Dephosphorylation of skimmed milk, ultra-filtered milk or micellar casein isolate

Abstract

The present invention relates to a process for the dephosphorylation of skimmed milk, ultra-filtered milk (UF milk) or micellar casein isolate (MCI) comprising (i) acidifying the skimmed milk, UF milk or MCI, preferably not below pH 6.0, preferably between 6.0 and 6.7, (ii) cooling the acidified skimmed milk, UF milk or MCI to a temperature between 0 DEG C and 15 DEG C, (iii) adding gluconate and / or maleate to the cooled skimmed milk, UF milk or MCI, and (iv) washing the skimmed milk, UF milk or MCI to remove phosphorus, thereby dephosphorylating the skimmed milk, UF milk or MCI, preferably to the extent that the total phosphorus content of the skimmed milk, UF milk or MCI is reduced by at least 20%, preferably 20-40%, more preferably 30-40%, compared to the material provided to step (i). The invention also relates to a dephosphorylated MPC, MPI or MCI obtainable by the process of the invention and to a liquid enteral nutritional composition comprising 2.0-3.0 kcal / ml of heat sterilization wherein 16 en%-35 en% is provided by a protein, the combination of the calorie content and the relative protein calorie content being selected such that 10-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein is present in the composition, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein. Wherein the protein comprises micellar casein (MC), whey protein (WP) and optionally caseinate (CAS) wherein at least 70 wt% MC and less than 15 wt% WP are present, based on the total protein content, and wherein the composition has a total amount of phosphorus of less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

Description

Technical Field

[0001] This invention pertains to the field of high-protein liquid enteral nutrition compositions, wherein micellar casein constitutes a major component. More specifically, this invention relates to the dephosphorylation of skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI). More specifically, this invention addresses the challenge of reducing the phosphorus content of skim milk, ultrafiltered milk (UF milk, including MPC or MPI), or micellar casein isolate (MCI), and relates to shelf-stable and heat-sterilized liquid enteral nutrition compositions containing 16 en%-35 en% protein and 2.0-3.0 kcal / ml, with a phosphorus content meeting the requirements for foods for special medical purposes (FSMP). Background Technology

[0002] This invention belongs to the field of high-protein liquid enteral nutrition compositions, which are ready-to-use compositions with low intake volume and rich in all essential minerals, vitamins, and trace elements. The use of high-protein nutritional supplements can reduce mortality and complication rates in malnourished patients. For malnourished patients, it is crucial to provide the necessary nutritional support and calories, as these cannot be achieved through normal eating habits alone. In particular, the use of high-protein nutritional supplements offers opportunities to improve the quality of life for malnourished patients and / or the elderly.

[0003] Many high-protein compositions are available on the market, such as the Compact protein series (Nutricia). The technical challenge is to produce compositions with increased protein density that remain shelf-stable (i.e., able to withstand the heat conditions necessary for heat sterilization to achieve shelf stability) without compromising texture and flavor, and utilizing high-quality protein. Micellar casein is a preferred candidate for such high-protein compositions; it serves as a benchmark for protein quality standards alongside whey protein, but unlike whey protein or caseinates, micellar casein can withstand heat treatment even at high volumes. Therefore, its use in high-protein and high-calorie compositions has been discovered.

[0004] However, currently commercially available high-protein liquid enteral nutrition compositions with extremely high micellar casein concentrations do not meet the requirements for Foods for Specific Medical Purposes (FSMPs) because these compositions exceed (i.e., exceed) the phosphorus levels required by FSMP regulations. Ca and P are essential components of micelles, with P concentration playing a particularly important role. In the context of this invention, the FSMP regulations are European Commission Directive 1999 / 21 / EC of 25 March 1999 concerning guidance on Foods for Specific Medical Purposes (FSMPs), the contents of which are incorporated herein by reference. A good example of such commercially available micellar casein products is MCI88 from FrieslandCampina, with a phosphorus level of 1500-1700 mg / 100g powder (and 85 g protein / 100g powder), i.e., 17.6-20 mg phosphorus / g protein.

[0005] To a large extent, phosphorus is derived from micellar casein. Casein provides about half of the milk phosphorus (22% organic, 32% inorganic), present at approximately 1 g P / kg milk [Source: Walstra, P., Wouters, JT, & Geurts, TJ (2006). Part 2: Processes, in Dairy science and technology. CRC press: Boc Raton, USA, pp. 207-272]. Casein contains two types of phosphorus: organic phosphorus and inorganic phosphorus. P is either part of colloidal calcium phosphate (inorganic P) or covalently bound to casein as a phosphate group (casein-P). Casein micelles typically contain both organic phosphorus (46%) and inorganic phosphorus (64%). Removing phosphorus from such micellar casein products is challenging because phosphorus, along with calcium, is a key factor in stabilizing the micellar structure, which is crucial for maintaining the stability of high-protein compositions. The core of casein micelles is bound by calcium phosphate nanoclusters (CCPs or casein phosphopeptides), and these CCP nanoclusters are important for maintaining the thermal stability of the casein micelle structure. Without CCPs, the micelles will disintegrate. Although phosphorus can be washed away, it is important to maintain the integrity of the micelle casein to remain largely intact in order to maintain product stability while retaining the increased protein concentration and reduced viscosity. Given these trade-offs, commercially available shelf-stable (i.e., heat-sterilized) high-protein liquid enteral nutrition compositions typically contain up to 300 mg / 100 mL of total phosphorus, which is significantly higher than the maximum level of 192 mg / 100 mL (or 30–80 mg / 100 kcal) required by FSMP. The majority of the total phosphorus (approximately 250 mg / 100 mL) comes from the micelle casein source, and only a small fraction of the total phosphorus (approximately 50 mg / 100 mL) originates from K₂PO₄. To meet the maximum value set by the FSMP requirements, P should be reduced by at least 23%, and it is clear that such a number can only be achieved when using an MCI that exhibits a reduced P level.

[0006] WO 2009 / 072885 discloses problems related to the use of micellar casein in the production of high-protein liquid enteral nutrition compositions, and further problems in the presence of acids (particularly citric acid) include the formation of calcium-acid complexes (such as calcium citrate). It is also recognized that a certain level of Ca ion activity is beneficial in maintaining the desired viscosity of the composition during processing (e.g., during pasteurization and / or sterilization). The viscosity of the composition during processing, including heat sterilization, is controlled by using a mixture of micellar casein and casein acid salts. WO 2013 / 129925 discloses a high-energy liquid enteral nutrition composition containing 6 to 20 g / 100 ml of protein, said protein comprising micellar casein and lactic acid. The object of this application is to reduce the viscosity of protein-dense compositions with increased levels of micellar casein by adding lactic acid instead of using citric acid.

[0007] US 2011 / 159163 describes a method for increasing the protein concentration in milk and reducing the mineral content in concentrated milk, using acidification and incubation at pH 5.9–6.3, followed by ultrafiltration at pH 6.4–7.0. Calcium and phosphorus achieve optimal solubility within this initial pH range. To avoid the growth of pathogenic bacteria during incubation, the temperature is controlled above 37°C or below 25°C. The pH is lowered using at least one acid selected from the group consisting of: citric acid, malic acid, lactic acid, acetic acid, phosphoric acid, gluconic acid, gluconic acid-δ-lactone, and / or hydrochloric acid. In examples, citric acid is used for pH adjustment, and ultrafiltration is performed at 50°C. The protein residue in these examples contains approximately 16 wt%–18 wt% protein, and 18–21 mg Ca and 13–13.5 mg P / g protein. While this method is intended for concentrated milk, these conditions are not suitable for achieving higher protein concentrations associated with milk protein concentrates and isolates.

[0008] WO 2015 / 156662 describes the use of transglutaminase (TG) to cross-link micellar casein, which helps stabilize milk heat even at high protein concentrations. The calcium and phosphorus content of cross-linked micellar casein can be reduced without destabilizing the casein. However, TG treatment is costly and difficult to implement on an industrial scale.

[0009] US 6,558,717 discloses a method for separating casein and calcium phosphate as separate products from milk, wherein carbon dioxide is contacted with the milk under pressure to precipitate casein. While the method refers to casein, it involves the production of caseinate, thereby separating it from calcium phosphate. Under these conditions, the micellar structure is preserved.

[0010] For the purpose of producing phosphorus-reduced bovine casein for use in the infant formula industry, McCarthy Noel et al. " The physical characteristics and emulsification properties of partially dephosphorylated bovine [beta]-casein [Physical Characteristics and Emulsifying Properties of Partially Dephosphorylated Bovine [β]-casein]” FOOD CHEMISTRY, Vol. 138(2) 2012, 1304-1311 describes the partial dephosphorylation of bovine β-casein by coagulation with rennet and cold dissolution of the resulting curd. Dephosphorylation was carried out using potato acid phosphatase at pH 6.5. After the reaction, free phosphate groups were removed by precipitation at pH 5 and centrifugation. The authors concluded that, unlike undephosphorylated β-casein which forms a continuous gel structure, partially dephosphorylated β-casein does not gel under acidic conditions.

[0011] Choi Inseob et al. Gluconic acid as a chelator to improve clarity of skim milk powder dispersions at pH 3.0 "[Glucolic Acid as a Chelating Agent to Improve Clarity of Skim Milk Powder Dispersions at pH 3.0]" Food Chem., 344, 2020 describes the use of gluconic acid to improve the clarity of 5 w / v% skim milk powder dispersions in acidic environments. When citric acid is used instead, the turbidity is lower, but the dispersion remains translucent. Glucolic acid is a better chelating agent, and the higher solubility of CCP in the whey phase supports this conclusion.

[0012] While high amounts and concentrations of micellar casein are preferred for the production of high-protein liquids, the drawback is that it leads to increased phosphorus concentrations, thus failing to meet FSMP requirements. Furthermore, removing phosphorus without destabilizing the micelles remains challenging. Therefore, there is a need in the art to reduce the phosphorus content in high-protein liquid enteral nutrition compositions with high micellar casein content to ultimately meet FSMP requirements. For energy-intensive high-protein products that primarily contain micellar casein as a protein source (e.g., at least 80 wt% of the protein is micellar casein), the phosphorus content should be less than 15 mg, preferably less than 14 mg, and even more preferably less than 13.3 mg phosphorus / g protein. Essentially, the lower the phosphorus content, the better, but without impairing the micellar casein structure; only under these limiting conditions can high-protein compositions meeting FSMP requirements for phosphorus (using a nitrogen coefficient of 6.25) with protein levels exceeding 12 g / 100 ml and up to 14.4 g / 100 ml be produced. Summary of the Invention: This invention provides a method for reducing the phosphorus content of skim milk, ultrafiltration milk (UF milk) (including milk protein concentrate (MPC) and milk protein isolate (MPI)) or micellar casein isolate (MCI), and the dephosphorylated product is a suitable component for producing shelf-stable (heat-sterilized) high-protein liquid enteral nutrition compositions that meet FSMP requirements, particularly in terms of phosphorus content (maximum value of 192 mg / 100 ml or 30-80 mg / 100 kcal according to FSMP Directive 1999 / 21 / EC).

[0014] Commercially available skim milk, UF milk, and MCI contain approximately 1700 mg P / 100 g powder (or 20 mg P / g protein), a level so high that it is difficult to increase the amount of these proteins in high-protein products. As illustrated in the examples, by acidifying to pH 6.0, cooling to 4°C, and using 70 mEq. gluconate, the dephosphorylation method of this invention enables a reduction to 75 mg P / 100 g powder (or 12.3 mg P / g protein), a reduction of 38.3%. The method of this invention is particularly suitable for concentrated protein applications such as MPC, MPI, and MCI.

[0015] Gluconate or maleate chelating agents bind to calcium, enabling the elution of phosphorus while stabilizing the micelle casein structure. The effects of selective acidification and specific chelating agents have been demonstrated in examples, and results show that chelating agents capable of reducing phosphorus while preserving a thermally stable micelle structure are limited to gluconate and maleate, with gluconate yielding the best results. Care should be taken to control pH to minimize or even avoid the formation of acid gels in the micelles. As noted, particularly good thermal stabilization has been achieved with gluconate and maleate, especially gluconate. Citrate is a common calcium chelating agent in the art, but it shows no ability to maintain micelle thermal stability, and the amount of phosphorus reduction is limited. Acetates have shown disappointing results, presumably due to low affinity for CCP, with no phosphorus reduction observed. Lactates have an even weaker calcium chelating affinity than acetates, so similar results are expected. Hoping not to be bound by any theory, the inventors believe that the differences between chelating agents and the success of maleates, especially gluconate, lie in how they affect the activity of calcium in solution and how these effects alter the casein-mineral balance. The chelating agent reduces free calcium ions, causing CCP to dissolve from casein and releasing certain casein proteins from the micelles, which in turn affects thermal stability. Based on the results provided, we believe that citrate causes complete dissolution of CPP from casein, and as explained above, these caseins disintegrate in the absence of CCP. On the other hand, lactate and acetate have no affinity for CCP.

[0016] Therefore, in a first aspect, the present invention provides a method for dephosphorylation of skim milk, ultrafiltration milk (UF milk) (including MPC and MPI) or MCI, the method comprising: (i) Acidified skim milk, UF milk, or MCI, preferably at a pH of not less than 6.0, (ii) Cool the skim milk, UF milk, or MCI to a temperature between 0°C and 15°C. (iii) Adding gluconate and / or maleate to cooled skim milk, UF milk, or MCI, and (iv) Wash the skim milk, UF milk, or MCI (step (iii)) to remove phosphorus, wherein washing is preferably performed using microfiltration and / or percolation. This dephosphorylates the skim milk, UF milk, or MCI.

[0017] Regarding "dephosphorylation," it should preferably be understood as a reduction in the total phosphorus content of the starting material (i.e., skim milk, UF milk, or MCI) by at least 20%, preferably 20%-40%, and more preferably 30%-40% (compared to the total phosphorus content of the starting material). Commercially available MCI contains approximately 1500-1700 mg / 100 g of total phosphorus, and in the case of approximately 85 / 100 g of protein, the amount of phosphorus in such MCI powder is typically about 18-20 mg / g of protein. Although the amount of micellar casein in other starting materials (skim milk, UF milk) may be lower than the amount calculated above for MCI, the same 18-20 mg / g of protein is typical for those starting materials because the only source of phosphorus in these other milk sources is micellar casein.

[0018] In a second aspect, the present invention relates to skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI) dephosphorylated with gluconate and / or maleate, which contains 20% to 40% less total phosphorus (total content) than the original skim milk, UF milk, or MCI not dephosphorylated according to the invention. The phosphorus content of the “dephosphorylated” skim milk, UF milk (including MPC, MPI), or MCI is preferably less than 15.0 mg phosphorus / g protein, more preferably less than 14.0 mg phosphorus / g protein, and even more preferably less than 13.3 mg phosphorus / g protein. While there is no lower limit, in practice, the amount of phosphorus is preferably greater than 10 mg phosphorus / g protein. Skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI) dephosphorylated with gluconate and / or maleate can and preferably is obtained by the method according to the invention and as described above and throughout the details of this application.

[0019] In another aspect, the present invention provides the use of dephosphorylated skim milk, UF milk, or MCI for the manufacture of heat-sterilized high-protein liquid enteral nutrition compositions comprising 2.0-3.0 kcal / ml, wherein 16 en%-35 en% is provided by protein, and wherein the protein comprises at least 70 wt% micelle casein based on total protein. In a related aspect, the present invention provides a heat-sterilized liquid enteral nutrition composition comprising 2.0-3.0 kcal / ml, wherein 16 en%-35 en% is provided by protein, wherein the protein comprises micellar casein (MC), whey protein (WP), and optionally caseinate (CAS), wherein there is at least 70 wt% MC and less than 15 wt% WP based on the total protein content, and wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal. The composition is preferably characterized at least by weight per calorie (mg / kcal), according to FSMP, i.e., 30-80 mg / 100 kcal, more preferably 30-72 mg / 100 kcal, and even more preferably 30-64 mg / 100 kcal. The composition further comprises gluconate and / or maleate. The presence of detectable amounts of gluconate and / or maleate in the composition indicates the use of dephosphorylated skim milk, UF milk, or MCI, which is necessary to achieve the reduced phosphorus content required to meet FSMP standards.

[0020] Within the aforementioned calorie limit of 2.0-3.0 kcal / ml and the protein calorie contribution range of 16 en%-35 en% will allow the composition to provide 8.0-26 g / 100 ml of protein. However, the term "high protein" preferably refers to a combination of calorie content and protein calorie content such that the composition contains 10-18 g / 100 ml of protein, preferably 12-18 g / 100 ml of protein. The challenge of FSMP compliance is particularly prominent at these higher protein concentrations. Attached image description: Figure 1: Effects of acidification on (a) stability (ζ potential), (b) bulk strength (particle size) and (c) viscosity.

[0022] List of preferred embodiments 1. A method for dephosphorylation of skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI), the method comprising: (i) Acidified skim milk, UF milk, or MCI, preferably with a pH not lower than 6.0, and preferably between 6.0 and 6.7. (ii) Cool the acidified skim milk, UF milk, or MCI to a temperature between 0°C and 15°C. (iii) Adding gluconate and / or maleate to cooled skim milk, UF milk, or MCI, and (iv) Wash the skim milk, UF milk, or MCI to remove phosphorus. This dephosphorylates the skim milk, UF milk, or MCI, preferably to the extent that the total phosphorus content of the skim milk, UF milk, or MCI is reduced by at least 20%, preferably 20%-40%, and more preferably 30%-40% compared to the material provided in step (i).

[0023] 2. The method according to Example 1, wherein the gluconate and / or maleate chelating agent is sodium gluconate, potassium gluconate, disodium maleate and / or dipotassium maleate, preferably sodium gluconate and / or potassium gluconate.

[0024] 3. A dephosphorylated skim milk, ultrafiltration milk (UF milk), or micellar casein isolate (MCI) that can be obtained by the method according to Example 1 or 2.

[0025] 4. A dephosphorylated skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI) comprising (added) gluconate and / or maleate, wherein the total phosphorus content of the skim milk, UF milk, or MCI is less than 15.0 mg P / g protein, more preferably less than 14.0 mg, and even more preferably less than 13.3 mg total phosphorus / g protein, wherein the dephosphorylated skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI) is preferably obtainable by means of the method according to Example 1 or 2.

[0026] 5. Use of dephosphorylated skim milk, UF milk, or MCI according to any one of Examples 3 or 4 for the manufacture of a heat-sterilized high-protein liquid enteral nutrition composition comprising 2.0-3.0 kcal / ml, wherein 16 en%-35 en% is provided by protein, and wherein the protein comprises at least 70 wt% micelle casein based on total protein, wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

[0027] 6. A heat-sterilized liquid enteral nutrition composition comprising 2.0-3.0 kcal / ml, wherein 16 en%-35 en% is provided by protein, a preferred combination of calorie content and relative protein calorie content, such that the composition contains 10-18 g / 100 ml protein, preferably 12-18 g / 100 ml protein, wherein the protein comprises micellar casein (MC), whey protein (WP) and optionally caseinate (CAS), wherein there is at least 70 wt% MC and less than 15 wt% WP based on the total protein content, and wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

[0028] 7. The liquid composition according to Example 6, wherein the total phosphorus content is 30-72 mg / kcal (i.e., at least 10% below the maximum FSMP value), preferably 30-64 mg / kcal (i.e., at least 20% below the maximum FSMP value).

[0029] 8. The liquid composition according to Example 6 or 7, wherein the protein provides 16% to 32% of the total energy content of the composition, more preferably 18% to 30% or even more preferably 20% to 28% or, preferably, in combination with a calorie content of 2.2-2.6 kcal / ml.

[0030] 9. The liquid composition according to any one of Examples 6-8, wherein the amount of protein is between 12 g / 100 ml and 18 g / 100 ml.

[0031] 10. The liquid composition according to any one of Examples 6-9, wherein the weight ratio of micelle casein to caseinate ranges from 90:10 to 60:40.

[0032] 11. The liquid composition according to any one of Examples 6-10, wherein the amount of whey protein is less than 10 wt% of the total protein.

[0033] 12. The liquid composition according to any one of Examples 6-11, wherein the composition comprises 75 mg P / 100g dry weight and / or 12.3 mg P / g protein.

[0034] 13. The liquid composition according to any one of Examples 6-12, further comprising at least two minerals of Na, K, Cl, Ca, and Mg in levels within the range (mg / 100 kcal) according to the table below, more preferably at least three, even more preferably at least four, and most preferably all minerals: Detailed implementation method: This invention provides a method for dephosphorylation of skim milk, ultrafiltration milk (UF milk), or micellar casein isolate (MCI). As a first step, the skim milk, UF, or MCI is acidified (i.e., pH < 7, but preferably not lower than 6.0). Below pH 6.0, acid formation is observed in the MCI, and the desired micellar structure is lost. In an example, such acid gel formation was observed at pH 5.8. (See reference for example.) Figure 1B .

[0036] While this method is applicable to the dephosphorylation of skim milk, ultrafiltered milk (UF milk) (including MPC and MPI), or MCI, the starting material is preferably UF milk (including MPC and MPI) or MCI. Ultrafiltered milk is a subclass of milk protein concentrate produced by passing milk under pressure through a thin, porous membrane to separate milk components according to size. Specifically, ultrafiltration allows smaller lactose, water, mineral, and vitamin molecules to pass through the membrane, while larger protein and fat molecules (key components in cheese preparation) are retained and concentrated. Preferred sources are MPC, MPI, and MCI because of the increased protein concentration therein. The protein concentration is preferably at least 60 wt%. Within this subgroup, considering the lower whey protein concentration of MCI, MCI is the preferred starting material. All of the above are commercially available.

[0037] Following the acidification step, the acidified mixture is cooled at a temperature between 0°C and 15°C, preferably between 2°C and 12°C, and more preferably between 2°C and 9°C. In this example, it is shown that when cooling from 20°C to a lower temperature, preferably within the above-mentioned range, the reduction in P is improved. Commercially available industrial-scale cooling is not preferred, and those skilled in the art can optimize the balance between the reduction in P and the need for excess resources required for cooling to achieve an appropriately reduced P level.

[0038] Unless otherwise expressly stated, the terms “phosphorus” and “total phosphorus” are used interchangeably throughout the application and should be understood as the sum of both organic and inorganic phosphorus.

[0039] Unless otherwise expressly stated, throughout this application, the term “FSMP” refers to Foods for Special Medical Purposes (FSMP) Directive 1999 / 21 / EC of 25 March 1999.

[0040] In the context of this invention, total phosphorus concentration can be measured using any conventional method in the art, such as inductively coupled plasma optical emission spectrometry (ICP-OES) used in this example.

[0041] Finally, gluconate and / or maleate (preferably at least gluconate) are added to the cooled mixture and washed to remove phosphorus. Preferably, gluconate and / or maleate are added in an amount of 0.05-0.5 g / g micellar casein (preferably 0.1-0.4 g / g micellar casein). More preferably, gluconate is added in an amount of 0.05-0.5 g / g micellar casein (preferably 0.1-0.4 g / g micellar casein).

[0042] Casein micelles exist in milk as polydisperse spherical complexes with an average diameter of 200 nm. Casein micelles possess heterogeneous, hydrated, and dynamic structures, exhibiting loose packing and high porosity. They are composed of different types of casein, namely α-casein... s1 -Casein, α s2 Casein micelles consist of casein, β-casein, and κ-casein, as well as colloidal calcium phosphate (CCP). CCP is essential for maintaining micelle structure: casein micelles dissociate when CCP is chelated or dissolved. High-pressure treatment, pH reduction, or calcium chelating agents can induce micelle dissociation. Casein micelles contain two types of phosphorus: organic and inorganic. Phosphates esterified to casein molecules via the hydroxyl groups of serine amino acids are generally called organic phosphates and account for approximately 23% of milk, while phosphates associated with casein molecules in the form of calcium phosphate nanoclusters are called inorganic phosphates and account for approximately 32% of milk. Some phosphorus molecules also exist in the whey phase, such as dissolved inorganic phosphates and organic phosphates in ester form.

[0043] Based on common knowledge to those skilled in the art, the term "micelle casein" (MC) is not an enzymatically (transglutaminase (TG)) cross-linked micelle casein. There is no step of enzymatically or actively covalently cross-linking micelles to prevent dissociation. Therefore, in the method of the present invention, there is no step of enzymatically and (actively) covalently cross-linking casein micelles, and destabilization and dissociation of micelles must be avoided in different ways. Furthermore, in the context of the present invention, and given the exclusion of any enzymatic cross-linking step in manufacturing, the MC in the compositions of the present invention is not a transglutaminase-crosslinked micelle casein (or in other words, the present invention relates to or is limited to non-TG-crosslinked micelle casein). Throughout the application, MPC, MPI, or MCI are not enzymatically (or covalently) cross-linked MPC, MPI, or MCI.

[0044] This invention aims to reduce the phosphorus content of skim milk, ultrafiltered milk (UF) or micelle casein isolate (MCI), and shelf-stable and heat-sterilized high-protein liquid enteral nutrition compositions by removing esterified organophosphates on serine amino acids and inorganic phosphates present in calcium phosphate nanoclusters. However, reducing phosphorus content is challenging because casein micelles can dissociate and disrupt their structure. The inventors have discovered methods for reducing the phosphorus content of skim milk, ultrafiltered milk (UF), or micelle casein isolate (MCI) while preserving the basic micelle structure; although these micelles may differ from micelle casein with normal phosphorus levels, this is reflected in the size reduction attributed to dephosphorylation and other processing steps. However, despite the above, it has been unexpectedly shown that when using the method according to the invention, the resulting structure is sufficiently stable to withstand the heat treatment necessary for the extended shelf life required by the products according to the invention.

[0045] Maleate and gluconate were found to play a crucial role in chelating phosphorus into and extracting phosphorus from micelles without significantly affecting the micelle structure. Calcium chelating agents are not new in the art, and those skilled in the art typically employ citrate or even lactate, both of which are widely used in high protein concentration applications. Chelating agents contribute to the disintegration of casein micelles, the reduction of the negative charge of κ-casein, and the release of free calcium. 2+ The interaction between ions and κ-casein has an effect. The benefit of this invention is found to be specific to the named chelating agent, while citrate cannot induce the desired reduction in P levels while preserving the micellar casein structure.

[0046] While good results were obtained with dipotassium maleate and disodium maleate, Table 1 shows that the best results were achieved with gluconate. More commercially available chelating agents are citrate and acetate, but these were found unsuitable for achieving the effects observed with potassium or sodium gluconate, as well as dipotassium maleate and disodium maleate. Lactate does not interact with CCPs in casein micelles and therefore does not play a role here (Source: de Kort, EJP (2012)). Influence of calcium chelators on concentrated micellar casein solutions: from micellar structure to viscosity and heat stability[The Effects of Calcium Chelating Agents on Concentrated Micellar Casein Solutions: From Micellar Structure to Viscosity and Thermal Stability]. Internal PhD, WU, Wageningen University. Page 129. On the other hand, gluconate and maleate not only enhance the consumption of phosphorus from casein micelles but also help maintain the intrinsic structure of casein micelles. Furthermore, unlike the other chelating agents mentioned, the addition of gluconate or maleate does not affect thermal stability. When using maleate, disodium maleate is preferred. In the context of this invention, embodiments involving the use of maleate as a chelating agent are not required to protect hydrogen maleate salts such as potassium hydrogen maleate and sodium hydrogen maleate.

[0047] Gluconate is the most preferred chelating agent, preferably sodium gluconate or potassium gluconate.

[0048] The acidification step is performed to increase the solubility of phosphorus in casein micelles. This is an important step because adjusting the pH affects the particle size, viscosity, and stability of casein micelles. Figure 1 shows a significant decrease in negative charge observed at pH between 6.7 and 6.0, while viscosity and particle size remain the same. No significant decrease in negative charge was observed at pH between 6.0 and 5.6, but a significant increase in particle size was observed. This is attributed to gel formation. At pH 5.4, a significant increase in both particle size and viscosity was observed due to the complete collapse of κ-casein. Therefore, during the acidification step, the pH is preferably between 6.0 and 7.0, more preferably between 6.0 and 6.7, and even more preferably between 6.1 and 6.5.

[0049] Washing to remove phosphorus is preferably carried out by microfiltration and / or percolation, wherein the dephosphorylation product is a residue.

[0050] The present invention further provides skim milk, ultrafiltered milk (UF milk) (including milk protein concentrate (MPC) and milk protein isolate (MPI)) or micellar casein isolate (MCI) dephosphorylated with gluconate and / or maleate, which contains 20% to 40% less total phosphorus compared with the corresponding untreated counterparts, the corresponding counterparts being further characterized by the absence of gluconate and / or maleate, and a phosphorus concentration (i.e., the sum of organic and inorganic phosphorus) less than 192 mg / 100 ml in compliance with FSMP regulations.

[0051] Preferably, the total phosphorus content of the high-protein composition is at least 10% lower than the FSMP maximum (i.e., less than 72 mg / 100 kcal), and preferably at least 20% lower than the FSMP maximum (i.e., less than 64 mg / 100 kcal). This is summarized in Table 1 below. Therefore, most preferably, a reduction in the original phosphorus concentration is desired between 30% and 40% (Table 1). While part of this reduction can be achieved by reducing the amount of phosphate commonly used in such compositions (preferably by avoiding the use of both phosphate and phosphoric acid), the desired reduction in total phosphorus levels compared to the FSMP presented in Table 1 can be achieved according to the invention by reducing the amount of phosphorus in a typical micellar casein source having 300 mg P / 100 ml compared to the original 300 mg P / 100 ml concentration in the starting material, preferably by at least 23%, more preferably by at least 31%, and most preferably by at least 38%. Utilizing those reductions in the P content of micellar casein achievable through the method of the invention, the total phosphorus level can be reduced below the FSMP maximum limit, and preferably to less than 90% or even less than 80% of the maximum total phosphorus content set by the FSMP.

[0052] Table 1: Preferred maximum phosphorus content of heat-sterilized high-protein liquid enteral nutrition compositions compared to commercial products and FSMP.

[0053] This application provides a heat-sterilized high-protein liquid enteral nutrition composition with reduced phosphorus content that meets FSMP requirements. In a preferred embodiment, the composition contains phosphorus in an amount of 32-78 mg / 100 kcal, preferably 36-76 mg / 100 kcal, even more preferably 38-74 mg / 100 kcal, and most preferably 40-70 mg / 100 kcal.

[0054] The term "liquid enteral nutrition composition" refers to an aqueous composition comprising protein, fat, and carbohydrates, which is administered orally or otherwise (typically via tube feeding) to the stomach or intestines of a patient. Oral administration is preferred. The viscosity is preferably less than 500 cP, more preferably less than 400 cP, and most preferably less than 300 cP, as measured at 20°C using a rotational viscometer with cone / plate geometry at a shear rate of 100 s⁻¹. The heat-sterilized high-protein liquid enteral nutrition composition according to the invention is designed to supplement a person's diet or provide complete nutritional support. Therefore, the composition according to the invention further comprises fat and carbohydrates, as well as preferably sources of vitamins and minerals and / or prebiotics. Preferably, the composition according to the invention is a complete nutritional composition.

[0055] The high-protein composition of the present invention is a packaged product, ready for transport and sale. In a preferred embodiment, it is a ready-to-use composition. It is heat-sterilized and preferably shelf-stable.

[0056] The term "heat-sterilized" refers to a food that has been heat-treated to destroy foodborne microorganisms and can be safely stored on a shelf at room temperature (typically for a period of at least 10 months). The composition is preferably a shelf-stable composition. The term "shelf-stable" as used herein refers to storage stability. A nutritional composition is shelf-stable if it remains stable against microbial spoilage and physical defects (such as cream separation, gelation, precipitation, etc.) for a certain period of time when stored at ambient temperature in sealed packaging. Preferably, when stored at ambient temperature (20°C) in sealed packaging, the nutritional composition has a shelf stability of at least one month after packaging, more preferably at least three months, even more preferably at least six months, and most preferably at least 12 months. The invention is not limited to specific sterilization conditions, and in fact, the invention enables high-protein foods to withstand a variety of heat sterilization conditions common in the art and well-known to those skilled in the art. However, a significant part of the challenge in achieving high-protein compositions in the art stems from the need for such heat treatment to reduce the microbial load to a level suitable for long-term product storage.

[0057] The total protein preferably provides 16% to 32% of the total energy content of the composition, more preferably 18% to 30% or even more preferably 20% to 28% or more. In preferred embodiments, each of these numbers is combined with a calorie content of 2.2 kcal / ml to 2.6 kcal / ml. Therefore, it is deduced that the amount of protein in the composition is preferably 8.8-20.8 g / 100 ml, more preferably 9.9-19.5 g / 100 ml, and most preferably 11-18.2 g / 100 ml. However, even more preferably, the amount of protein is between 12 g / 100 ml and 18 g / 100 ml. It is at these higher protein concentrations that phosphorylated protein (i.e., a lower protein concentration per gram of protein) provides an advantage over commercially available high-protein compositions (which do not meet FSMP guidelines when using extremely high amounts of MC).

[0058] The liquid high-protein composition according to the invention is characterized by a high amount of micelle casein, preferably 70 wt%-95 wt%, more preferably 75 wt%-95 wt%, and most preferably 80 wt%-95 wt% of all proteinaceous substances. The term "micelle casein" refers to casein in milk with a structure known in the art, comprising different caseins, namely αS1, β, αS2, and κ caseins. As mentioned above, the size of the micelles can vary. In unprocessed milk, micelles can vary between about 100 nm and 500 nm, but after dephosphorylation using the method of the invention, although the range of micelle size variation may be wider, the micelle structure is essentially preserved.

[0059] Preferably, according to the FSMP recommended levels summarized in the table below (which is a selection from Table 2 of the above EU directive), the composition according to the invention further comprises at least two minerals of Na, K, Cl, Ca, and Mg in levels conforming to the ranges in the table below, more preferably at least three, even more preferably at least four, and most preferably all minerals:

[0060] According to one embodiment, the composition comprises 30-175 mg sodium / 100 kcal. In a preferred embodiment, the composition preferably comprises 30-140 mg / 100 kcal, more preferably 32-100 mg / 100 kcal, and even more preferably 34-80 mg / 100 kcal of sodium. Alternatively, the composition comprises 70-250 mg sodium / 100 ml of liquid composition, preferably 80-230 mg sodium / 100 ml of liquid composition.

[0061] The composition preferably contains potassium. In a preferred embodiment, the composition contains 80-295 mg potassium / 100 kcal. Specifically, the composition contains 85-250 mg / 100 kcal, more preferably 90-200 mg / 100 kcal, and even more preferably 95-150 mg / 100 kcal of potassium. Alternatively, the composition preferably contains 180-400 mg potassium / 100 ml of liquid composition, more preferably 200-380 mg potassium / 100 ml of liquid composition. For the purpose of lowering phosphorus levels, it is preferred that the potassium is not provided in the form of potassium phosphate. Suitable forms may be potassium gluconate and / or dipotassium maleate.

[0062] The dephosphorylation process does not affect, nor is it influenced by, the presence of calcium in the composition. Typically, the calcium concentration in skim milk, UF milk, or MCI is approximately 30 mg / g protein. Preferably, the Ca level is maintained within the FSMP standard, i.e., between 35 mg Ca / 100 kcal and 175 mg Ca / 100 kcal.

[0063] The composition preferably contains chlorine (Cl) in an amount of 30-175 mg / 100 kcal, more preferably 35-150 mg / 100 kcal, and even more preferably 40-140 mg / 100 kcal. Alternatively, the composition contains 100-380 mg chlorine per 100 ml of liquid composition, preferably 110-350 mg chlorine per 100 ml of liquid composition.

[0064] According to one embodiment of the invention, a shelf-stable / heat-sterilized liquid enteral nutrition composition comprises 12-16 g / 100 ml protein and 2.0-3.0 kcal / ml, preferably 2.2-2.6 kcal / ml, wherein the protein comprises micellar casein (MC), whey protein (WP), and optionally caseinate (CAS), wherein there is at least 70 wt% MC and less than 15 wt% WP based on the total protein content, the composition preferably comprises 7-12 g / 100 ml fat and 18-30 g / 100 ml digestible carbohydrates, and wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, more preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

[0065] Given the potential negative impact of WP on viscosity in heat-sterilized compositions, when WP is included in the compositions of the present invention, it should preferably be controlled to a reduced amount, i.e., less than 15 wt% of total protein, most preferably less than 10 wt%. WP can also be provided in whole form and / or hydrolyzed form. The above is the sum of both hydrolyzed WP and whole WP. The measure of the degree of hydrolysis of whey protein is the "degree of hydrolysis" (DH). DH is defined as the percentage of the total number of peptide bonds in the protein cleaved during hydrolysis. The DH of a protein can be determined, for example, by a trinitrobenzenesulfonic acid (TNBS) procedure as known in the art (Adler-Nissen, J.Agr. Food Chem. 1979, 27(6), 1256). When whey protein is hydrolyzed, the whey protein source may already contain a certain (small) amount of peptide moiety prior to the hydrolysis process. The value of the degree of hydrolysis as described herein is corrected according to the presence of this peptide moiety in the whey protein source; in other words, the value of DH is corrected according to the natural DH of the whey protein. Therefore, in this document, DH refers to additional hydrolysis obtained via a purposeful hydrolysis process. When the composition contains hydrolyzed whey protein, it preferably has a degree of hydrolysis in the range of 1% to 25%, more preferably in the range of 5% to 25%. As mentioned above, the degree of hydrolysis used herein is corrected for the natural degree of hydrolysis of the whey protein source (i.e., the whey protein used to prepare the hydrolyzed whey protein).

[0066] Given the chosen micellar casein source and the desired reduction in whey protein levels, the composition may also contain a limited amount of caseinate. Most MCI sources have a WP content of less than 10%, preferably less than 5 wt%, based on total protein. A combination of micellar casein and caseinate is particularly preferred when the source providing the micellar casein contains more than 10 wt% whey protein (such as MPC or MPI (micellar casein to whey protein weight ratio of 80:20)). Caseinate can be added to the micellar casein to control (reduce) the amount of whey protein in the protein fraction as described above. Thus, in one embodiment of the invention, the weight ratio of micellar casein to caseinate ranges from 90:10 to 75:25. Sodium caseinate, magnesium caseinate, potassium caseinate, calcium caseinate, and any mixtures thereof or combinations thereof (such as sodium caseinate / potassium caseinate and sodium caseinate / magnesium caseinate) are used as sources of caseinate. Preferably, calcium caseinate or caseinate containing Ca are not used, because micellar casein already contains sufficient calcium. Furthermore, sodium caseinate / potassium caseinate provides a better flavor.

[0067] The composition preferably comprises: -16 en% -35 en% protein, -30%-55% of digestible carbohydrates, and -30 en%-55 en% fat, Preferably, the combination of calorie content and protein (en%) is selected such that the amount of protein is between 10 g / 100 ml and 18 g / 100 ml, and most preferably between 12 g / 100 ml and 18 g / 100 ml. As used throughout this application, "en%" refers to the percentage of total energy of the composition. Therefore, it refers to energy percentage, which represents the relative amount by which the components contribute to the total calorie value of the composition. The amount of energy provided by protein, fat, and carbohydrates can be estimated, for example, using the Atwater factor, where: protein (and amino acids) is 4 kcal / g (17 kJ / g), digestible carbohydrates are 4 kcal / g, and fat is 9 kcal / g (37 kJ / g).

[0068] The liquid composition preferably contains 7-12 g / 100 ml of fat and 18-30 g / 100 ml of digestible carbohydrates. 。

[0069] The liquid nutritional composition according to the invention preferably comprises fat, which provides 30% to 55% of the total energy content of the composition. Preferably, the composition according to the invention comprises vegetable fat, preferably rapeseed oil, sunflower oil, corn oil, soybean oil, low-erucic acid rapeseed oil, or a combination thereof.

[0070] Depending on the desired benefits, the fat may comprise medium-chain triglycerides (MCTs, primarily 8 to 10 carbon atoms long), long-chain triglycerides (LCTs), or any combination of both. Preferably, the fat comprises 30 wt% to 60 wt% animal or algal fat, 40 wt% to 70 wt% vegetable fat, and optionally 0 to 20 wt% MCT, based on the total fat of the composition. If animal fat is present, it preferably comprises a low amount of milk fat, i.e., less than 6 wt%, particularly less than 3 wt%. In particular, a mixture of corn oil, egg yolk oil, and / or low-erucic acid rapeseed oil and a specific amount of marine oil is used. Egg yolk oil, fish oil, and algal oil are preferred sources of non-vegetable fats.

[0071] Especially for compositions intended for oral administration, to prevent the formation of off-flavors and reduce fishy aftertaste, it is recommended to select ingredients with relatively low levels of docosahexaenoic acid (DHA) (i.e., less than 6 wt% by fat, preferably less than 4 wt%). Preferably, the marine oil containing DHA is present in the compositions according to the invention in an amount of less than 25 wt% by fat, preferably less than 15 wt%. On the other hand, for maximum health benefits, the inclusion of eicosapentaenoic acid (EPA) is highly desirable. The amount of EPA is preferably between 4 wt% and 15 wt% by fat, more preferably between 8 wt% and 13 wt%. The weight ratio of EPA:DHA is advantageously at least 6:4, for example between 2:1 and 10:1.

[0072] Furthermore, the liquid nutritional compositions according to the invention may advantageously contain emulsifiers. Known emulsifiers, such as lecithin, can be used, and emulsifiers generally contribute to the energy content of the fat in the composition.

[0073] The liquid nutritional composition according to the invention comprises digestible carbohydrates that provide 30% to 55% of the total energy content of the composition. Suitable digestible carbohydrates are glucose, fructose, sucrose, lactose, trehalose, palaginose, corn syrup, malt, maltose, isomaltose, partially hydrolyzed corn starch, maltodextrin, glucose oligosaccharides, and polysaccharides.

[0074] Preferably, the digestible carbohydrates include trehalose or isomaltulose. Trehalose / isomaltulose reduces sweetness compared to sucrose, as their relative sweetness is sucrose (100), trehalose (45), and isomaltulose (40-50). Furthermore, due to their similar chemical structures, the measured viscosity is similar regardless of which of these three is used as the digestible carbohydrate. However, in the high-protein compositions of the present invention, the experts prefer trehalose over sucrose because the former has lower perceived viscosity. Therefore, trehalose is the preferred carbohydrate choice because it produces lower (perceived) viscosity, does not exhibit an undesirable Maillard reaction, and has approximately half the sweetness of sucrose. In one embodiment of the invention, the digestible carbohydrate comprises trehalose, wherein the amount of trehalose is 20 wt% to 60 wt% of the digestible carbohydrate, more preferably 20 wt% to 45 wt%, and even more preferably 25 wt% to 45 wt% of the digestible carbohydrate; in the most preferred embodiment, the remainder is provided by maltodextrin of DE 16-20.

[0075] In another aspect, the invention further relates to the use of the compositions described herein for the prevention or treatment of malnutrition in persons in need (preferably malnourished persons and / or preferably older persons at least 50 years of age). Additionally, the invention relates to a (non-therapeutic) method of providing nutrition to persons in need (preferably malnourished persons and / or preferably older persons at least 50 years of age), comprising the enteral (preferably oral) administration of the liquid nutritional composition according to the invention.

[0076] It should be understood that various changes and modifications to the currently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the invention and without diminishing its advantages.

[0077] Example Example 1 - MCI was tested using different types of calcium chelating agents (Table 2). pH and T also varied.

[0078] MCI A micellar casein isolate solution was prepared in demineralized water at 60°C by dissolving 105 g (7% (w / v)) of powdered MCI-88 from FrieslandCampina. To dissolve the powdered MCI, the solution was placed in a preheated (60°C) water bath (Julabo GmbH, Boven-Leeuwen, Netherlands) and stirred at 1000 rpm using a top-mounted stirrer (IKA, Stauffen, Germany). When complete dispersion of the powder was observed, the dispersion was placed in the 60°C water bath for 1 hour with continuous stirring at 200 rpm. Next, the dispersion was homogenized for 5 minutes at 25,000 rpm using an ultra-turrax (IKA, Oude Vijvers, Netherlands). The protein dispersion was then further homogenized using a GEA Pony NS2006L pilot-scale two-stage homogenizer (Parma, Italy) at a first valve pressure of 300 bar and a second valve pressure of 50 bar. To confirm complete dissolution of the particles in solution, particle size distribution was measured using a Mastersizer 3000 (Malvern Analytical Ltd., Malvern, UK). Additionally, dry matter was measured using a CEM moisture analyzer (Mettler Toledo, Tiel, Netherlands). The samples were stored overnight at 4°C to reach equilibration.

[0079] The MCI solution was acidified to pH 6.0 (± 0.05), and a chelating agent with a concentration of 70 mEq K / L was added. The chelating agent was selected from potassium gluconate, disodium maleate, and potassium citrate.

[0080] Table 2. Overview of the concentrations (in mmol / L and mEq K / L) of different calcium chelating agents

[0081] K = functional group Store the sample overnight at 4°C to reach equilibration. The next day, readjust the pH to 6. Then, add demineralized water until a protein concentration of 5% (w / v) is achieved based on dry matter measurements. Next, store the sample at 5°C for 1 hour to reach equilibration. Apply a similar method to all other chelating agent concentrations.

[0082] The same experiment was conducted without any chelating agent to investigate the effects of pH and cooling temperature (alternatively, operating at ambient temperature). The effect of pH is plotted in Figure 1. Measurements at 20°C are given in Table 3.

[0083] Perform microfiltration and / or percolation to obtain percolate.

[0084] The amount of phosphorus in the starting material and the residue was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). The starting material contained 1500-1700 mg / 100 g MCI88, and the amount of P in the starting material was 17.6-20 mg at 85 g protein / 100 g.

[0085] To measure protein particle size, particle size distribution was measured using a Mastersizer 3000 (Malvin Analytical Ltd., Malvin, UK). Data were processed using Mastersizer 3000 software v3.80 (Malvin Analytical Ltd., Malvin, UK). All measurements were performed in triplicate.

[0086] Zeta potentials were measured using a Zetasizer Nano Z (Malvern Analytical Ltd., Malvern, UK) equipped with a 4 mW He-Ne laser. Measurements were performed using a 1.5 mL disposable folded capillary Zetasizer Nano cell (DTS1060, Malvern Instruments, Worcestershire, UK). Samples were diluted 100-fold in the supernatant after ultracentrifugation. Analysis was performed at ambient cell temperature and 100 V. Data were processed using Zetasizer software (Malvern Analytical Ltd., Malvern, UK).

[0087] Viscosity was measured at 20°C using a rotational viscometer with a cone / plate geometry at a shear rate of 100 s⁻¹.

[0088] Composition A To produce a heat-sterilized liquid enteral nutrition composition, dephosphorylated MCI-88 (osmolality) (prepared using 70 mEq potassium gluconate at pH 6.0 and cooled to 4°C) was used as the starting material. The osmolality was stirred using a magnetic stirrer and preheated to 55°C using a hot plate stirrer (imLab IKA plate, Odwifels, Netherlands). Sodium caseinate and sugar (both in powder form) were first mixed and then added to the osmolality. Maltodextrin was added while dissolving. The minerals were premixed in water at a 1:10 ratio before adding citrate and calcium chloride. Similarly, magnesium and citric acid were premixed and subsequently added to the mixture while stirring. The remaining minerals were added directly to the mixture one at a time until dissolved. A mixture of low-erucic acid rapeseed oil and lecithin was first blended and preheated to 65°C. Then, the oil mixture was added and homogenized at 25,000 rpm for 5 minutes using an ultra-turrax (IKA, Odwijksmälsch, Netherlands) to prevent phase separation. Each sample (residue and total product) was transferred to pressure-resistant DURAN culture tubes to prevent product boiling.

[0089] Next, the samples were placed in a temperature-controlled oil bath (Ulrob AG, Bowen-Lewiven, Netherlands) and heated at 127°C. The samples were inverted during the heat treatment. The heat solidification time (HCT) was measured visually after heating for 5, 10, and 20 minutes. After 20 minutes, the samples were cooled to 5°C with ice packs.

[0090] result Table 3 below shows the phosphorus levels of the MCI compositions and composition A: Table 3: Phosphorus Reduction Through the Use of Different Calcium Chelating Agents

[0091] No decrease in P was observed.

[0092] Although experiments were also conducted using acetate, no effect was observed.

[0093] Citrate had no effect on reducing P, and no micellar structure was observed after treatment.

[0094] The best results were obtained using maleate (disodium) salt and gluconate. Treatment with gluconate resulted in the most significant decrease in phosphorus (P) in micelle casein, and the micelle casein structure was preserved, particularly in a stable state, under the condition of gluconate.

[0095] Figure 1 shows the zeta potential (A), particle size (B), and viscosity (C) of the micelle casein isolate under pH and cooling conditions. No chelating agents were used in those experiments. Figure 1A and Figure 1B Both clearly show that micelle structure is lost below pH 6, even if this may not be the case. Figure 1C This was directly observed in the viscosity curves. pH values ​​below 6.0 led to (partial) aggregation of casein micelles. Furthermore, it indicated damage to the internal casein micelles, manifested as casein molecule dissolution (especially β-casein), which increased at pH 6.0 and worsened with decreasing pH.

[0096] The results of the thermal stability test for composition A are given in Table 4 below: Table 4: Thermal stability of samples containing different calcium chelating agents

[0097] Example 2A: Heat-sterilized liquid enteral nutrition composition This is the formulation of a 2.4 kcal / ml product with 0.58 kcal / ml protein (24 en%), 0.83 kcal / ml fat (35 en%), and 0.99 kcal / ml carbohydrates (41 en%). The total dry matter content is 37.3%, with 10.2% protein and 6.57% fat.

[0098] In this formulation, the total P is 165 mg / 100 ml or 68.7 mg P / 100 kcal.

[0099] Example 2B: Heat-sterilized liquid enteral nutrition composition The formulation of Example 2A has the following target mineral levels, all of which are at least 20% lower than the maximum values ​​set by the FSMP. These target numbers are provided in conjunction with the FSMP range and observations of corresponding high-protein compositions with high micellar casein content currently on the market.

[0100] Table 5. Target values ​​of minerals compared to those found in high-protein compositions with high micellar casein content. Values ​​presented as mg / 100 ml (2.4 kcal / ml product).

[0101] Total P (mg) / 100 ml product; 50 mg inorganic P from K2PO4 and 250 mg organic P from MCI. Example 3: Selection of minerals Following the formulation of Example 2, various minerals were experimented with. Table 6 shows an overview of the different minerals tested in the process of attempting to replace monosodium phosphate during casein micelle remodeling, seeking a way to reduce phosphorus while retaining the desired micelle casein structure necessary for preparing high-protein compositions.

[0102] Table 6. Selection of Minerals .

Claims

1. A method for dephosphorylating skim milk, ultrafiltered milk (UF milk), or micellar casein isolate (MCI), the method comprising: (i) Acidified skim milk, UF milk, or MCI, preferably with a pH not lower than 6.0, and preferably between 6.0 and 6.

7. (ii) Cool the acidified skim milk, UF milk, or MCI to a temperature between 0°C and 15°C. (iii) Adding gluconate and / or maleate to cooled skim milk, UF milk, or MCI, and The skim milk, UF milk, or MCI is washed to remove phosphorus, wherein the washing is preferably carried out using microfiltration and / or percolation, thereby dephosphorylating the skim milk, UF milk, or MCI, preferably to the extent that the total phosphorus content of the skim milk, UF milk, or MCI is reduced by at least 20%, preferably 20%-40%, more preferably 30%-40%, compared to the material provided in step (i).

2. The method according to claim 1, wherein the gluconate and / or maleate chelating agent is sodium gluconate, potassium gluconate, disodium maleate and / or dipotassium maleate, preferably sodium gluconate and / or potassium gluconate.

3. A dephosphorylated milk protein concentrate (MPC), milk protein isolate (MPI), or micellar casein isolate (MCI) obtainable by the method according to claim 1 or 2, having a protein content of at least 60 wt%.

4. A dephosphorylated milk protein concentrate (MPC), milk protein isolate (MPI), or micellar casein isolate (MCI) comprising (added) gluconate and / or maleate, wherein the MCI has a total phosphorus content of less than 15.0 mg P / g protein, more preferably less than 14.0 mg, and even more preferably less than 13.3 mg total phosphorus / g protein, and a protein content of at least 60 wt%, and wherein the dephosphorylated MPC, MPI, or MCI is preferably obtainable by means of the method according to claim 1 or 2.

5. Use of dephosphorylated MPC, MPI, or MCI according to any one of claims 3 or 4 for the manufacture of a heat-sterilized high-protein liquid enteral nutrition composition comprising 2.0-3.0 kcal / ml, wherein 16 en%-35 en% is provided by protein, and wherein the protein comprises at least 70 wt% micelle casein based on total protein, wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

6. A heat-sterilized liquid enteral nutrition composition comprising a calorie content of 2.0-3.0 kcal / ml and a relative protein calorie content defined as 16 en%-35 en% provided by protein, wherein the calorie content and the relative protein calorie content are preferably selected such that the composition contains 10-18 g / 100 ml protein, more preferably 12-18 g / 100 ml protein, wherein the protein comprises micellar casein (MC), whey protein (WP) and optionally caseinate (CAS), wherein there is at least 70 wt% MC and less than 15 wt% WP based on the total protein content, and wherein the total phosphorus content of the composition is less than 192 mg / 100 ml and / or 30-80 mg / 100 kcal, preferably at least 30-80 mg / 100 kcal, most preferably less than 192 mg / 100 ml and 30-80 mg / 100 kcal.

7. The liquid composition according to claim 6, wherein the total phosphorus content is 30-72 mg / kcal (i.e., at least 10% lower than the maximum value for Foods for Special Medical Purposes (FSMP), preferably 30-64 mg / kcal (i.e., at least 20% lower than the maximum value for FSMP).

8. The liquid composition according to claim 6 or 7, wherein the protein provides 16% to 32% of the total energy content of the composition, more preferably 18% to 30% or even more preferably 20% to 28% or, preferably, in combination with a calorie content of 2.2-2.6 kcal / ml.

9. The liquid composition according to any one of claims 6-8, wherein the amount of protein is between 12 g / 100 ml and 18 g / 100 ml.

10. The liquid composition according to any one of claims 6-9, wherein the weight ratio of micelle casein to caseinate is in the range of 90:10 to 75:

25.

11. The liquid composition according to any one of claims 6-10, wherein the amount of whey protein is less than 10 wt% based on total protein.

12. The liquid composition according to any one of claims 6-11, wherein the composition comprises 75 mg P / 100 g dry weight and / or 12.3 mg P / g protein.

13. The liquid composition according to any one of claims 6-12, further comprising at least two minerals of Na, K, Cl, Ca, and Mg in levels within the range (mg / 100 kcal) according to the table below, more preferably at least three, even more preferably at least four, and most preferably all minerals: 。

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