Process for the separation of casein and whey protein from milk

Abstract

The invention relates to a process for the separation of whey protein and casein from milk wherein a) through addition of an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2 to the milk; and / or b) by injection of gaseous CO2 into the milk, to obtain an acidified milk which is subjected to filtration. The invention further relates to a micellar casein isolate (MCI) obtainable by the process and a nutritional composition comprising the MCI obtainable by the process.

Description

[0001] Process for the separation of casein and whey protein from milk

[0002] Field of the Invention

[0003] The invention is in the field of separation of casein and whey protein from milk and production of low mineral casein products.

[0004] Background

[0005] Animal milk has a high nutritional value. It comprises a high content of high-quality proteins, i.e. caseins and whey proteins, and lactose which are relevant to human (medical) nutrition. Animal milk is widely used in the preparation of human (medical) nutritional products and is thus a high-value ingredient. For example, animal milk is used in the preparation of dairy products, such as yogurts and cheeses, but also in the preparation of infant formula, as well as other medical nutritional products, such as products for bed-ridden patients, or patients with special nutritional needs.

[0006] However, the ratio of the components in animal milk is not suitable for the preparation of several products for human consumption. For example, the ratio caseins to whey proteins is too high for use in infant formula. In the context of human (medical) nutritional products, the content of minerals is also critical. Consumption of mineral in amounts above the recommended dietary allowance (RD A) is not recommended, especially in subjects who are suffering from a disease and consuming medical nutritional products. In medical nutritional products, the content of minerals is strictly regulated and must comply with the requirements of food for special medical purposes (FSMP) legislation. In the context of the present invention, with FSMP legislation it is the European Commission guideline on Food for Special Medical Purposes (FSMP) directive 1999 / 21 / EC of 25 March 1999 is intended, its contents herein incorporated by reference. According to the FSMP legislation, the required content of minerals is 50 - 250 mg Calcium, 25 - 90 mg P, 20 - 60 mg Sodium, 5 - 15 mg Magnesium, 60 - 145 mg Potassium per 100 kcal in products for infants; and 35 - 175 mg Calcium, 30 - 80 mg P, 30 - 175 mg Sodium, 7.5 - 25 mg Magnesium, and 80 - 295 mg Potassium per 100 kcal in products for adults. The content of minerals in compositions comprising proteins derived from animal milk is usually higher than recommended for human consumption, as it exceeds the requirements of FSMP. Moreover, there is an interest in the art to formulate high-protein nutritional compositions comprising relatively high concentrations of micellar casein isolate or whey protein concentrate. For example, administration of high-protein supplements lowers the mortality rate and complications of patients who are elderly and / or malnourished as a result of diseases such as cancer. Compositions that are both high in protein and low in minerals, can be achieved only by using protein sources which are low in minerals.

[0007] However, minerals are not easily separated when processing milk; as both Calcium (Ca) and Phosphorous (P) are essential constituents of casein micelles. Around 30 mg of Ca2+ions are present in 1 g of casein micelles [source: Walstra, P.; Wouters, J. T ; & Geurts, T.J. (2006). Part 1 : Processes, in Dairy science and technology. CRC press: Boc Raton, USA p. 3-203], Casein micelles also provide about half of the P of milk (22% organic, 32% inorganic), there being about 1 g P per kg milk [source: Walstra, P., Wouters, J. T., & Geurts, T.J. (2006). Part 2: Processes, in Dairy science and technology. CRC press: Boc Raton, USA p. 207-272],

[0008] Thus, separation processes to isolate micellar casein from milk tend to also isolate minerals bound therein, especially Ca and P. Ca and P removal from micellar casein is a delicate matter, since Ca and P are a key factor in stabilizing the micellar structure and their removal may impact protein stability as well as heat stability. Indeed, at high temperatures the micellar structure of casein may be impacted, with the micelles shrinking and dissociating on one end, or on the opposite end aggregating and coagulating. Since this implies changes in micelles size, particle size distribution as measured by dynamic light scattering (DLS) is a suitable technique to measure heat stability [source: Beliciu, C. M. & Moraru, C. I. “Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering ” J. Dairy Sci. (2009) 92: 1829-1839], For example, micellar casein concentrates were found to aggregate at temperatures above 110 °C at pH 6.5-6.7 [source: Sauer, A. & Moraru C.I. “Heat stability of micellar casein concentrates as affected by temperature and pH” J. Dairy Sci. (2012) 95; 11 : 6399-6350], Most human (medical) nutritional composition undergo sterilization steps involving heat treatment, e.g. UHT. Therefore, heat-stable micellar casein isolates are preferred in such formulations. Accounting for all these requirements, there is a need of separating high value components such as caseins, whey proteins and lactose from milk, while reducing the amounts of minerals in the casein fraction and maintaining micellar casein’s heat stability. WO 2013 / 068653 teaches a method for producing a milk product, in which casein and whey protein are separated by a combination of microfiltration and ultrafiltration. WO 2013 / 068653 does not address the mineral content or any issues in high-protein compositions related therewith.

[0009] WO 2019 / 213135 teaches a process for the separation of phospholipids from milk by using ultrafiltration and microfiltration. The starting material of WO 2019 / 213135 is buttermilk or butter serum, and not milk.

[0010] WO 2019 / 104169 teaches a process for purifying a whey protein composition (exome) by ultrafiltration, microfiltration and CO2 treatment. WO 2019 / 104169 does not teach separation of a casein fraction with low mineral content.

[0011] WO 2010 / 011129 teaches a process for preparing milk protein concentrate. US 6,558,717 teaches a method of isolating casein from milk. Neither WO 2010 / 011129 nor US 6,558,717 teach isolating casein and whey protein with mineral content low enough to make nutritional products complying with RD A and FSMP.

[0012] US 2018 / 343880 describes a process for the separation of whey protein and casein from milk wherein the milk is subjected to microfiltration to obtain a whey -protein enriched permeate stream and a micellar casein-enriched retentate stream wherein the retentate is acidified using CO2 or organic acids to solubilise minerals and reduce the mineral content of micellar casein concentrate. US 2018 / 343880 does not teach the heat stability of its concentrate.

[0013] US 2019 / 320672 describes a process for producing e.g. infant formulas, containing milk protein and milk saccharide with a reduced mineral content, by using electrodialysis for the removal of polyvalent ions on the nano-filtered retentate. US 2019 / 320672 does not teach acidification of the milk or mineral removal from micellar casein.

[0014] It is desirable to develop a process that can efficiently separate casein and whey protein from milk and at the same time yield heat-stable products with low content of minerals, particularly P and Ca, more preferably also Na, Mg and K, in order to make nutritional products which comply with RD A and FSMP. Summary of the invention

[0015] In the process according to the invention, milk is acidified by addition of an acidic aqueous solution comprising a weak organic acid and / or dissolved gaseous CO2, or by injection of gaseous CO2 into the milk, before a microfiltration step. Without wishing to be bound to a theory, it is believed that the acidification lowers the pH of milk thus increasing the solubility of minerals. Preferably the pH of the acidified milk is in the range from 6.3 to 6.5. The process works particularly well when injecting CO2 into the milk before microfiltration. It is important that the stream or fraction comprising the micellar casein does not go below a pH value of 6.3 as that may lead to disintegration of the micellar casein, and gel formation.

[0016] Micellar casein’s low solubility ensures micellar casein is separated in the retentate of the microfiltration step, while the minerals’ improved solubility ensures minerals and whey protein are separated in the permeate of the microfiltration step. The micellar casein-enriched stream is further treated (i.e. purified) in a ultrafiltration step wherein it is further washed with diafiltration water. The remaining minerals are collected in the permeate of the ultrafiltration step, while a micellar casein isolate (MCI) is obtained as retentate of the ultrafiltration step.

[0017] In a preferred embodiment, acidification is achieved by injecting CO2 into the milk before the microfiltration step. When and where the CO2 is injected, e.g. directly into the milk before the microfiltration, in the retentate of the microfiltration, or in the diafiltration water of the microfiltration and / or ultrafiltration, influences the resulting mineral content and heat stability. In a preferred embodiment, CO2 is injected into the milk before microfiltration.

[0018] At the same time, the temperature of the microfiltration step is optimised to more than 30 °C, preferably more than 40 °C, preferably below 60 °C to ensure sufficient solubility of CO2 in the milk on one hand, and low solubility of micellar casein on the other.

[0019] The inventors have surprisingly found that by (acidifying the milk by) adding an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2, preferably wherein the acidic aqueous composition is acidified diafiltration water comprising a weak organic acid, and / or acidified by injecting CO2 into the milk, before microfiltration, and running the microfiltration at a temperature of more than 30 °C, preferably more than 40°C, preferably below 60 °C, the following was achieved: • the mineral content in the resulting micellar casein isolate (MCI) was controlled, particularly at least Ca and P levels, more preferably also Na, Mg, and K levels. With controlled mineral content it is intended that the MCI of the invention contains less than 2600 mg Ca and less than 1650 mg P per 100 g dry weight of the MCI, more preferably also less than 110 mg Na, less than 115 mg Mg and less than 270 mg K per 100 g dry weight of the MCI; and / or the MCI of the invention contains less than 29.15 mg Ca and less than 19.0 mg P per g of protein, preferably also less than 1.25 mg Na, less than 1.33 mg Mg, and less than 3.10 mg K per g of protein;

[0020] • the resulting MCI was heat stable, particularly had a heat coagulation time (HCT) of at least 5, preferably at least 10 minutes at 120 °C, most preferably at least 20 minutes at 120°C.

[0021] The same results were not obtained when the streams were acidified by injecting CO2 at different points of the process (Comparative Examples 1 and 2).

[0022] Hence, an advantage of the process of the invention is obtaining a MCI with low or reduced mineral content, particularly at least a reduced Ca and P mineral content, but more preferably also a reduced Na, Mg and K mineral content. Mineral content is relevant in a number of applications and strictly monitored in medical nutrition and in early life products. The required content of minerals according to the FSMP legislation is summarized in the table below, which is an extract of table 2 of EU Directive 1999 / 21ZEC of 25 March 1999, concerning requirements for individuals above 10 years old, i.e. adults.

[0023] For adults, FSMP requirements are:

[0024] The MCI of the invention comprises less than 29.15 mg Ca and less than 19.0 mg P per g of protein, most preferably less than 17.5 mg P per g protein. The MCI preferably also comprises less than 1.25 mg Na, less than 1.33 mg Mg, and less than 3.10 mg K per g of protein. Thus, the MCI of the invention can be advantageously used to prepare (medical) nutritional products for adults or early life products complying with FSMP regulation. For example, the MCI of the invention can be used as the sole protein source to prepare a high protein nutritional product with a content of protein up to 30 en.% based on total energy of the composition, which is a high protein content in the field. In such compositions, the amount of Ca and P provided by the MCI is around 40 mg Ca and 30 mg P per 100 kcal, which are well within the FSMP requirements, leaving room for adding other minerals- containing ingredients.

[0025] A further advantage of the process of the invention is obtaining a MCI with high heat stability. The inventors have surprisingly found that MCI obtained from the process of the invention had a heat coagulation time (HCT) of at least 20 minutes at 127 °C. Heat stability is an important factor for further processing of the MCI in nutritional products, as most nutritional products undergo heat treatment, for example for sterilization purposes. Hence, the MCI of the invention can be advantageously used in (medical) nutritional products which undergo heat treatments, e.g. UHT.

[0026] Therefore, in a first aspect the invention concerns a process for the separation of whey protein and casein from milk (and from one another) comprising: i. acidifying the milk by i(a). adding an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2; and / or i(b) injecting gaseous CO2 into the milk, to obtain an acidified milk, preferably with a pH in the range from 6.3 to 6.5; and ii. subjecting the acidified milk to microfiltration, to obtain a whey protein-enriched stream as permeate and a micellar casein-enriched stream as retentate, wherein the microfiltration is carried out at a temperature of more than 30 °C, preferably more than 40 °C, preferably below 60 °C; and iii. adding diafiltration water to the casein-enriched stream and subjecting the mixture to ultrafiltration to obtain a micellar casein isolate (MCI) as retentate (and a waste stream as permeate).

[0027] Steps i) and ii) are preferably carried out without any intermediate processing steps. The acidification is preferably achieved by: i(a) adding an acidic aqueous composition comprising a weak organic acid and dissolved gaseous CO2; and / or by i(b) injecting gaseous CO2 into the milk. As outlined herein, step i(b) is most preferred. The process of the invention is for separating whey protein and casein from milk as well as for separating whey protein and casein from one another.

[0028] A most preferred embodiment of the invention concerns a process for the separation of whey protein and casein from milk comprising: i. acidifying milk by injection of gaseous CO2 into the milk to obtain an acidified milk with a pH in the range of 6.3 - 6.5; and ii. subjecting the acidified milk to microfiltration, to obtain a whey protein-enriched permeate stream and a micellar casein-enriched retentate stream, wherein the microfiltration is carried out at a temperature in the range of 30 - 60 °C, preferably in the range of 40 - 60°C; and iii. adding diafiltration water to the casein-enriched stream and subjecting the mixture to ultrafiltration, to obtain a micellar casein isolate (MCI) as retentate and a waste stream as permeate.

[0029] Herein it is preferred that an amount of 0.55 to 2.04 cm3, more preferably 0.66 to 1.74 cm3, most preferably 0.77 to 1.54 cm3of gaseous CO2 is injected per 1 gram of milk; and wherein preferably gaseous CO2 is injected into the milk at a time that is not more than 30 minutes, more preferably not more than 15 minutes before the acidified milk is subjected to microfiltration.

[0030] It is further preferred that step i) and ii) are carried out without any intermediating steps.

[0031] Preferably the process is for the production of a micellar casein isolate (MCI) with low minerals content and high heat stability, preferably wherein the MCI is in liquid form (LMCI).

[0032] In a second aspect, the invention concerns a micellar casein isolate (MCI), preferably in liquid form, comprising a. protein in an amount of 75 - 99 wt.%, preferably 80 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of at least 88 wt.%, preferably at least 90 wt.%, more preferably at least 94 wt.% based on dry weight of total protein in the MCI; c. less than 2600 mg / 100 g, preferably less than 2529 mg / 100 g, more preferably less than 2480 mg / lOOg calcium based on dry weight of the MCI, and / or less than 29.15 mg, preferably less than 28.67 mg calcium per g of protein in the MCI; d. less than 1650 mg / 100 g, preferably less than 1618 mg / 100 g, more preferably less than 1510 mg / lOOg phosphorous based on dry weight of the MCI, and / or less than 19.0 mg, preferably less than 18.33 mg phosphorous per g of protein in the MCI; wherein the MCI has a heat coagulation time (HCT) of at least 5, preferably at least 10 minutes at 120 °C, most preferably at least 20 minutes at 120°C.

[0033] In a third aspect, the invention concerns the use of the micellar casein isolate of the invention for the preparation of adult medical nutrition products, particularly high protein products typically comprising at least 10 g / 100 ml protein.

[0034] Thus, the invention further concerns a nutritional composition comprising

[0035] 10 - 20 g protein per 100 ml and / or wherein protein provides 14 - 30 % of the total energy of the composition, and wherein the protein comprises 5 - 20 g MCI of the invention per 100 ml and / or 2 - 8 g MCI of the invention per 100 kcal and / or wherein at least 60 wt% of the protein is provided by the MCI of the invention, and wherein preferably the nutritional composition has an energy density of 150 to 350 kcal / 100 ml and the Ca and P levels, more preferably also the Na, Mg and K levels comply with the table below:

[0036] The composition is preferably a heat-sterilized liquid composition.

[0037] The MCI preferably has a P mineral level less than 17.5 mg per g protein. List of preferred embodiments

[0038] 1. A process for the separation of whey protein and casein from milk comprising: i. acidifying milk a) through addition of an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2 to the milk; and / or b) by injection of gaseous CO2 into the milk, to obtain an acidified milk; and ii. subjecting the acidified milk to microfiltration, to obtain a whey protein-enriched permeate stream and a micellar casein-enriched retentate stream, wherein the microfiltration is carried out at a temperature of more than 30 °C; and iii. adding diafiltration water to the casein-enriched stream and subjecting the mixture to ultrafiltration, to obtain a micellar casein isolate (MCI) as retentate and a waste stream as permeate.

[0039] 2. The process according to claim 1, wherein the acidified milk has a pH of 6.3 - 6.5.

[0040] 3. The process according to claim 1 or 2, wherein the aqueous composition of step (i) has a pH of 3.0 - 5.0.

[0041] 4. The process according to anyone of the preceding claims, wherein the diafiltration water of step (iii) has a pH of 3.0 - 5.0.

[0042] 5. The process according to claim 4, wherein a weak organic acid and optionally gaseous CO2 are dissolved in the diafiltration water of step (iii).

[0043] 6. The process according to any one of the preceding claims, wherein the microfiltration is carried out at a temperature of 30 to 65 °C, more preferably 40 to 60 °C, even more preferably 45 to 60 °C, most preferably 45 to 55°C.

[0044] 7. The process according to any one of the preceding claims, wherein the weak organic acid is selected from citric acid, lactic acid, malic acid, tartaric acid, ascorbic acid, or combinations thereof, preferably citric acid.

[0045] 8. The process according to any one of the preceding claims, wherein the acidification is achieved by: i(a) adding an acidic aqueous composition comprising a weak organic acid and dissolved gaseous CO2; and / or by i(b) injecting gaseous CO2 into the milk.

[0046] 9. The process according to any one of the preceding claims, wherein acidification is achieved by injection of gaseous CO2 in the milk, wherein the gaseous CO2 is injected into the milk at a time that is not more than 30 minutes, more preferably not more than 15 minutes, most preferably not more than 5 minutes before the milk is subjected to microfiltration.

[0047] 10. The process according to any one of the preceding claims, further comprising adding diafiltration water to the whey protein enriched stream obtained in step (ii) and subjecting the mixture to ultrafiltration to obtain a whey protein concentrate (WPC).

[0048] 11. A micellar casein isolate (MCI), preferably obtainable by the process of any one of claims 1 to 10, comprising a. protein in an amount of 75 - 99 wt.%, preferably 80 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of at least 88 wt.%, preferably at least 90 wt.%, more preferably at least 94 wt.% based on dry weight of total protein in the MCI; c. less than 2600 mg / 100 g, preferably less than 2529 mg / 100 g calcium based on dry weight of the MCI; and / or less than 29.15 mg, preferably less than 28.67 mg calcium per g protein; d. less than 1650 mg / 100 g, preferably less than 1618 mg / 100 g phosphorous based on dry weight of the MCI; and / or less than 19.0, preferably less than 18.33 mg phosphorous per g protein; wherein the MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes at 120 °C, most preferably at least 20 minutes at 120 °C.

[0049] 12. The micellar casein isolate (MCI) according to claim 11, wherein the MCI further comprises e. less than 270 mg / 100 g, preferably less than 265 mg / 100 g potassium based on dry weight of the MCI; and / or less than 3.10 mg, preferably less than 3.0 mg potassium per g protein; and f. less than 115 mg / 100 g, preferably less than 112 mg / 100 g magnesium based on dry weight of the MCI; and / or less than 1.33 mg, preferably less than 1.27 mg magnesium per g protein; and g. less than 110 mg / 100 g, preferably less than 106 mg / 100 g sodium based on dry weight of the MCI; and / or less than 1.25 mg, preferably less than 1.20 mg sodium per g protein.

[0050] 13. A nutritional composition comprising 10 - 20 g protein per 100 ml and / or wherein protein provides 14 - 30 % of the total energy of the composition, and wherein the protein comprises 5 - 20 g micellar casein isolate (MCI) according to claim 11 or 12 per 100 ml and / or 2 - 8 g micellar casein isolate (MCI) according to claim 11 or 12 per 100 kcal. 14. The nutritional composition according to claim 13, having an energy density of 150 to 350 kcal / 100 ml, wherein at least the Ca and P levels, more preferably also the Na, Mg and K levels are within the ranges according to the table below:

[0051] 15. Use of the whey protein concentrate (WPC) obtainable by the process according to claim 10, for the preparation of infant formula, follow-on formula or growing up milk.

[0052] Detailed description

[0053] Definitions

[0054] The term “dissolved gaseous CO2” as used herein refers to gaseous CO2 which is injected or bubbled into an aqueous composition or milk and becomes dissolved in such aqueous composition or milk by forming species such as bicarbonate (HCO3 ) and carbonate (CO32).

[0055] The term “diafiltration water” as used herein refers to (demineralized) water used during a (dia)filtration step and aids in purification of the desired compound such as micellar casein.

[0056] The term “protein concentrate” as used herein refers to a composition comprising at least 40 wt.% protein based on total dry weight of the composition.

[0057] The term “protein isolate” as used herein refers to a composition comprising at least 80 wt.% protein based on total dry weight of the composition.

[0058] The term “enriched” as used herein refers to the content of the enriched component, based on dry weight, is increased in one stream compared to another stream. Consequently, the term “casein or whey protein-enriched stream” as used herein refers to a stream comprising a content of casein or whey protein based on dry weight of the stream which is higher compared to the content of casein or whey protein in the unprocessed milk from which the stream is derived, based on dry weight of milk.

[0059] The term “volume concentration factor” or “VCF” as used herein refers to the factor at which a liquid composition is concentrated upon filtration, i.e. the total volume of the incoming stream prior to filtration divided by the total volume of the retentate after filtration, irrespective of the total solid content. Thus, when 5 L of a liquid composition is fractioned over an microfiltration membrane into a permeate of 4 L and a retentate of 1 L, this microfiltration process operates with a VCF of 5 / 1 = 5. VCFs are determined on the actual volume in the incoming stream, thus excluding optionally added water.

[0060] Unless explicitly mentioned otherwise, the terms “phosphorous” and “total phosphorous” are used interchangeably throughout the application. The term “phosphorous” (abbreviated sometimes to P) as used herein refers to the sum of both organic and inorganic phosphorous.

[0061] The terms “heat stability” and “thermal stability” as used herein are synonyms and used interchangeably throughout the application and refer to the ability of a material or system to resist changes in its properties or structure when exposed to variations in temperature. In the art, the correlation between heat stability and coagulation time is used for the determination of heat stability of milk fractions [source: “Heat-Induced Changes in Milk” International Dairy Federation (1995) Chapter 12], Herein, heat stability is defined as heat coagulation time (HCT). The “heat coagulation time” or “HCT” of a substance, e.g. milk or micellar casein isolate (MCI), is the time from immersion of a sample of such substance in an oil bath at a definite temperature until the appearance of visible coagulation. A suitable example of a laboratory test to determine HCT is heating a sample of milk or MCI, sealed in a narrow glass tube, in a temperature-controlled oil bath. The HCT is the time at which the onset of visible coagulation in the sample is observed, see e.g. Holm et al., Heat Coagulation of Milk, Journal of Dairy Science, Chapter I, 1931. Alternatively, particle size distribution, as measured according to ISO 22412:2017 or by dynamic light scattering (DLS) as described by Beliciu & Moraru (“Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering.” J. Dairy Sci. (2009) 92: 1829-1839), can be used to determine coagulation of the sample.

[0062] The term “heat-sterilized” refers to foods that are treated by heat to destroy foodborne microorganisms and are safely stored at room temperature on the shelf, typically for a period of at least 10 months. The composition is preferably a shelf-stable composition. The term “shelf-stable” herein refers to storage stability. A nutritional composition is shelf-stable if it is storage stable at ambient temperature with respect to microbiological spoilage and physical defects like creaming, gelation, precipitation, etc., for a certain amount of time. Preferably, the nutritional composition has a shelf-stability of at least one month, more preferably at least 3 months, even more preferably at least 6 months and most preferably at least 12 months after packaging, when stored in a sealed packaging at ambient temperature (20 °C). The invention is not limited to specific sterilization conditions, and in fact the invention renders it possible to subject the MCI to heat sterilization conditions which are common practice in the field, and which belong to the skilled person’s common general knowledge. However, in the field a significant part of the problem of achieving high protein compositions rests in the need for such heat treatment in order to reduce the microbial load to levels that the product can be shelved for extended periods.

[0063] The term “total casein” or “casein” as present in the LCMI as used herein refers to the amount of micellar casein comprising the sum of any type of casein present in micellar form, for example a-casein + P-casein + K-casein and so on.

[0064] Unless expressly mentioned otherwise, throughout the application the term “FSMP” as used herein refers to Food for Special Medical Purposes (FSMP) directive 1999 / 21ZEC of 25 March 1999, its contents herein incorporated by reference.

[0065] The terms “microfiltration” and “ultrafiltration” as used herein have meanings that are part of the skilled person’s common general knowledge. “Microfiltration” or “MF” refers to a filtration process over a membrane with pores of > 0.2 pm. “Ultrafiltration” or “UF” refers to a filtration process over a membrane with pores of 3 to 200 nm.

[0066] The term “en%” as used herein refers to % of total energy of the composition. It thus refers to energy percentage representing the relative amount that a constituent contributes to the total caloric value of the composition. The amounts of energy provided by proteins, fats and carbohydrates can be estimated e.g. using Atwater factor wherein: 4 kcal per gram (kcal / g) (17 kJ / g) for protein (and amino acids), 4 kcal / g for digestible carbohydrates and 9 kcal / g (37 kJ / g) for fat. Unless expressly mentioned otherwise, throughout the application, when the pH of the acidified milk or the micellar casein stream is given, it is the pH when measured at 10 wt% dry matter content.

[0067] Process

[0068] The process of the invention is for the separation of whey protein and casein from milk (and from one another) and comprises i. acidifying the milk by i(a). addition of an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2; and / or i(b). by injection of gaseous CO2 into the milk; ii. subjecting the acidified milk to a microfiltration step, to obtain a whey protein- enriched stream as permeate and a micellar casein-enriched stream as retentate, wherein the microfiltration is carried out at a temperature of more than 30 °C; and iii. adding diafiltration water to the casein-enriched stream and subjecting the mixture to ultrafiltration, to obtain a micellar casein isolate (MCI) as retentate (and a waste stream as permeate). In step (i) it is preferred to acidify using gaseous CO2 either by dissolving into the acidic composition comprising a weak organic acid, or by injection into the milk; the latter is preferred.

[0069] The acidified milk of step (i) preferably has a pH value in the range from 6.3 to 6.5. Preferably, the mixture comprising the micellar casein enriched stream of step (iii) has a pH value in the range from 6.3 to 6.9.

[0070] The milk used in the process of the invention is preferably selected from skimmed milk, low- fat milk, full-fat milk, or any combinations thereof. Preferably the milk is pasteurised milk. Preferably the milk is cow, sheep or goat milk, preferably cow milk. There is no limit to the milk source but preferably the micellar casein: whey weight ratio is in the range from 65:35 to 85: 15.

[0071] The process preferably further comprises optional steps. Preferably the process comprises a pre-treatment step of the milk before the acidification step, and / or a ultrafiltration step of the whey protein-enriched stream obtained from the microfiltration. The steps are further detailed here below.

[0072] Acidification step

[0073] In the acidification step, milk is acidified by i(a) addition of an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2; and / or i(b) injection of gaseous CO2 into the milk, to obtain acidified milk. Acidification by i(b) is most preferred. Preferably the acidified milk has a pH of 6.3 to 6.5. It is preferred that acidification of the milk does not go below a pH of 6.3 as it may disrupt the micellar casein structure in the milk. Hence, preferably the casein remains soluble.

[0074] Preferably the weak organic acid is selected from citric acid, lactic acid, malic acid, tartaric acid, ascorbic acid, or combinations thereof, preferably citric acid. Preferably the weak organic acid is a chelating acid.

[0075] Preferably the acidic aqueous composition comprises diafiltration water. The acidic aqueous composition is preferably obtained by dissolving a weak organic acid and / or gaseous CO2 in diafiltration water to obtain acidified diafiltration water.

[0076] Preferably the acidic aqueous composition has a pH of 3.0 - 5.0. Preferably an amount of weak organic acid of 50 - 150 g / 100 g of diafiltration water retentate is used.

[0077] When the acidic aqueous composition is added to the milk, preferably the acidic aqueous composition is added to the milk at a time that is not more than 30 minutes, more preferably not more than 15 minutes before the milk is subjected to microfiltration.

[0078] When gaseous CO2 is injected directly into the milk, preferably gaseous CO2 is injected into the milk at a time that is not more than 30 minutes, more preferably not more than 15 minutes before the acidified milk is subjected to microfiltration. Preferably the gaseous CO2 is injected into the milk at a flow of 2 to 20 kg / h, more preferably 3 to 15 kg / h, most preferably 5 to 10 kg / h. Preferably an amount of 0.55 to 2.04 cm3, more preferably 0.66 to 1.74 cm3, most preferably 0.77 to 1.54 cm3of gaseous CO2 is injected per 1 gram of milk. In a further preferred embodiment, an amount of 0.2 to 1.0 g of gaseous CO2 / kg milk is used, more preferably 0.3 to 0.9 g / kg milk, most preferably 0.4 to 0.8 g of gaseous CO2 is used per kg of milk.

[0079] Preferably an amount of gaseous CO2 is injected to reach a pH of the acidified milk of 6.3 - 6.5.

[0080] Microfiltration step

[0081] In the microfiltration step the milk is filtered over a microfiltration membrane to yield a casein-enriched stream as retentate and a whey protein-enriched stream as permeate. The microfiltration can be carried out with any apparatus known in the art.

[0082] Preferably the acidified milk is subjected to microfiltration within 1 hour from the acidification, more preferably within 30 minutes, most preferably within 15 minutes.

[0083] The microfiltration is performed over a membrane that enables fractionation of casein and whey proteins. Preferably such a membrane has a porosity of 0.05 - 0.35 pm. Preferably, a ceramic membrane or a spiral wound (organic) membrane is used. When a ceramic membrane is used, it is preferred that the porosity is 0.05 - 0.30 pm, and when a spiral wound organic membrane is used, it is preferred that the porosity is 0.10 - 0.35 pm.

[0084] The microfiltration is carried out at a temperature of more than 30 °C. Preferably the microfiltration is carried out at a temperature of 30 to 65 °C, more preferably 40 to 60 °C, even more preferably 45 to 60 °C, most preferably 45 to 55 °C.

[0085] Preferably the microfiltration is carried out at 0 - 5 bar, more preferably 0.05 - 2 bar.

[0086] The microfiltration preferably operates with a volume concentration factor (VCF) of 1.5 - 8, more preferably 2 - 4.

[0087] The minerals and whey protein are separated in the permeate of the microfiltration step. Hence, the pH of the acidified milk is preferably in the range of 6.3 - 6.5 during the microfiltration. After microfiltration, the pH of the casein-enriched stream does not have to remain in the range of 6.3 - 6.5 but may increase, for example to above 6.9. Though, in a preferred embodiment, the casein-enriched stream has a pH in the range of 6.3 - 6.9, when measured at 10 wt% dry matter content. Optionally microfiltration is enhanced with diafiltration. Diafiltration is accomplished by diluting the milk with an amount of water and subjecting the diluted milk to microfiltration. The amount of diafiltration water used preferably ranges from 10 to 500 wt.%, more preferably from 50 to 200 wt.%, most preferably from 70 to 150 wt.% based on total weight of the milk. The diafiltration water is added to the milk at once, or the total amount of diafiltration water is added in several fractions. After each addition of water to the milk, the diluted incoming milk is subjected to microfiltration. In the microfiltration step, the milk is fractioned into a casein-enriched stream and a whey protein-enriched stream. The casein- enriched stream is a liquid composition comprising a protein fraction comprising casein, which is enriched in casein compared to the casein content in the milk. The whey protein- enriched stream is a liquid composition comprising a protein fraction comprising whey protein, which is enriched in whey protein compared to the whey protein content in the milk. Thus, the casein-enriched stream is enriched in casein, i.e. has a higher casein content based on dry matter compared to the milk, while the whey protein-enriched stream is enriched in whey proteins, i.e. has a higher whey proteins content based on dry matter compared to the milk.

[0088] Preferably the casein-enriched stream comprises casein in an amount of at least 85 wt.%, more preferably at least 88 wt.% based on total protein weight in the casein-enriched stream. Preferably the casein-enriched stream comprises whey protein in an amount of less than 15 wt.%, more preferably less than 10 wt.%, most preferably less than 5 wt%. based on total protein weight in the casein-rich stream. Alternatively, the weight ratio of casein to whey protein is at least 8: 1.

[0089] Preferably the whey protein-enriched stream comprises whey protein in an amount of at least 90 wt.%, more preferably at least 95 wt.% based on total protein weight in the whey protein- enriched stream. Preferably the whey protein-enriched stream comprises casein in an amount of less than 15 wt.%, more preferably less than 10 wt.%, most preferably less than 5 wt%. based on total protein weight in the whey protein-enriched stream.

[0090] Ultrafiltration of the casein-enriched stream

[0091] The process of the invention comprises an ultrafiltration step of the casein-enriched stream obtained as retentate from the microfiltration, wherein diafiltration water is added to the casein-enriched stream and the mixture is subjected to ultrafiltration to yield a micellar casein isolate (MCI) as a retentate (and a waste stream as a permeate).

[0092] Preferably no gaseous CO2 is injected directly into the casein-enriched stream, after microfiltration but prior to ultrafiltration.

[0093] The ultrafiltration of the casein-enriched stream can be carried out with any apparatus known in the art. The ultrafiltration of the casein-enriched stream preferably employs a membrane having a molecular weight cut-off of at most 25 kDa, more preferably at most 20 kDa, most preferably at most 15 kDa, and preferably of at least 2.5 kDa, more preferably at least 5 kDa. Preferably the ultrafiltration of the casein-enriched stream operates at a VCF of 0.5 - 5.0, more preferably of 1.0 - 3.5. Preferably the ultrafiltration of the casein-enriched stream is carried out at a temperature of 1 - 15 °C, more preferably 2 - 10 °C.

[0094] Ultrafiltration of the casein-enriched stream is enhanced with diafiltration. Diafiltration is accomplished by diluting the casein-enriched stream with water and subjecting the mixture to ultrafiltration. The amount of diafiltration water used preferably ranges from 50 to 500 wt.%, more preferably from 100 to 350 wt.%, most preferably from 130 to 250 wt.% based on total weight of the casein-enriched stream to which the water is added. Preferably the total amount of diafiltration water is added in several fractions to the casein-enriched stream. After each addition of water to the casein-enriched stream, the diluted casein-enriched stream is subjected to ultrafiltration.

[0095] In a further embodiment, the diafiltration water used in the ultrafiltration step can be acidic diafiltration water comprising a weak organic acid and optionally dissolved gaseous CO2. Acidification of the diafiltration water used in the ultrafiltration step is optional and is dependent on the mineral content of the casein enriched stream obtained from microfiltration wherein the pH of casein enriched stream is preferably in the range from 6.3 to 6.9. Hence, if the pH of the casein enriched stream obtained from microfiltration is higher than 6.9, preferably acidic diafiltration water is added to the casein enriched stream to obtain a mixture with a pH in the range from 6.3 to 6.9 that is subsequently subjected to ultrafiltration.

[0096] When the acidic diafiltration water is obtained by dissolving a weak organic acid and optionally gaseous CO2 in diafiltration water, preferably the diafiltration water has a pH of 3.0 - 5.0. Preferably the weak organic acid is selected from citric acid, lactic acid, malic acid, tartaric acid, ascorbic acid, or combinations thereof, preferably citric acid. Preferably an amount of weak organic acid of 50 - 150 g / 100 g of diafiltration water is used. It is preferred that there is 50 - 150 g weak organic acid selected from citric acid, lactic acid, malic acid, tartaric acid, ascorbic acid, or combinations thereof, preferably citric acid, per 100 g of diafiltration water. Preferably an amount of 0.1 to 1.0 g of gaseous CO2 / kg casein enriched stream is used, more preferably 0.1 to 0.9 g gaseous CO2 / kg casein enriched stream, most preferably 0.1 to 0.8 g of gaseous CO2 is used per kg of casein enriched stream.

[0097] Preferably, the gaseous CO2 is injected into the diafiltration water at a time that is not more than 30 minutes, more preferably not more than 15 minutes, most preferably not more than 5 minutes before the casein-enriched stream is subjected to ultrafiltration.

[0098] In the most preferred embodiment, the acidic diafiltration water of step (iii) is obtained by dissolving a weak organic acid and gaseous CO2 in the diafiltration water.

[0099] The ultrafiltration of the casein-enriched stream yields a micellar casein isolate (MCI) as retentate and a composition comprising minerals, lactose, residual whey proteins (waste stream) as permeate. Preferably the MCI is in liquid form (LMCI).

[0100] Optional pretreatment of milk

[0101] Preferably the process of the invention comprises steps in which the milk is pretreated before being subjected to acidification and microfiltration. Preferably the process comprises a milk pretreatment step before the acidification step selected from one or more of cream separation, heat treatment, and spore removal. Preferably the process comprises all of cream separation, heat treatment, and spore removal.

[0102] When present, the cream separation involves removing the fat portion of milk by any means known in the art, such as centrifugal separation.

[0103] When present, the heat treatment can be any one of microwave heating, direct steam injection, pasteurization, such as HTST, ESL or UHT, or sterilization, such as dry heat or moist heat sterilization, preferably pasteurization at a temperature of maximum 75°C for 30 seconds. When present, the spore removal involves removal of bacteria from the milk by any means known in the art, such as UV treatment, or bactofuge (high-speed centrifugation) or bacterial filtration, preferably by microfiltration using a pore size of around 0.8 pm.

[0104] Optional ultrafiltration of the acidified whey protein-enriched stream

[0105] Preferably the process of the invention comprises ultrafiltration of the whey protein-enriched stream obtained as permeate from the microfiltration, wherein the whey protein-enriched stream is further processed. In the ultrafiltration of the whey protein-enriched stream step, the whey protein-enriched stream is filtered over an ultrafiltration membrane to yield a whey protein concentrate (WPC) as retentate and a stream containing minerals and lactose (lactose stream) as permeate.

[0106] The ultrafiltration of the whey protein-enriched stream can be carried out with any apparatus known in the art. The ultrafiltration of the whey protein-enriched preferably employs a membrane having a molecular weight cut-off of at most 25 kDa, more preferably at most 10 kDa, and preferably of at least 2.5 kDa, more preferably at least 5 kDa. Preferably the ultrafiltration of the whey protein-enriched stream operates at a VCF of 30 - 80, more preferably of 40 - 60. Preferably the ultrafiltration of the whey protein-enriched stream is carried out at a temperature of 1 - 15 °C, more preferably 2 - 10 °C, most preferably 3 - 5 °C.

[0107] In one embodiment, ultrafiltration of the whey protein-enriched stream is enhanced with diafiltration. Diafiltration is accomplished by diluting the whey protein-enriched stream with an amount of diafiltration water and subjecting the diluted whey protein-enrich stream to ultrafiltration. The amount of diafiltration water used preferably ranges from 50 to 500 wt.%, more preferably from 100 to 350 wt.%, most preferably from 130 to 250 wt.% based on total weight of the whey protein-enriched stream. Preferably the total amount of diafiltration water is added in several fractions to the whey protein-enriched stream. After each addition of water to the casein-enriched stream, the diluted casein-enriched stream is subjected to ultrafiltration.

[0108] The ultrafiltration of the acidified whey protein-enriched stream yields a whey protein concentrate (WPC) as retentate and a composition comprising minerals and lactose (lactose stream) as permeate. Preferably the WPC is in liquid form (LWPC). Micellar casein isolate

[0109] The process of the invention as described above, yields a micellar casein isolate (MCI). The MCI is obtained as retentate of the ultrafiltration of the casein-enriched stream. Preferably the MCI is in liquid form (LMCI).

[0110] Preferably the MCI comprises protein in an amount of at least 75 wt.%, more preferably at least 80 wt.%, most preferably at least 85 wt.% based on total dry weight of the MCI. Preferably the MCI comprises protein in an amount of 75 - 99 wt.%, more preferably 80 - 97 wt.%, most preferably 85 - 95 wt.% based on total dry weight of the MCI.

[0111] The protein fraction of the MCI comprises very little whey proteins and is high in casein concentration. Preferably the protein fraction of the MCI comprises less than 10 wt.%, more preferably less than 5 wt.%, even more preferably less than 2 wt.%, most preferably less than 0.5 wt.% whey protein based on the weight of the protein fraction of the MCI. Consequently, preferably the protein fraction of the MCI comprises at least 90 wt%, more preferably at least 95 wt%, even more preferably at least 98 wt%, most preferably at least 99.5 wt% micellar casein based on the weight of the protein fraction of the MCI.

[0112] The content of total solids in the MCI preferably ranges from 5 to 30 wt.%, more preferably ranges from 7 to 30 wt.%, most preferably from 15 to 20 wt.%, based on total weight of the MCI. Preferably the MCI has a pH of 6.3 - 6.9 measured at 10 wt% dry matter content.

[0113] The process of the invention results in a low or reduced calcium mineral content. The MCI comprises Calcium (Ca) in an amount of less than 2600 mg / 100 g, preferably less than 2550 mg / 100 g, more preferably less than 2529 mg / 100 g, more preferably less than 2480 mg / lOOg based on dry weight of the MCI. Characterized in terms of protein, the MCI comprises Ca in an amount of less than 29.15 mg, preferably less than 28.67 mg per g of protein. Worded differently, the MCI comprises Ca in an amount in the range from 1900 to 2600 mg / 100 g, preferably in the range from 1950 to 2550 mg / 100 g, more preferably in the range from 2000 to 2529 mg / 100 g based on dry weight of the MCI; and / or the MCI comprises Ca in an amount in the range from 22.40 to 29.15 mg, preferably in the range from 22.67 to 28.67 mg per g of protein. The process of the invention results in reduced phosphorous mineral content. The MCI comprises phosphorous (P) in an amount of less than 1650 mg / 100 g, preferably less than 1630 mg / 100 g, more preferably less than 1618 mg / 100 g, more preferably less than 1510 mg / lOOg based on dry weight of the MCI. Characterized in terms of protein, the MCI comprises P in an amount of less than 19.0 mg, preferably less than 18.33 mg per g of protein. In a most preferred embodiment, the MCI comprises P in an amount less than 17.5 mg per g protein. Worded differently, the MCI comprises P in an amount in the range from 1300 to 1650 mg / 100 g, preferably in the range from 1310 to 1630 mg / 100 g, more preferably in the range from 1324 to 1618 mg / 100 g based on dry weight of the MCI; and / or the MCI comprises P in an amount in the range from 14.50 to 19.0 mg, preferably in the range from 15.0 to 18.33 mg per g of protein, more preferably in the range from 15.0 to 17.5 mg per g protein.

[0114] In addition to the reduced Ca and P levels, preferably the MCI also comprises Potassium (K) in an amount of less than 270 mg / 100 g, or worded differently, comprises K in an amount in the range of 120 and 270 mg / 100 g, more preferably in the range from 125 to 268 mg / 100 g, even more preferably in the range from 130 to 265 mg / 100 g, most preferably in the range from 135 to 265 mg / 100 g based on dry weight of the MCI; and / or preferably the MCI comprises K in an amount of less than 3.10 mg, more preferably less than 3.0 mg per g protein, worded differently preferably the MCI comprises K in an amount in the range from 1.45 to 3.10 mg, more preferably from 1.53 to 3.00 mg K per g protein.

[0115] Preferably the MCI comprises Magnesium (Mg) in an amount of less than 115 mg / 100 g, or worded differently, comprises Mg in an amount in the range from 50 to 115 mg / 100 g, more preferably in the range from 60 to 115 mg / 100 g, most preferably in the range from 71 to 112 mg / 100 g based on dry weight of the MCI; and / or preferably the MCI comprises Mg in an amount of less than 1.33 mg, more preferably less than 1.27 mg per g protein or worded differently preferably the MCI comprises Mg in an amount of 0.75 to 1.33 mg, more preferably in the range of 0.80 to 1.27 mg per g protein.

[0116] Preferably the MCI comprises Sodium (Na) in an amount of less than 110 mg / 100 g or worded differently in the range from 15 to 110 mg / 100 g Na, more preferably in the range from 18 to 106 mg / 100 g based on dry weight of the MCI; and / or preferably the MCI comprises Na in an amount of less than 1.25, more preferably less than 1.20 mg per g protein or worded differently preferably the MCI comprises Na in an amount in the range from 0.15 to 1.25 mg, more preferably in the range from 0.20 to 1.20 mg per g protein.

[0117] In a more preferred embodiment, the MCI comprises 22.67 - 28.67 mg Ca and 15.00 - 18.33 mg P per g protein. In a most preferred embodiment, the MCI comprises 22.67 - 28.67 mg Ca, 15.00 - 18.33 mg P, 0.20 - 1.20 mg Na, 0.80 - 1.27 mg Mg, and 1.53- 3.00 mg K per g protein.

[0118] The MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes, more preferably at least 15 minutes, most preferably at least 20 minutes at 120 °C. Preferably the MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes, more preferably at least 15 minutes, most preferably at least 20 minutes at 130 °C.

[0119] In a preferred embodiment, the product of the invention is a micellar casein isolate (MCI), preferably in liquid form comprising a. protein in an amount of 80 - 97 wt.%, preferably 85 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of at least 88 wt.%, preferably at least 90 wt.%, more preferably at least 94 wt.% based on dry weight of total protein in the MCI; c. less than 2600 mg / 100 g, preferably less than 2529 mg / 100 g, more preferably less than 2480 mg / lOOg Ca based on dry weight of the MCI; and / or less than 29.15 mg, preferably less than 28.67 mg Ca per g protein; d. less than 1650 mg / 100 g, preferably less than 1618 mg / 100 g, more preferably less than 1510 mg / lOOg P based on dry weight of the MCI; and / or less than 19.0 mg, preferably less than 18.33 mg P per g protein; wherein the MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes, more preferably at least 15 minutes, most preferably at least 20 minutes at 120°C.

[0120] In a preferred embodiment, the product of the invention is a liquid micellar casein isolate (MCI), comprising a. protein in an amount of 75 - 99 wt.% based on total dry weight of the MCI; b. total casein in an amount of 88 - 99 wt.%, based on dry weight of total protein in the MCI; c. in the range from 1900 to 2600 mg / 100 g Ca based on dry weight of the MCI; and / or in the range from 22.40 to 29.15 mg Ca per g protein; d. in the range from 1300 to 1650 mg / 100 g P based on dry weight of the MCI; and / or in the range from 14.50 to 19.0 mg P per g protein; and wherein the MCI further comprises in the range from 15 to 110 mg / 100 g Na based on dry weight of the MCI; and / or in the range from 0.15 to 1.25 mg Na per g protein; in the range from 50 to 115 mg / 100 g Mg based on dry weight of the MCI; and / or in the range from 0.75 to 1.33 mg Mg per g protein; in the range from 120 to 270 mg / 100 g K based on dry weight of the MCI; and / or in the range from 1.45 to 3.10 mg K per g protein.

[0121] Herein, it is preferred that the MCI has a pH of 6.3 - 6.9 measured at 10 wt% dry matter content of the MCI.

[0122] In a more preferred embodiment, the product of the invention is a liquid micellar casein isolate (MCI) comprising a. protein in an amount of 80 - 97 wt.% based on total dry weight of the MCI; b. total casein in an amount of 90 - 99 wt based on dry weight of total protein in the MCI; c. in the range from 1950 to 2550 mg / 100 g calcium based on dry weight of the MCI; and / or in the range from 22.67 to 28.67 mg calcium per g protein; d. in the range of in the range from 1310 to 1630 mg / 100 g phosphorous based on dry weight of the MCI; and / or in the range from 15.0 to 18.33 mg phosphorous per g protein; and wherein the MCI further comprises in the range from 18 to 106 mg / 100 g Na based on dry weight of the MCI; and / or in the range from 0.20 to 1.20 mg Na per g protein; in the range from 60 to 115 mg / 100 g Mg based on dry weight of the MCI; and / or in the range from 0.80 to 1.27 mg Mg per g protein; in the range from 125 to 268 mg / 100 g K based on dry weight of the MCI; and / or in the range from 1.53 to 3.00 mg K per g protein.

[0123] Herein, it is preferred that the MCI has a pH of 6.3 - 6.9 measured at 10 wt% dry matter content of the MCI. In a most preferred embodiment, the product of the inventions is a liquid micellar casein isolate (MCI) comprising a. protein in an amount of 85 - 95 wt% based on total dry weight of the MCI; b. total casein in an amount of 94 to 99 wt. % based on dry weight of total protein in the MCI; c. in the range from 22.67 to 28.67 mg calcium per g protein; d. in the range from 15.0 to 17.5 mg phosphorous per g protein; and wherein the MCI further comprises in the range of 0.20 to 1.20 mg Na per g protein; and in the range of 0.80 to 1.27 mg Mg per g protein; and in the range of 1.53 to 3.00 mg K per g of protein.

[0124] Herein, it is preferred that the MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes at 120 °C, most preferably at least 20 minutes at 120 °C.

[0125] Herein, it is preferred that the MCI has a pH of 6.3 - 6.9 measured at 10 wt% dry matter content of the MCI.

[0126] Preferably, in any of the above embodiments, the MCI is heat sterilised.

[0127] Whey protein concentrate

[0128] The process of the invention yields a whey protein-enriched stream as permeate of the microfiltration or a whey protein concentrate (WPC) as retentate of the optional ultrafiltration step of the whey protein-rich stream. Preferably the WPC is in liquid form (LWPC).

[0129] Preferably the whey protein-enriched stream or WPC comprises protein in an amount of at least 75 wt.%, more preferably at least 80 wt.%, most preferably at least 85 wt.% based on total dry weight of the whey protein-enriched stream or WPC. Preferably the whey protein- enriched stream or WPC comprises protein in an amount of 80 to 99 wt.%, more preferably 85 to 97 wt.% based on total dry weight of the whey protein-enriched stream or WPC.

[0130] The protein fraction of the whey protein-rich stream or WPC comprises very little casein and is high in whey proteins. Preferably the protein fraction of the whey protein-enriched stream or WPC comprises less than 10 wt.%, more preferably less than 5 wt.%, most preferably less than 1 wt.% casein based on the weight of the protein fraction of the whey protein-enriched stream or WPC. Preferably the protein fraction of the whey protein-enriched stream or WPC comprises at least 75 wt.%, more preferably at least 80 wt.%, most preferably at least 85 wt.% whey proteins based on the weight of the protein fraction of the whey protein-enriched stream or WPC. Preferably the whey protein-enriched stream or WPC comprises 75 to 99 wt.%, more preferably 80 to 97 wt.%, most preferably 85 to 95 wt.% whey proteins on total dry weight of the whey protein-enriched stream or WPC.

[0131] The content of total solids in the whey protein-enriched stream or WPC preferably ranges from 5 to 30 wt.%, more preferably ranges from 7 to 30 wt.%, most preferably from 17 to 24 wt.%, based on total weight of the whey protein-enriched stream or WPC.

[0132] Application

[0133] The MCI or WPC of the invention may be commercialised directly or may be further used for any suitable application, for example preparation of dairy products. Preferably the MCI or WPC of the invention are used for the preparation of (medical) nutritional products, such as infant formula, follow-on formula, growing up milk or adult medical nutritional products.

[0134] Preferably the WPC obtainable / obtained by the process of the invention is used for the preparation of infant formula, follow-on formula or growing up milk. The term ‘infant formula’ is well-defined and controlled internationally and consistently by regulatory bodies. In particular, CODEX STAN 73 - 1981 “Standard For Infant Formula and Formulas For Special Medical Purposes Intended for Infants” is widely accepted. It recommends for nutritional value and formula composition, which require the prepared milk to contain per 100 ml not less than 60 kcal (250 kJ) and no more than 70 kcal (295 kJ) of energy. FDA and other regulatory bodies have set nutrient requirements in accordance therewith. The herein used “infant formula” and “infant milk formula” are interchangeable.

[0135] In the field of adult medical nutrition, there is a great interest in reducing the amounts of minerals in the diet provided to patients. For example, the recommended daily allowance (RDA) of calcium for adults depends on several factors, including pathologies, pregnancy, life stage (see for example “Recommended Dietary Allowances: 10thEdition” National Research Council (US) Subcommittee on the Tenth Edition of the Recommended Dietary Allowances, Washington (DC): National Academies Press (US); 1989). Generally, the calcium RDA for adults is based on an estimate of 200 to 250 mg / day of obligatory loss and an estimated absorption rate of 30 to 40%. as discussed above, in the context of food for special medical purposes (FSMP), the required content of minerals are codified in EU Directive 1999 / 21 / EC of 25 March 1999.

[0136] The MCI of the invention is high in protein content and low in calcium and phosphorous content. Therefore, the MCI of the invention, preferably obtainable / obtained by the process of the invention, is preferably used in the preparation of adult medical nutritional products. Examples of adult medical nutritional products include but are not limited to nutritional products for patients suffering from neurodegenerative disorders, Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, sarcopenia, dysphagia, cancer, cachexia, stroke, diabetes, epilepsy, frailty or malnutrition.

[0137] Nutritional composition

[0138] Associated with the above applications, the nutritional composition of the invention comprises

[0139] 10 - 20 g protein per 100 ml and / or wherein protein provides 14 - 30 % of the total energy of the composition, wherein the protein comprises 5 - 20 g MCI of the invention per 100 ml and / or 2 - 8 g MCI of the invention per 100 kcal.

[0140] Preferably the nutritional composition has an energy density of 150 to 350 kcal / 100 ml.

[0141] It is preferred that the Ca and P levels comply with the table below:

[0142] Preferably the composition also has Na, Mg and K levels comply with the table below:

[0143] The protein in the nutritional composition is at least partly provided by the MCI of the invention, preferably at least 50 wt.% of the protein, more preferably at least 70 wt.% of the 1 protein, more preferably at least 90 wt.% of the protein. In a preferred embodiment, all of the protein in the nutritional composition is provided by the MCI of the invention.

[0144] In a preferred embodiment, the nutritional composition of the invention comprises 10 - 20 g protein per 100 ml and wherein the protein comprises 5 - 20 g MCI of the invention per 100 ml and wherein the composition has an energy density of 150 to 350 kcal / 100 ml and wherein at least the Ca and P levels in the nutritional composition, more preferably also the Na, Mg and K levels are within the ranges according to the table below: wherein the MCI comprises a. protein in an amount of 75 - 99 wt.% based on total dry weight of the MCI; b. total casein in an amount of 88 - 99 wt.%, based on dry weight of total protein in the MCI; c. in the range from 22.40 to 29.15 mg Ca per g protein; d. in the range from 14.50 to 19.0 mg P per g protein; and preferably wherein the MCI further comprises in the range from0.15 to 1.25 mg Na per g protein; in the range from 0.75 to 1.33 mg Mg per g protein; and in the range from 1.45 to 3.10 mg K per g protein.

[0145] In a more preferred embodiment, the nutritional composition comprises 10 - 20 g protein per 100 ml and wherein 90 - 100 wt.% of the protein is provided by the MCI of the invention, wherein the composition has an energy density of 150 to 350 kcal / 100 ml and wherein the Ca and P levels and also the Na, Mg and K levels are within the ranges according to the table below: wherein the MCI comprises a. protein in an amount of 80 - 97 wt.% based on total dry weight of the MCI; b. total casein in an amount of 90 - 99 wt based on dry weight of total protein in the MCI; c. in the range from 22.67 to 28.67 mg calcium per g protein; d. in the range froml5.0 to 18.33 mg phosphorous per g protein; and wherein the MCI further comprises in the range from 0.20 to 1.20 mg Na per g protein; in the range from 0.80 to 1.27 mg Mg per g protein; and in the range from 1.53 to 3.00 mg K per g protein.

[0146] In a most preferred embodiment, the nutritional composition comprises 10 - 20 g protein per 100 ml and wherein 95 - 100 wt.% of the protein is provided by the MCI of the invention, wherein the composition has an energy density of 150 to 350 kcal / 100 ml and wherein the Ca and P levels and also the Na, Mg and K levels are within the ranges according to the table below: wherein the MCI comprises a. protein in an amount of 85 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of 94 to 99 wt. % based on dry weight of total protein in the MCI; c. in the range of 22.67 to 28.67 mg calcium per g protein; d. in the range of 15.0 to 17.5 mg phosphorous per g protein; and wherein the MCI further comprises in the range of 0.20 to 1.20 mg Na per g protein; in the range of 0.80 to 1.27 mg Mg per g protein; and in the range of 1.53 to 3.00 mg K per g of protein. The nutritional composition is preferably a liquid ready-to-drink, heat-sterilized composition.

[0147] Preferably the nutritional composition is an adult medical nutritional product, preferably selected from nutritional products for patients suffering from neurodegenerative disorders, Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, sarcopenia, dysphagia, cancer, cachexia, stroke, diabetes, epilepsy or malnutrition. This means that the product preferably complies with FSMP mineral levels for adults as described herein.

[0148] In a preferred embodiment, the nutritional composition has an energy density of 150 to 350 kcal / 100 ml, wherein protein provides 14 - 30 % of the total energy of the composition, wherein the composition comprises

[0149] 10 - 20 g protein per 100 ml, and

[0150] 5 - 20 g MCI per 100 ml and / or 2 - 8 g MCI per 100 kcal, wherein the MCI comprises a. protein in an amount of 75 - 97 wt.%, preferably 80 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of at least 88 wt.%, preferably at least 90 wt.%, more preferably at least 94 wt.% based on dry weight of total protein in the MCI; c. Ca in an amount of less than 29.15 mg, preferably less than 28.67 mg per g protein; d. P in an amount of less than 19.0 mg, preferably less than 18.33 mg per g protein, more preferably less than 17.5 mg per g protein; e. preferably less than 3.10 mg K, less than 1.25 mg Na, and less than 1.33 mg Mg per g protein, wherein the MCI is heat stable when heated up to 120 °C for 5 minutes, measured by a lack of change in particle size distribution as measured according to ISO 22412:2017, wherein at least the Ca and P levels, more preferably also the Na, Mg and K levels comply with the table below:

[0151] The nutritional composition is preferably a liquid ready-to-drink, heat-sterilized composition.

[0152] Examples EXAMPLE 1

[0153] Example 1 shows the process according to the invention, wherein acidification was performed by injecting gaseous CO2 directly into the milk to reach a pH of 6.5 in the acidified milk before being subjected to microfiltration.

[0154] Methods

[0155] Skimmed milk was injected with gaseous CO2 at an amount set to 0.65g / kg milk to reach a pH of about 6.5 and was subjected to ceramic microfiltration at the conditions reported in Table 1.

[0156] Table 1. Condition of the ceramic microfiltration.

[0157] The pH of the acidified milk and of diafiltration water was checked with a pH-meter as known in the art.

[0158] The microfiltration yielded a whey protein rich stream as a permeate and a casein protein rich stream as a retentate. The retentate, comprising the micellar casein fraction, was then further mixed with diafiltration water (pH value of 4.1) and the mixture was subjected to ultrafiltration following the conditions reported in Table .

[0159] Table 2. Conditions of the ultrafiltration. The ultrafiltration yielded a liquid micellar casein isolate (LMCI) as retentate and a salts-rich stream as permeate. The total solid, protein content, lactose content and mineral content of the LMCI were measured using the MilkoScan.

[0160] The heat stability of the LMCI was tested by submerging the samples in an oil bath at 127 °C for 5, 10, 15 and 20 min with constant agitation. The particle size distribution before and during heat treatment was measured according to ISO 22412:2017.

[0161] Results

[0162] Mineral content

[0163] Compared to Comparative Example 2, wherein CO2 injection is performed directly on the retentate comprising the casein fraction, the mineral content of the process according to the invention was reduced and reached the specifications necessary for the further use of the LMCI in the preparation of medical nutrition. In this regard, the amount of Ca present in the MCI reached below an amount of less than 29.15 mg per g protein and the amount of P reached below an amount of 19.0 mg per g protein.

[0164] Heat stability

[0165] After heat treatment, the LCMI was visually stable. To quantify the heat stability, particle size distribution was measured at different times (heat coagulation time): unheated, after 5 minutes, 10 minutes, 15 minutes and 20 minutes. An increase in size indicates coagulation, which may result in protein aggregation that can create a gel-like consistency. A decrease in size would imply disassembly of the casein micelles. Both situations indicate poor heat stability of the LMCI. If particle size distribution is unchanged by the heat treatment, it is an indication that the LMCI is heat stable. HCT is the time at which the onset of visible coagulation in the sample was observed, see e.g. Holm et al., Heat Coagulation of Milk, Journal of Dairy Science, Chapter I, 1931. The obtained LMCI reached a heat coagulation time (HCT) of up to 20 minutes at 127 °C. In addition, heat stability is determined by swelling or aggregation of particles as assessed by visual observation.

[0166] Taken together, the results indicate that the process of the invention is effective in removing minerals from LMCI and obtaining an LMCI with heat stability. Similar results are expected for weak organic acids such as citric acid. EXAMPLE 2

[0167] Example 2 shows the process according to the invention, wherein the milk was acidified by injection of CO2 into the milk or by addition of diafiltration water comprising citric acid before microfiltration to obtain acidified milk with a pH of 6.5.

[0168] Methods

[0169] Samples were prepared according to Table 3.

[0170] Table 3. Samples of Example 2

[0171] In batches A and B, skimmed milk was injected with CO2 and then mixed with diafiltration water (pH value of the diafiltration water is about 7) to reach a pH of 6.5 in the acidified milk. In batch C, citric acid was dissolved in diafiltration water to reach a pH of 4.1. The acidified diafiltration water was subsequently mixed with skimmed milk to obtain the acidified mixture. The skimmed milk-diafiltration water mixture was subjected to ceramic microfiltration at the conditions reported in Table 2. The microfiltration yielded a whey protein-rich steam as permeate and casein-rich stream as retentate. Further diafiltration water (pH 7.0) was added to the retentate and the mixture was subjected to ultrafiltration following the conditions reported in Table 3. The ultrafiltration yielded a liquid micellar casein isolate (LMCI) as retentate and a salts-rich stream as permeate.

[0172] The mineral content of the LMCI was measured according to Example 1. The heat stability of the LMCI was challenged with heat treatment according to Example 1. The size distribution and pH were measured according to Example 1.

[0173] Results The content of minerals was reduced in MCI obtained from each batch. Batch A - C all had a Ca content of less than 2600 mg / 100 g based on dry weight of the MCI and had less than 1650 mg / 100g ofP.

[0174] Batch A endured heat treatment with less than 4 - 6% change in particle size (based on D90) up to 15 minutes. Batches B and C have lower heat stability than Batch A, but satisfactory to resist further processing steps and have a 7 - 9 % change in particle size based on D90 . All batches remained heat-stable and reached a HCT up to 20 minutes at 127°C.

[0175] Taken together, the results showed that the process of the invention wherein CO2 was injected directly into the milk (Batches A and B) is effective in removing minerals. The obtained LMCI has a satisfactory heat stability. Injection of CO2 directly into the milk has the further advantage of a simpler process setup compared to the addition of citric acid to diafiltration water.

[0176] EXAMPLE 3

[0177] Milk has been subjected to microfiltration and ultrafiltration to yield casein-enriched and whey-protein enriched streams wherein a CO2 acidification was implemented into the method. A comparison has been made between the methods wherein:

[0178] (1) CO2 is injected into the casein-rich stream obtained as the retentate of microfiltration (MFR);

[0179] (2) CO2 is injected into the casein-rich stream obtained as the retentate of microfiltration and injected into the diafiltration water used in ultrafiltration;

[0180] (3) CO2 is injected directly into the milk before microfiltration.

[0181] The conditions of microfiltration and ultrafiltration were according to Table 1 and 2 respectively. Reference is made to Example 1 for measurement on the composition, pH, heat stability and particle size distribution.

[0182] Methods

[0183] ( 1 ) COAnj ection into the casein-rich stream obtained as retentate of microfiltration

[0184] CO2 was injected directly into the casein-rich stream obtained as the retentate of microfiltration, which was then subjected to ultrafiltration. Skimmed milk was mixed with diafiltration water (pH 7.0) and subjected to ceramic microfiltration at conditions according to Table 1 (pore size = 0.1 pm). The retentate rich in casein was injected with 4 kg / hour CO2 to reach a pH of 6.5 and then subjected to a multiple loop UF unit of 10 kDa with the usage of diafiltration water (pH 7.0). The conditions of ultrafiltration are listed in Table 2.

[0185] (2) CO2 injection into the casein-rich retentate and into the diafiltration water used in microfiltration

[0186] Skimmed milk was mixed with diafiltration water (pH 7.0) and subjected to ceramic microfiltration at conditions according to Table 1 (pore size = 0.1 pm). There was no acidification prior to microfiltration, the pH was in the range of 6.9- 7.0. The retentate rich in casein was injected with CO2 to reach a pH of 6.5 and then subjected to a multiple loop UF unit of 10 kDa with the usage of diafiltration water which has been injected with CO2 (pH is 4.1) and split through the multiple loops.

[0187] (3) CO2 injection into the milk before microfiltration

[0188] Acidification was performed by injecting gaseous CO2 at 6 kg / hour directly into the milk to reach a pH of 6.5 in the acidified milk before being subjected to microfiltration. The method was performed according to example 1.

[0189] Results

[0190] Heat stability was evaluated by determining whether the particle size distribution of the obtained LMCI remains unchanged after heat treatment. Both methods using CO2 injection on into the MFR and directly into the milk resulted in a heat stable LMCI by visual observation while method (2) using CO2 injection into the MFR and diafiltration water of UF resulted in a heat unstable LCMI by visual observation of swelling and aggregated particles due to the uncontrolled acidification by the diafiltration water through the multiple loops.

[0191] Further measurements on the mineral content of the heat-stable LMCI according to method (1) and (3) were performed. The specification requirements of protein (14.5-16%) and lactose content (0.45-0.70%) based on total content of the LMCI were met. The average was taken from the measurements which has been shown in Table 4.

[0192] Table 4. Sample results of LMCI obtained from methods (1) and (3).

[0193] The method according to the invention thus achieves a lower calcium and phosphorous content and offers a more efficient process. In particular, the timing of acidification is not arbitrary. If acidification is performed only after completion of microfiltration, minerals that could otherwise have been removed via the permeate remain associated with the casein in the retentate, thereby necessitating an additional separation step.

[0194] COMPARATIVE EXAMPLE 1

[0195] Milk is subjected to microfiltration and ultrafiltration to yield casein-enriched and wheyprotein enriched streams, but without acidifying the milk prior to microfiltration. To investigate the optimal location for the CO2 injection, CO2 is injected in the diafiltration water of the ultrafiltration step to reach a pH of about 4.1.

[0196] Methods

[0197] Skimmed milk is mixed with diafiltration water and subjected to ceramic microfiltration at conditions according to Table 5. There is no acidification prior to microfiltration, the pH is in the range of 6.9- 7.0. The retentate rich in casein is then subjected to a multiple loop UF unit of 10 kDa (loops 1 - 4). Diafiltration water is injected with CO2 (pH is 4.1) and split through the multiple loops.

[0198] Table 5. Conditions of the ceramic microfiltration.

[0199] Results

[0200] This setup of multiple loops with acidified diafiltration water leads to heat unstable LMCI due to the uncontrolled acidification by the diafiltration water through the multiple loops. The heat coagulation time (HCT) of the obtained LMCI is less than 5 minutes at 127°C. It is verified that during the first and second loop the UF retentate has a pH of 6.5 but from the third and fourth loop, the retentate is very sensitive to the addition of acidified water, pH decreases rapidly to 6.1 and even lower than 6 and casein is irreversibly damaged on heat stability because the aggregated casein structure becomes more susceptible to denaturation and structural changes when exposed to heat, resulting in reduced heat stability and decreased functionality in dairy products.

[0201] Adjusting the concentration of CO2 added per loop to ensure a better pH was not feasible as it would involve a high complexity setup.

[0202] COMPARATIVE EXAMPLE 2

[0203] Milk is subjected to microfiltration and ultrafiltration to yield casein-enriched and wheyprotein enriched streams, but without acidifying the milk prior to microfiltration. To investigate the optimal location for the CO2 injection, in comparative example 2 CO2 is injected directly into the casein-rich stream obtained as the retentate of microfiltration, which is then subjected to ultrafiltration.

[0204] Methods

[0205] Skimmed milk is mixed with diafiltration water (pH 7.0) and subjected to ceramic microfiltration at conditions according to Table 5 (pore size = 0.1 pm). The retentate rich in casein is injected with CO2 to reach a pH of 6.5 and then subjected to a multiple loop UF unit of 10 kDa with the usage of diafiltration water (pH 7.0).

[0206] Results This setup leads to acceptable heat stable LMCI with a HCT of more than 5 minutes at 127°C but with little impact on the mineral content due to the short contact time between the microfiltration retentate and the CO2, resulting in a LMCI with Calcium and Phosphorous content being above 29.15 mg per g protein and above 19.0 mg per g protein, respectively.

Claims

Claims1. A process for the separation of whey protein and casein from milk comprising: i. acidifying milk a) through addition of an acidic aqueous composition comprising a weak organic acid and / or dissolved gaseous CO2 to the milk; and / or b) by injection of gaseous CO2 into the milk, to obtain an acidified milk; and ii. subjecting the acidified milk to microfiltration, to obtain a whey protein-enriched permeate stream and a micellar casein-enriched retentate stream, wherein the microfiltration is carried out at a temperature of more than 30 °C; and iii. adding diafiltration water to the casein-enriched stream and subjecting the mixture to ultrafiltration, to obtain a micellar casein isolate (MCI) as retentate and a waste stream as permeate.

2. The process according to claim 1, wherein the acidified milk has a pH of 6.3 - 6.5.

3. The process according to claim 1 or 2, wherein the aqueous composition of step (i) has a pH of 3.0 - 5.0.

4. The process according to anyone of the preceding claims, wherein the diafiltration water of step (iii) has a pH of 3.0 - 5.0.

5. The process according to claim 4, wherein a weak organic acid and optionally gaseous CO2 are dissolved in the diafiltration water of step (iii).

6. The process according to any one of the preceding claims, wherein the microfiltration is carried out at a temperature of 30 to 65 °C, more preferably 40 to 60 °C, even more preferably 45 to 60 °C, most preferably 45 to 55°C.

7. The process according to any one of the preceding claims, wherein the weak organic acid is selected from citric acid, lactic acid, malic acid, tartaric acid, ascorbic acid, or combinations thereof, preferably citric acid.

8. The process according to any one of the preceding claims, wherein acidification is achieved by injection of gaseous CO2 in the milk, wherein the gaseous CO2 is injected into the milk at a time that is not more than 30 minutes, more preferably not more than 15 minutes, most preferably not more than 5 minutes before the milk is subjected to microfiltration.

9. The process according to any one of the preceding claims, further comprising adding diafiltration water to the whey protein enriched stream obtained in step (ii) and subjecting the mixture to ultrafiltration to obtain a whey protein concentrate (WPC).

10. A micellar casein isolate (MCI), preferably obtainable by the process of any one of claims 1 to 9, comprising a. protein in an amount of 75 - 99 wt.%, preferably 80 - 95 wt.% based on total dry weight of the MCI; b. total casein in an amount of at least 88 wt.%, preferably at least 90 wt.%, more preferably at least 94 wt.% based on dry weight of total protein in the MCI; c. less than 2600 mg / 100 g, preferably less than 2529 mg / 100 g, more preferably less than 2480 mg / lOOg calcium based on dry weight of the MCI; and / or less than 29.15 mg, preferably less than 28.67 mg calcium per g protein; d. less than 1650 mg / 100 g, preferably less than 1618 mg / 100 g, more preferably less than 1510 mg / lOOg phosphorous based on dry weight of the MCI; and / or less than 19.0, preferably less than 18.33 mg phosphorous per g protein; wherein the MCI has a heat coagulation time (HCT) of at least 5 minutes, preferably at least 10 minutes at 120 °C, most preferably at least 20 minutes at 120 °C.

11. The micellar casein isolate (MCI) according to claim 10, wherein the MCI further comprises e. less than 270 mg / 100 g, preferably less than 265 mg / 100 g potassium based on dry weight of the MCI; and / or less than 3.10 mg, preferably less than 3.0 mg potassium per g protein; and f. less than 115 mg / 100 g, preferably less than 112 mg / 100 g magnesium based on dry weight of the MCI; and / or less than 1.33 mg, preferably less than 1.27 mg magnesium per g protein; andg. less than 110 mg / 100 g, preferably less than 106 mg / 100 g sodium based on dry weight of the MCI; and / or less than 1.25 mg, preferably less than 1.20 mg sodium per g protein.

12. A nutritional composition comprising 10 - 20 g protein per 100 ml and / or wherein protein provides 14 - 30 % of the total energy of the composition, and wherein the protein comprises 5 - 20 g micellar casein isolate (MCI) according to claim 11 or 12 per 100 ml and / or 2 - 8 g micellar casein isolate (MCI) according to claim 11 or 12 per 100 kcal.

13. The nutritional composition according to claim 12, having an energy density of 150 to350 kcal / 100 ml, wherein at least the Ca and P levels, more preferably also the Na, Mg and K levels are within the ranges according to the table below:

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