72% whey protein from milk raw materials in demineralized NF-retentate. During MF filtration, approximately 54% of the polyvalent ions (Ca, Mg and P) were transferred to the MF retentate.

Getting the product in the form of infant formula.

155 kg of Demineralized NF-retentate was mixed with 42 kg of skimmed milk, 17.6 kg of vegetable fat mixture and 8.1 kg of GOS syrup with 71% dry matter. This mixture was pasteurized, evaporated and spray dried to give 60 kg of final infant formula powder with an 81/19 milk/casein whey protein ratio and an energy value of 2160 kJ per 100 g of powder. The compositions of skimmed milk, raw milk, demineralized NF-retentate and infant formula are shown in table. 2.

- 27 040098

table 2

Compositions of skimmed milk, milk raw materials, demineralized NF-retentate and infant formula

Component Unit Dairy raw materials Skimmed milk Demineralized NF-retentate Food fusion for kids Protein % 6.4 3.2 3.6 11.7 Protein representing casein % 4.9 2.4 0.2 2.1 whey protein % 1.5 0.8 3.5 9.6 NPN*6.25 % 0.2 0.2 0.2 0.6 Lactose % 4.7 4.6 14.3 45.2 Fat % 0.1 <0.1 <0.1 29.3 Ash substances % 1.0 0.7 0.6 2.0 Dry matter % 12.5 8.8 19.0 97.0 Calcium % 0.20 0.12 0.11 0.36 Magnesium % 0.02 0.01 0.01 0.04 Phosphorus % 0.14 0.09 0.09 0.30 Sodium % 0.05 0.05 0.03 0.12 Potassium % 0.17 0.16 0.10 0.37 Chlorine % 0.11 0.10 0.06 0.24 GOS 4.8

Typically additional organic functional ingredients such as, for example, vitamins, nucleotides and polyunsaturated fatty acids (PUFAs), etc. added to infant formula.

Obtaining a product based on organic milk containing calcium phosphate.

kg of the MF retentate obtained from the inorganic polyvalent ion reduction step was evaporated and spray dried to obtain 0.7 kg of a mineral-containing organic milk product containing a significant amount of calcium, magnesium and phosphorus. This product can be used to enrich all types of organic food with milk minerals and especially calcium, magnesium and phosphorus.

Conclusion.

From both Example 2 and Example 3, it can be concluded that the combination of microfiltration, nanofiltration and subsequent removal of inorganic, polyvalent ions (for example, by mineral precipitation) surprisingly provides an effective alternative to the combination of microfiltration, ultrafiltration and nanofiltration. In general, the present invention makes it possible to avoid the separation of milk saccharide and whey protein in the whey protein containing stream that is obtained after the microfiltration step, and thus a much simpler process is obtained.

Example 4 Preparation of Low Mineral Organic Lactose Concentrate (LMLC).

This example describes the preparation of low mineral organic lactose concentrate (LMLC), which is used in example 5.

source of milk.

2000 kg of Pasteurized (73°C/15 s) organic skimmed milk (milk source) was preheated to 10°C. The pH of the milk source was 6.7.

The preheated milk source was subjected to batch ultrafiltration (UF) on a GR73PE polymer membrane from Alfa Laval (Denmark), which has a pore size corresponding to 10 kDa at 10° C. and a transmembrane pressure (TMP) of 4.0 bar. After collecting 1000 l of UF-permeate, the filtration was completed. 1000 kg of UF retentate and 1000 kg of UF permeate were collected. Collected 49% lactose and 45% NPN from milk source in UF-permeate and collected >99% casein and whey protein from milk source in UF-retentate.

The protein-free UF permeate was concentrated by nanofiltration (NF) and subjected to NF/diafiltration on a batch basis at 10° C. on an NF245 membrane from DOW Chemical using a TMP of 19 bar. After collecting 730 L of NF-permeate, diafiltration was started by adding RO-filtered tap water to the NF-retentate at the same flow rate as the NF-permeate flow rate. After adding 3,000 L of RO-filtered tap water, the diafiltration was terminated. The diafiltered NF-retentate is finally con

- 28 040098 centered to provide 270 kg of NF-retentate. The pH of the NF retentate was about 6.7. The concentrated NF retentate contained 98% lactose and 48% NPN from the UF permeate. During the NF/DIA process, approximately 73% of the monovalent ions (Na, K and Cl) and approximately 10% of the polyvalent ions (Ca, Mg and P) of the UF permeate were transferred to the NF permeate.

The concentrated NF-retentate was then heated to 80°C and kept at this temperature for 45 min, after which it was cooled to 10°C. This heat treatment caused calcium and magnesium salts to precipitate and the precipitated salts were removed by batch MF filtration at 10° C. on a Tami 1.4 μm ceramic membrane with a total TMP gradient. When 240 L of permeate was collected, the retentate was diafiltered with 30 L of RO-filtered tap water, which was added at the same flow rate as the permeate flow rate. A total of 270 kg of MF permeate (referred to as low mineral lactose concentrate (LMLC)) was collected. Collected 98% lactose and 45% NPN from UF-permeate in LMLC. During MF filtration, approximately 61% of the polyvalent ions (Ca, Mg and P) were transferred to the MF retentate.

Application of LMLC in infant formula.

This LMLC can be used as a source of lactose in infant formula due to its low mineral content and high lactose content in dry matter up to 93%. The compositions of the milk source (organic skimmed milk) and LMLC are shown in Table 1. 3.

Table 3 ______Compositions of raw milk and demineralized NF-retentate______

Component Unit Skimmed milk LMLC Protein % 3.4 0.3 The protein that is casein % 2.6 <0.1 whey protein % 0.9 <0.1 NPN*6.25 % 0.2 0.3 Lactose % 4.8 17.1 Fat % <0.1 <0.1 Ash substances % 0.7 0.4 Dry matter % 9.2 18.3 Calcium % 0.12 0.03 Magnesium % 0.01 <0.01 Phosphorus % 0.09 0.08 Sodium % 0.05 0.04 Potassium % 0.16 0.14 Chlorine % 0.10 0.09

Example 5 Preparation of an infant formula product.

Getting the product in the form of infant formula.

157 kg of LMCL from example 4 was mixed with 80 kg of raw milk from example 3 (source of casein), 155 kg of demineralized NF-retentate from example 3 and 27.3 kg of vegetable fat. This mixture was pasteurized, evaporated and spray dried to give 99 kg of final infant formula powder with a whey protein/casein ratio of 63/37 and an energy value of 2145 kJ per 100 g of powder. The compositions of milk raw materials from example 2, demineralized NF-retentate from example 3, LMLC from example 4 and infant formula are shown in table. 4.

- 29 040098

Table 4

Compositions of milk raw materials, demineralized NF-retentates and infant formula

Component Unit Concentrated milk raw materials (note 3) Low Mineral Lactose Concentrate (Note 4) Demineralized NF-retentate (Note 3) Food fusion for kids Protein % 6.3 0.3 3.6 11.2 The protein that is casein % 4.8 <0.1 0.2 4.1 whey protein % 1.5 0.3 3.5 7.1 NPN*6.25 % 0.2 0.3 0.2 0.9 Lactose % 4.7 17.1 14.3 53.3 Fat % 0.1 <0.1 <0.1 27.6 Ash substances % 1.0 0.4 0.6 2.3 Dry matter % 12.5 18.31 19.0 97.1 Calcium % 0.20 0.02 0.11 0.36 Magnesium % 0.02 <0.01 0.01 0.04 Phosphorus % 0.14 0.08 0.09 0.38 Sodium % 0.05 0.04 0.03 0.16 Potassium % 0.17 0.14 0.10 0.51 Chlorine % 0.11 0.09 0.06 0.33

Typically, additional functional ingredients such as, for example, vitamins, nucleotides, oligosaccharides and polyunsaturated fatty acids (PUFAs), etc., are added to the infant formula.

Conclusion.

From this example and example 4, it can be concluded that low mineral nutritional products such as infant formula can be effectively obtained by combining concentrated skim milk (from example 3), low mineral lactose concentrate (from example 4) and demineralized NF-retentate (from example 3).

Example 6 Preparation of an organic ingredient for infant formulas.

Preparation of an organic ingredient for use in organic infant formula products.

218 kg of the Dairy Raw Material from Example 2 was mixed with 360 kg of the demineralized NF-retentate from Example 2. The mixture was evaporated, pasteurized and spray dried to give 92 kg of an organic infant formula ingredient with a whey protein/casein ratio of 60/40, which can be be used to obtain the final product in the form of infant formula by adding vegetable fat and/or cream to the ingredient. The compositions of milk raw materials, demineralized NF-retentate and ingredient for infant formula are shown in table. 5.

Table 5

Compositions of milk raw materials, demineralized NF-retentate and ingredient for infant formulas

Component Unit Dairy raw material Demineralized NF-retentate Ingredient for infant formula Protein % 3.5 2.1 16.4 Protein representing casein % 2.6 0.1 6.6

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whey protein % 0.9 2.0 9.9 NPN*6.25 % 0.2 0.2 1.2 Lactose % 4.8 15.6 72.2 Fat % <0.1 <0.1 0.1 Ash substances % 0.7 0.5 3.7 Dry matter % 9.3 18.7 95.0 Calcium % 0.12 0.08 0.60 Magnesium % 0.01 0.01 0.07 Phosphorus % 0.09 0.08 0.54 Sodium % 0.05 0.02 0.20 Potassium % 0.16 0.06 0.62 Chlorine % 0.10 0.04 0.40

Conclusion.

From this example, it can be concluded that the combination of demineralized NF-retentate (an example of a demineralized whey protein product containing milk saccharide according to the present invention) and a casein source (e.g. skimmed milk) are attractive ingredients for nutritional products. such as, for example, infant formula products.

Example 7. Obtaining a demineralized product based on whey protein containing milk saccharide.

Obtaining an ingredient for use in infant formula products.

155 kg of the Demineralized NF Retentate from Example 3 was pasteurized, evaporated and spray dried to give 30 kg of a demineralized whey protein product containing milk saccharide with a whey protein/casein ratio of 95:5, which can be used to obtain the final an infant formula product by adding a source of casein, vegetable fat and/or cream to the ingredient. The compositions of the demineralized NF retentate and the infant formula ingredient are shown in Table 1. 6.

Table 6

Compositions of milk raw materials, demineralized NF-retentate and ingredient

for infant formula Component Unit Demineralized NFretentate Demineralized product based on whey protein containing milk saccharide Protein % 3.7 18.5 Protein representing casein % 0.2 0.8 whey protein % 3.5 17.6 NPN*6.25 % 0.2 0.9 Lactose % 14.3 71.5 Fat % <0.1 <0.1 Ash substances % 0.6 2.8 Dry matter % 19.0 95.0 Calcium % 0.11 0.53 Magnesium % 0.01 0.06 Phosphorus % 0.09 0.46 Sodium % 0.03 0.16 Potassium % 0.10 0.50 Chlorine % 0.06 0.32

Conclusion.

From this example, it can be concluded that demineralized NF-retentate (an example of a demineralized whey protein product containing milk saccharide according to the present invention) as such is an attractive ingredient for obtaining nutritional products, such as, for example, products in the form infant formula and other products in which

- 31 040098 the use of a combination of milk saccharides and undenatured whey protein is reasonable.

Example 8 Preparation of infant formula based on organic skimmed milk.

source of milk.

1500 kg Pasteurized (73°C/15 s) organic skimmed milk (milk source) was preheated to 10°C. The pH of the milk source was about 6.7.

Concentration of the milk source.

The preheated milk source was concentrated by batch filtration with a GR73PE polymer membrane from Alfa Laval (Denmark) having a pore size corresponding to 10 kDa at 10° C. and a transmembrane pressure (TMP) of 4.0 bar. After collecting 1000 liters of conc. permeate filtration was stopped. Collected 500 kg of conc. retentate and 1000 kg of conc. permeate. Collected 67% lactose and 65% NPN from the milk source in conc. permeate and collected >99% casein and whey proteins from the milk source in conc. retentate.

Nanofiltration conc. permeate for lactose purification.

Conc. the permeate was concentrated by nanofiltration (NF) and subjected to NF/diafiltration on a batch basis at 10°C on an NF-FF membrane from DOW Chemical using a TMP of 19 bar. The first 300 kg of NF-permeate was collected to be used later to dilute conc. retentate. After collecting 440 L of NF-permeate, diafiltration was started by adding RO-filtered tap water to the NF-retentate at the same flow rate as the NF-permeate flow rate. After adding 1680 L of RO-filtered tap water, NF filtration was stopped and 560 kg of NF retentate was provided. The concentrated NF retentate contained 98% lactose and 47% NPN from conc. permeate. During the NF/DIA process, about 53% monovalent ions (Na, K and Cl) and about 5% polyvalent ions (Ca, Mg and P) conc. permeate was transferred to NF-permeate.

Additional purification of lactose obtained from concentrated permeate.

The concentrated NF-retentate, mainly containing water, lactose, polyvalent ions and the remaining monovalent ions, was then heated to 80°C and kept at this temperature for 45 min, after which it was cooled to 10°C. This heat treatment caused the precipitation of salts containing phosphorus, calcium and magnesium, and the precipitated salts were removed by MF in batch mode at 10°C on a ceramic 1.4 μm membrane from Tami with a total TMP gradient. When 520 L of permeate was collected, the retentate was diafiltered with 40 L of RO-filtered tap water, which was added at the same flow rate as the permeate flow rate. A total of 560 kg of MF permeate (referred to as low mineral lactose concentrate, LMLC) was collected. Collected 97% lactose and 47% NPN from conc. permeate in LMLC. During MF filtration, approximately 40% of the polyvalent ions (Ca, Mg and P) were removed into the MF retentate.

Dairy raw materials.

Milk raw materials were obtained by mixing 500 kg of the above conc. retentate with 300 kg of the above NF-permeate and the mixture was preheated to 52°C. 800 kg Milk raw materials contained 9.3% dry matter, including 5.6% wt./weight. protein and 2.7% wt./weight. lactose. The pH of the raw milk was about 6.7.

Microfiltration of dairy raw materials.

The preheated milk raw material was subjected to microfiltration (MF) in batch mode on a polymeric FR membrane from Synder Filtration (USA), which is characterized by a pore size corresponding to 800 kDa at 50°C and with a transmembrane pressure (TMP) of 0.45 bar. After collecting 300 L of permeate, diafiltration was started by adding the above NF-permeate from the nanofiltration with conc. permeate into the present MF-retentate at the same flow rate as the flow rate of the MF-permeate. After adding 2000 l of NF-permeate, the filtration was stopped. Collected 500 kg of MF-retentate and 2300 kg of MF-permeate. Collected 96% lactose and 75% total whey protein from raw milk in MF-permeate and collected 97% micellar casein from milk in MF-retentate.

Nanofiltration of MF-permeate.

The MF permeate was concentrated by nanofiltration (NF) and subjected to NF/diafiltration on a batch basis at 10° C. on an NF-FF membrane from DOW Chemical as follows.

After collecting 300 L of NF-permeate, the NF-process was started to deliver NF-permeate as a diluent for MF/DIA of raw milk at the same flow rate as the flow rate of MF-permeate. Initially, the TMP was 6 bar and it was raised to 15 bar.

When MF/DIA was stopped, NF continued with concentration and diafiltration of the MF permeate. First, the MF-permeate was concentrated to 9% dry matter. NF/diafiltration was then started by adding RO-filtered tap water to the NF retentate at the same flow rate as the NF permeate flow rate. After adding 900 L of RO-filtered tap water, the diafiltration was terminated. The diafiltered NF-retentate was finally concentrated to provide 180 kg of NF-retentate. The concentrated NF-retentate contained 95% lactose and 62% whey protein from milk raw materials.

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Procedure for reducing the amount of inorganic polyvalent ions in whey protein-containing NF concentrate.

The concentrated NF-retentate was heated to 65°C and kept at this temperature for 40 min, after which it was cooled to 10°C. This heat treatment caused the calcium, magnesium and phosphorus salts to precipitate, and the precipitated salts were removed by centrifugation. The resulting supernatant was called demineralized NF-retentate. 94% lactose and 61% total whey protein from milk raw materials were collected in demineralized NF-retentate, which is an example of a demineralized whey protein product containing milk saccharide according to the present invention.

Getting the product in the form of infant formula.

An infant formula product can be obtained from the above product streams by mixing 180 kg of demineralized NF-retentate with 70 kg of the above conc. retentate (concentrated skimmed milk), 374 kg LMLC, 33.2 kg mixture of vegetable fats and 15.1 kg GOS syrup containing 71% dry matter. This mixture was pasteurized, evaporated and spray dried to give 118 kg of final infant formula powder with a whey protein/casein ratio of 62/38 and an energy value of 2070 kJ per 100 g of powder. Compositions of skimmed milk (milk source), conc. retentate (used both as a source of casein for infant formula and milk raw material for MF-fractionation), LMLC, demineralized NF-retentate and infant formula are shown in table. 7.

Table 7

Compositions of organic skimmed milk (milk source), conc. retentate (used both as the milk raw material for MF fractionation and as a source of casein for infant formula), LMLC (low mineral lactose concentrate) demineralized NF retentate and infant formula_____

Component Unit Skimmed milk Conc. retentate LMC M Demineralized NF retentate Food fusion for kids Protein % 3.02 8.97 0.14 3.60 11.20 The protein that is casein % 2.24 6.91 <0.0 5 0.11 4.25 total whey protein % 0.68 2.06 0.14 3.49 6.10 NPN*6.25 % 0.17 0.18 0.14 0.20 0.85 Lactose % 4.24 4.24 7.38 11.23 47.30 Fat % 0.05 0.15 <0.0 5 <0.05 28.10 Ash substances % 0.69 1.14 0.45 0.90 3.46 Dry matter % 8.07 14.66 8.84 16.61 97.00 Calcium % 0.11 0.26 0.02 0.10 0.36 Magnesium % 0.01 0.02 0.01 0.02 0.08 Phosphorus % 0.08 0.19 0.05 0.12 0.44 Sodium % 0.03 0.03 0.03 0.05 0.20 Potassium % 0.14 0.16 0.15 0.26 0.97 Chlorine % 0.08 0.07 0.02 0.03 0.15 GOS 4.5

As indicated above, additional organic functional ingredients such as, for example, vitamins, nucleotides and polyunsaturated fatty acids (PUFAs), etc., are usually added to infant formula.

Conclusion.

It has been shown that an organic infant formula product can be obtained by MF fractionation without the use of ultrafiltration for whey protein containing streams that are obtained after MF fractionation of milk raw materials. The method has been shown to provide a high yield of whey protein and lactose from milk raw materials and still provide a sufficient degree of demineralization that is suitable for obtaining nutritional

-33 040098 products such as infant formula. The procedure for reducing the amount of polyvalent inorganic ions by mineral precipitation has proven to be particularly useful and provides a significant simplification of the process relative to prior art methods.

An even higher whey protein yield can be obtained with a higher MF/DIA in step b). The level of monovalent ions in the above infant formula product or the above demineralized NF-retentate can also be further reduced by washing out more monovalent ions during the NF/DIA in step c).

Example 9 Preparation of an infant formula based on organic low-citrate skimmed milk using electrodialysis.

source of milk.

67.962 kg Pasteurized (73°C/15s) Organic Skimmed Milk.

Preconcentration of milk raw materials by means of ultrafiltration (UF).

The milk source was subjected to continuous ultrafiltration (UF) on an HFK131 polymer membrane from Koch (USA) with a cut-off value of 10 kDa at 10° C. and a transmembrane pressure (TMP) of 3.5 bar. The degree Brix of the UF retentate was adjusted to 17.6 with a control valve controlled by a refractometer. 27.202 kg of retentate and 41.950 kg of permeate were collected, giving a concentration factor (CF) of 2.53.

Concentration of UF permeate by nanofiltration (NFI).

The UF permeate was adjusted to pH 5.8 with CO ₂ and concentrated by continuous nanofiltration (NF) at 10° C. on a DOW Chemical NF245 membrane using a TMP of 19 bar. The degree Brix of the NF retentate was adjusted to 23.0 with a control valve controlled by a refractometer. 7.350 kg of retentate was collected and the permeate was discarded.

Demineralization of the NFI retentate by electrodialysis (EDI).

NF-retentate from NFI was demineralized in a batch mode using an electrodialysis (ED) P15 EWDU 1x EDR-II/250-0.8 unit from MEGA (Czech Republic) at 10°С. The ED unit was equipped with Ralex CM(H)-PES cation exchange membranes and Ralex AM(H)-PES anion exchange membranes.

The electrolyte used for the flows at the electrodes contained 15.6 g/l NaNO ₃ .

The citrate permeability selectivity for AM(H)-PES anion exchange membranes was evaluated to be significantly greater than 0.01.

The ED process was stopped when the ratio between conductivity (cm/cm) and degree Brix (conductivity divided by degree Brix) reached 0.034 in the dilute solution (product), corresponding to a decrease in conductivity of about 85% (from 2.78 to 0.424 mS). Collected 6,615 kg of demineralized NF-retentate (lactose) and cooled to 6°C. The composition of the lactose used to standardize the lactose in the final product can be seen in Table 1. 8.

When the ED was stopped, the concentrate stream had the following characteristics:

ash materials: 1.83% w/w;

amount of citrate: 1.61% w/w;

amount of Ca: not measured;

amount of Mg: 0.054% w/w;

amount of Cl: 0.04% w/w;

amount of Na: 0.106% w/w;

amount of K: 0.331% w/w; and the amount of P: 0.14% wt./wt.

A demineralized dilute solution (milk saccharide demineralized product) has been shown to typically contain 0.02 g sialyllactose/100 g.

Microfiltration (filtration with the removal of microorganisms) of the UF retentate.

The UF retentate was preheated to 55°C and filtered through a 1.4 micron ceramic isoflux membrane from TAMI (France) continuously at 50°C to give a TMP starting at 0.5 bar and increasing to 0.8 bar . Microfiltration was carried out with a concentration factor (CF) of 40.

27.100 kg of Permeate (MPC) was cooled to 6°C and collected. The composition of MPC, which was used as a source of casein in the final product, can be seen in table. 8. Filtered to remove microorganisms, the UF retentate was used as a milk raw material for protein MF fractionation.

Fractionating protein microfiltration of raw milk.

27,000 kg Chilled milk raw material (filtered to remove UF retentate microorganisms) was preheated to 55°C and subjected to continuous microfiltration (MF) using a polymeric MF membrane with a pore size of approximately 0.1 micron and a narrow pore size distribution at 50° C. and with a transmembrane pressure (TMP) of 0.45 bar. 500% Diafiltration water was added with continuous filtration in a 4 loop filtration unit. CF was 1.0 during filtration. 27,500 kg of MF-retentate was cooled to 6°C and collected. This retentate contained >99% micellar casein proteins and 20% globular proteins.

- 34 040098 local serum from filtered with the removal of microorganisms UF-retentate. The permeate was cooled to 10° C. and simultaneously concentrated as described in the next section.

Concentration of MF-permeate by nanofiltration (NFII).

The MF permeate was concentrated by continuous nanofiltration (NF) at 10°C on an NF245 membrane from DOW Chemical using a TMP of 19 bar. The degree Brix of the NF retentate was adjusted to 27.0 with a control valve controlled by a refractometer. Collected 5,450 kg of retentate and the permeate was heated to 50°C and used as water for diafiltration in the previous section on MF. Excess permeate was discarded.

Demineralization of NFII retentate by electrodialysis (EDII).

The NF-retentate from NFII was demineralized in batch mode in an electrodialysis unit (ED) P15 EWDU 1x EDR-II/250-0.8 at 10°C. The ED unit was equipped with Ralex CM(H)-PES cation exchange membranes and Ralex AM(H)-PES anion exchange membranes. The ED process was stopped when the ratio between conductivity and Brix reached 0.028 in the dilute solution (product), which corresponded to about 82% reduction in conductivity (from 3.02 mS/cm to 0.541 mS/cm). 4.905 kg of Demineralized NF-retentate (demineralized whey protein concentrate containing milk saccharide, SPC) was collected and cooled to 6°C. The composition of the SPC that was used for the whey protein source in the final product can be seen in Table 1. 8.

As shown, SPC typically contained 0.02 g sialyllactose/100 g.

When the ED was stopped, the concentrate stream had the following characteristics:

ash materials: 1.45% w/w;

amount of citrate: 1.3% w/w;

amount of Ca: 0.286% w/w;

amount of Mg: 0.05% w/w;

amount of Cl: not measured;

amount of Na: 0.127% w/w;

amount of K: 0.19% w/w; and the amount of P: 0.073% wt./wt.

Preparation of the product as a liquid base for infant formula.

10.0 kg Demineralized NFII retentate (SPC), 4.5 kg microorganism-filtered UF retentate (MPC) and 8.5 kg demineralized NFI retentate (lactose) were mixed by gentle mixing in a 50 L stainless steel container . This formula can be used as a base for infant formula as it contains all the casein, whey and lactose proteins needed in infant formula with 65% whey protein and 60% total dry matter in infant formula. The composition of the product in the form of a liquid base for infant formula is shown in table. 8.

Obtaining a powdered base for infant formula.

5.0 kg of Liquid Infant Formula Base was freeze dried in a Telstar, Lyobeta Micrositelab 3.0 freeze dryer to give 1.0 kg of powder. The composition of the powder base for infant formula is shown in table. 8.

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Table 8

Chemical composition of 3 ingredients (lactose, MPC and WPC) and liquid and powder infant formula base

Unit Lactose NFI-ret. MRS UV-ret. SPC NFII-ret. Base for infant formula Liquid Powder Protein g/100 g 0.25 8.18 4.69 3.76 18.63 Casein g/100 g 0.00 6.54 0.00 1.32 6.52 whey protein g/100 g 0.25 1.64 4.69 2.44 12.11 Whey Protein vs. Total Protein g/100 g 100 20 100 65 65 Lactose g/100 g 19.35 4.81 15.72 15.10 74.21 Fat g/100 g <0.04 0.12 <0.04 <0.04 0.12 Dry matter g/100 g 20.21 14.51 21.19 19.44 96.00 Ash substances g/100 g 0.19 1.16 0.15 0.35 1.85 pH 5.54 6.68 6.00 6.43 6.58 Citrate g/100 g 0.16 0.23 0.17 0.17 0.81 Calcium g/100 g 0.031 0.249 0.054 0.087 0.411 Magnesium g/100 g 0.007 0.017 0.011 0.011 0.053 Chloride g/100 g 0.04 0.08 0.04 0.04 0.08 Sodium g/100 g 0.010 0.036 0.014 0.017 0.080 Potassium g/100 g 0.012 0.165 0.013 0.043 0.207 Phosphorus g/100 g 0.029 0.177 0.027 0.059 0.280 Copper mg/kg <0.1 <0.1 <0.1 <0.1 0.26 Zinc mg/kg <0.5 9.6 <0.5 1.8 12.0 Iodine mg/kg <0.05 0.16 <0.05 <0.05 <0.05 Selenium mg/kg <0.005 0.035 0.018 0.015 0.035 Molybdenum mg/kg 0.006 0.073 0.107 0.062 0.327 Manganese mg/kg <0.1 <0.1 <0.1 <0.1 <0.1 Urea mg/100 g 15.3 18.5 7.0 11.9 55.6 Vitamin B2 mg/100 g 0.40 0.25 0.616 0.497 2.06 Vitamin B5 mg/100 g 1.02 0.44 0.23 0.56 2.80 Vitamin Wb mg/100 g 0.061 0.045 0.063 0.050 0.258 Vitamin B8 mcg/100 g <1 <1 <1 <1 4.25 Vitamin B12 mcg/100 g <0.25 0.97 0.79 0.53 2.48 Choline mg/kg 340 119 265 259 1310 Cholesterol mg/100 g <1 11.9 <1 1.2 4.3 Myoinositis mg/100 g 11.4 4.42 8.58 8.65 42.1

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Carnitine mg/kg 80.6 19.9 58.4 58.3 291 Serene g/16 g N - 5.76 4.71 4.95 4.68 Glutamic acid g/16 g N - 22.13 17.95 19.12 18.36 Proline g/16 g N - 9.99 5.01 6.91 6.87 Glycine g/16 g N - 1.92 1.92 1.86 1.81 Alanine g/16 g N - 3.47 5.14 4.31 4.11 Valine g/16 g N - 6.58 5.29 5.59 5.42 Isoleucine g/16 g N - 5.32 5.50 5.13 5.36 Leucine g/16 g N - 10.32 13.13 11.46 10.95 Tyrosine g/16 g N - 4.87 2.92 3.48 3.35 Phenylalanine g/16 g N - 5.06 3.65 4.18 4.09 Lysine g/16 g N - 8.84 11.3 9.65 9.29 G istidine g/16 g N - 2.90 2.22 2.34 2.31 Arginine g/16 g N - 3.58 2.49 2.69 2.73 Aspartic acid g/16 g N - 8.09 12.41 10.00 9.66 Threonine g/16 g N - 4.56 5.29 4.71 4.56 tryptophan g/16 g N - 1.42 2.41 1.95 1.88 Cysteine g/16 g N - 0.71 2.69 1.81 1.93 Methionine g/16 g N - 2.70 2.35 2.15 2.47 Sum g/16 g N - 108.2 106.38 102.59 99.84

Conclusion.

The present inventors have observed indications that prior art infant formulas based on MF fractionation of milk contain an unexpectedly high amount of citrate. The present inventors investigated the reasons for this and found that citrate is not eliminated from whey protein streams or lactose-containing streams by NF-based demineralization, unless the NF pore size is chosen such that lactose is also removed.

However, the present inventors have found that by using electrodialysis and selecting electrodialysis membranes that allow passage not only of chloride and phosphate but also of citrate, the amount of citrate can be reduced without wasting lactose, which is a valuable carbohydrate for infant formulas. The present invention provides an efficient process for preparing low citrate infant formula bases and final low citrate infant formula while avoiding the separation of milk saccharides from the whey protein stream.

The present invention also has the distinct advantage that fluctuations in citrate content in raw milk are reduced and resulting infant formulas have a more stable citrate content. Citrate has been shown to affect the bioavailability of, for example, iron, calcium, magnesium and zinc (Glahn et al., Fairweather-Tait). Thus, the present invention provides the possibility of obtaining infant formulas that provide infants with a more uniform bioavailability of the above metal ions.

Links.

APV Membrane filtration and related molecular separation technologies published by APV Systems, 2000, ISBN 87-88016 757.

Fairweather-Tait et al., Iron and Calcium Bioavailability of Fortified Foods and Dietary Supplements, Nutrition Reviews®, Vol. 60, no. 12, November 2002: 360-367.

Glahn et al. _f Decreased Citrate Improves Iron Availability from Infant Formula:

Application of an In Vitro Digestion/Caco-2 Cell Culture Model, J. Nutr. 128:257-264, 1998.

Sata 2004 Ion Exchange Membranes Preparation, characterization, modification and application, Toshikatsu Sata, The Royal Society of Chemistry, 2004, ISBN 0-85404-590-2.

Tanaka 2015

Ion exchange membranes Fundamentals and Applications, Yoshinobu Tanaka, ^2nd edition, Elsevier, 2015, ISBN: 978-0-44463319-4.

Claims (18)

CLAIM 1. A method for producing a nutritional product comprising a demineralized whey protein product containing milk saccharide and a source of casein, the method comprising the steps a) providing milk raw materials; b) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration, thereby obtaining micellar casein enriched MF retentate and whey protein enriched MF permeate; c) subjecting the MF permeate to nanofiltration (NF) or nanofiltration/diafiltration using a membrane that allows passage of monovalent ions but retains milk saccharide, thereby obtaining NF retentate and NF permeate; d) subjecting the NF retentate to electrodialysis, thereby obtaining a demineralized whey protein product containing milk saccharide, which is characterized by a reduced level of calcium, magnesium and phosphorus; e) adding a casein source to a demineralized whey protein product containing milk saccharide to form a nutritional product. 2. The method according to claim 1, where the raw milk contains a total amount of milk saccharide in the range of 1-10% wt./weight. 3. The method according to any of the previous paragraphs, where the raw milk contains a total amount of proteins in the range of 1-12% wt./weight. 4. The method according to any one of the preceding claims, wherein the milk raw material contains or even consists of whole milk, skimmed milk, low fat milk or concentrated milk. 5. The method according to any one of the preceding claims, wherein step a) comprises the step of ultrafiltration (UF) and optionally UF/diafiltration of the milk source to produce UF milk retentate and UF milk permeate, wherein at least a portion of the UF milk retentate is used in quality of raw milk. 6. The method according to any of the previous paragraphs, where the NF-retentate contains a total amount of sodium of not more than 0.4% wt./weight. all solids; the total amount of potassium is not more than 1.3% wt./weight. all solids; and the total amount of chlorine is not more than 0.8% wt./weight. all solids. 7. The method according to any one of the preceding claims, wherein the pH of the NF retentate is in the range of 5.5-7.0. 8. The method according to any one of the preceding claims, wherein the anion exchange membrane used for electrodialysis has a citrate selective permeability coefficient of at least 0.01. 9. The method according to any one of the preceding paragraphs, where the demineralized whey protein product containing milk saccharide contains a total amount of calcium of not more than 1.0% wt./weight. all solids; the total amount of magnesium is not more than 0.1% wt./weight. all solids; and the total amount of phosphorus is not more than 0.8% wt./weight. all solids. 10. The method according to claim 9, where the demineralized product based on whey protein containing milk saccharide contains citrate in an amount of not more than 1% wt./weight. all solids. 11. The method according to any one of the preceding claims, wherein the casein source is one or more of milk, in particular concentrated milk or milk powder, milk protein concentrate, beta-casein isolate, micellar casein isolate, caseinate, or combinations thereof. 12. The method according to any one of the preceding claims, wherein the casein source is concentrated milk in the form of UF milk retentate and/or MF milk retentate. 13. The method according to any one of the preceding claims, wherein the demineralized whey protein product containing milk saccharide and the casein source are mixed to achieve a weight ratio of whey protein and casein in the range of 50:50-70:30. 14. The method according to any of the preceding claims, wherein the nutritional product is an organic product. 15. The method according to any one of the preceding claims, wherein step e) further comprises adding one or more functional ingredients to a demineralized whey protein product containing milk saccharide to form a nutritional product. 16. The method according to any one of the preceding claims, further comprising step f) converting the nutritional product into a powder. 17. A method for producing a demineralized whey protein product containing milk saccharide, the method comprising the steps i) provision of milk raw materials; ii) subjecting the milk raw material to microfiltration (MF) or microfiltration/diafiltration with - 38 040098 thereby obtaining MF-retentate and MF-permeate; iii) subjecting the MF permeate to nanofiltration or nanofiltration/diafiltration, thereby obtaining NF retentate and NF permeate; iv) subjecting the NF retentate to electrodialysis, thereby obtaining a demineralized whey protein product containing milk saccharide. 18. The method of claim 17 further comprising step v) drying the demineralized whey protein product containing milk saccharide.