Method for preparing casein hydrolysate

Abstract

Abstract The present invention relates to a method for preparing a casein hydrolysate by hydrolysis of casein with endogenous plasmin, and to the casein hydrolysate obtained by said method. In particular, the present invention relates to a method for preparing a casein hydrolysate without the addition of exogenous enzymes, so as to obtain an organic casein hydrolysate.

Description

Technical Field

[0001] The present invention relates to a method for preparing a casein hydrolyzate prepared by hydrolyzing casein with endogenous plasmin, and to a casein hydrolyzate obtainable by said method. In particular, the present invention relates to a method for preparing a casein hydrolyzate, which method comprises heat-treating a solution comprising casein and endogenous plasmin at a pH, temperature and time sufficient to obtain a casein hydrolyzate. The solution comprising casein and endogenous plasmin comprises at least 80% by weight of casein on total solids and up to 10% by weight of whey protein on total protein content.

Background Art

[0002] The composition of human milk is the biological standard for infant nutrition, and human milk is uniquely suitable for human infants both in its nutritional composition and in its non-nutritional bioactive factors that promote survival and healthy growth. The World Health Organization (WHO) recommends exclusive breastfeeding for the first six months of life and is working to support and promote breastfeeding among mothers worldwide. However, if a mother is unable to breastfeed or chooses not to breastfeed, it is necessary to provide the infant with a suitable alternative nutritional composition such as a prepared milk formula. Therefore, it is necessary to prepare infant nutrition agents other than human milk, such as prepared milk formulas, that provide a protein composition similar to that of human milk.

[0003] The proteins of human milk are divided into two groups: whey proteins and casein proteins. Approximately 40% of the proteins in human milk are casein and 60% are whey proteins. The main proteins of human milk are casein proteins that are homologous to bovine beta-casein, alpha-lactalbumin, lactoferrin, immunoglobulin IgA, lysozyme, and serum albumin. Alpha-lactalbumin, lactoferrin and serum albumin are whey proteins.

[0004] The casein composition of human milk and cow's milk differs in that human milk is mostly composed of beta and kappa-casein, while α-S1 casein is found in human milk but only in trace amounts (3-500 μg / mL) and therefore unlikely to function as an amino acid source.

[0005] Human milk also contains various endogenous enzymes such as plasmin, cathepsin, and elastase, and studies have shown that the proteolytic activity of some of these enzymes in human milk results in the production of hundreds of peptides.

[0006] Pepsin, the main gastric protease enzyme, is present in the neonatal stomach by 26 weeks of gestation and is known to function optimally at an acidic pH, denaturing at pH 7. However, because infant stomachs produce little acid, it has been found that in full-term infants, the stomach pH remains between 5 and 7 for several weeks postpartum and for up to one hour after digestion. This, combined with the buffering capacity of milk, leads to the hypothesis that pepsin-induced proteolysis, which can result in suboptimal amino acid uptake, hardly occurs in the infant stomach. The action of endogenous milk proteases on casein may compensate for the deficiency of pepsin activity in the neonatal gastrointestinal tract, thereby enabling the utilization of amino acids from casein not only functionally but also as a nutrient source.

[0007] Therefore, human milk contains hydrolyzed milk proteins such as hydrolyzed casein, and there is a need in the art for methods to prepare such casein hydrolysates.

[0008] As mentioned above, the action of endogenous protease enzymes in human milk acts in the mammary glands, and among these enzymes, plasmin is the main contributing factor. Prior art has argued that 32% of the peptides identified in human milk originate from β-casein and 10% from αS-1 casein. Furthermore, prior art has argued that the dominance of peptides in human milk is due to the action of plasmin on β-casein. Prior art has also argued that human milk contains more peptides than cow's milk (1.19 × 10⁶ peptides in cow's milk). 11In contrast, human milk is 7.62 × 10 11 This is indicated by a higher total ionic strength. This further strengthens the fact that there is a need for improved infant formula with a higher amount of plasmin-derived casein peptides, and therefore closer to those found in human milk.

[0009] Casein hydrolysates prepared from the enzymatic hydrolysis of casein proteins are known in the art, but all known methods for preparing protein hydrolysates enzymatically use the addition of exogenous proteases. For example, U.S. Patent Application Publication 2018 / 0160715A1 discloses a method for preparing protein hydrolysates for use in pediatric nutritional compositions. U.S. Patent Application Publication 2018 / 0160715A discloses the hydrolysis of casein by adding exogenous proteases such as trypsin (trypsin-like), chymotrypsin (chymotrypsin-like), pepsin and / or plasmin to a casein-containing slurry.

[0010] However, it is desirable to avoid the use of exogenous enzymes in order to reduce regulatory requirements associated with the use of exogenous proteases and enable the development of organically prepared milk powder products.

[0011] Therefore, an improved method for preparing casein hydrolysates is advantageous, and in particular, a more efficient method for preparing casein hydrolysates without adding exogenous enzymes is advantageous. Furthermore, a method for preparing casein hydrolysates having a peptide profile similar to that of human milk, for example, a casein hydrolysate having a peptide accumulation similar to that of human milk, is also advantageous. [Overview of the Initiative]

[0012] Therefore, an object of the present invention relates to preparing casein hydrolysates by avoiding the use of exogenous proteases. An object of the present invention is to provide an optimized method for preparing casein hydrolysates by hydrolyzing casein with endogenous plasmin such that casein is rapidly and efficiently hydrolyzed without gelation, aggregation, or precipitation of the casein hydrolysates during storage and for at least one year. Furthermore, an object of the present invention is to prepare casein hydrolysates having peptide accumulations similar to those in human milk.

[0013] Furthermore, an object of the present invention is to provide a casein hydrolysate in which a portion of the peptide has demonstrated biological activity, such as antibacterial activity or ACE inhibitory activity.

[0014] The inventors have surprisingly discovered that casein hydrolysates can be obtained rapidly and efficiently by using endogenous plasmin, even without the use of exogenous enzymes. Remarkably, in systems containing large amounts of casein, such as micellar casein isolates, endogenous plasmin was found not to be inactivated during heat treatment, even after a wide range of heat treatments, such as ultra-high temperature heat treatment at temperatures above 120°C for 3-5 seconds. Thus, the present invention enables downstream sterile incubation of micellar casein isolates at optimal pH and temperature for plasmin activity, without the risk of specific microorganisms. Furthermore, it has been found that casein hydrolysates can be prepared rapidly and efficiently without the addition of exogenous enzymes by optimizing hydrolysis parameters such as pH, temperature, and components present in the feed used for hydrolysis (casein, whey protein, and calcium components, etc.). This novel method for preparing casein hydrolysates also has the advantage of peptide accumulation being similar to that of breast milk. Therefore, for example, by using a casein hydrolysate obtained by hydrolysis of micellar casein isolate (MCI) with endogenous plasmin, it would be possible to obtain a formula milk product in which the amount of casein-derived peptides is much closer to the amount observed in human milk than in commercially available formula milk products today.

[0015] The term "peptide accumulation" refers to the relative increase in the peak area of ​​peptides over the hydrolysis process in a sample, as given by HPLC. Peptide accumulation is observed in the HPLC chromatograph at 12–14 minutes, 16.5 minutes, and 17–18 minutes. In other words, peptide accumulation occurs as a function of time and plasmin activity. The amino acids in the peptides present in the casein hydrolysate of this invention are not identical in amino acid sequence to those in human milk peptides, but they better reflect human milk in terms of peptide accumulation. The accompanying loss of intact casein protein is calculated in the same manner by the decrease in the peak area of ​​a given casein as a function of time, as determined by HPLC.

[0016] The present invention also enables the preparation of organic casein hydrolysates when the liquid source of casein and plasmin, such as micellar casein isolate, is an organic substance.

[0017] Therefore, one aspect of the present invention is a method for preparing a casein hydrolysate: i) A step of providing a solution comprising casein and endogenous plasmin, wherein the solution comprises casein comprising at least 80% by weight of the total solids, and the solution comprises whey protein comprising up to 10% by weight of the total protein content, ii) A step to adjust the pH of the solution in step i) to pH 7.2 to 9, iii) A step of subjecting the pH-adjusted solution to the first heat treatment step to inactivate microorganisms, iv) The solution from step iii) is subjected to a second heat treatment at a temperature of 25°C to 45°C for at least 6 hours to obtain a casein hydrolysate. Regarding methods including

[0018] Another aspect of the present invention relates to natural casein hydrolysates that can be obtained by the method according to the present invention.

[0019] Yet another aspect of the present invention is to provide a natural casein hydrolyzate that does not contain added exogenous enzymes.

[0020] Yet another aspect of the present invention is to use the natural casein hydrolyzate according to the present invention in a nutritional composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1] FIG. 1 shows the effect of the temperature of the first heat treatment step on the generation of plasmin-derived peptides in an 8% w / v MCI solution. FIG. 1 shows plasmin-derived peptides eluting at 12.00 minutes to 14.00 minutes by RP-HPLC. [Figure 2] FIG. 2 shows the effect of the temperature of the first heat treatment step on the generation of plasmin-derived peptides in an 8% w / v MCI solution. FIG. 2 shows plasmin-derived peptides eluting at 17.00 minutes to 18.00 minutes by RP-HPLC. [Figure 3] FIG. 3 shows the loss of intact beta-casein determined by RP-HPLC. The y-axis is normalized to beta-casein at t = 0 just prior to incubation. [Figure 4] FIG. 4 shows the loss of intact alpha-S1-casein determined by RP-HPLC. The y-axis is normalized to alpha-S1-casein at t = 0 just prior to incubation. [Figure 5] FIG. 5 shows the accumulation of peptides by heat treatment at 130 °C, 135 °C and 140 °C for 3 - 4 seconds. Plasmin-derived peptides elute at 12.00 minutes to 14.00 minutes by RP-HPLC. The heat treatment was either on the same day as the treatment (Day 1) or after storage at 5 °C for 24 hours (Day 2). The y-axis is the normalized peak area, i.e., the peak area at t = 0, and subsequent data points on the y-axis are relative values to this. [Figure 6]Figure 6 shows the accumulation of peptides by heat treatment at 130 °C, 135 °C and 140 °C for 3 to 4 seconds. Peptides derived from plasmin elute at 17.00 minutes to 18.00 minutes by RP-HPLC. The heat treatment was either on the same day as the treatment (Day 1) or after storage at 5 °C for 24 hours (Day 2). [Figure 7] Figure 7 shows the change in plasmin activity in heat-treated MCI on Day 1 and Day 2, that is, after storage at 5 °C for 24 hours. [Figure 8] Figure 8 shows a representative RP-HPLC chromatogram (overlay) of plasmin-induced hydrolysis of casein. [Figure 9] Figures 9A and 9B show the identified peptides of α-S1-casein after hydrolysis of MCI. [Figure 10] Figures 10A and 10B show the identified peptides of α-S2-casein after hydrolysis of MCI. [Figure 11] Figures 11A and 11B show the identified peptides of β-casein after hydrolysis of MCI. [Figure 12] Figure 12 shows the molecular weight distribution of the casein hydrolysate of the present invention. [Figure 13] Figure 13A shows the loss of intact alpha-S1 casein over time determined by RP-HPLC for samples with different pH values. The y-axis is normalized to alpha-S1 casein at t = 0 immediately before incubation. Figure 13B shows the loss of intact beta-casein over time determined by RP-HPLC for samples with different pH values. The y-axis is normalized to beta-casein at t = 0 immediately before incubation.

[0022] Next, the present invention will be described in more detail below. Detailed Description of the Invention

[0023] definition Before discussing the present invention in more detail, the following terms and expressions are first defined. Every reference to a single feature or limitation of the present invention, unless otherwise specified or explicitly implied to the contrary by the context in which the reference is made, encompasses the corresponding multiple features or limitations, and vice versa.

[0024] All percentages referred to herein are weight percentages unless otherwise specified. Furthermore, the terms “dry weight” and “dry weight basis” refer to the same concept and are used interchangeably.

[0025] For example, the term "w / w," as in 1% w / w, refers to a composition containing 1% by weight of the compound.

[0026] In the context of this invention, the term "solution" means a product having a water content of at least 60% by weight, for example, at least 70% by weight, preferably at least 80% by weight. Preferably, the water content in the "solution" of this invention is 80-96% by weight, particularly 85-95% by weight.

[0027] In connection with the present invention, the term "w / v," as in the amount of casein in a solution, means the weight percentage of casein per unit volume of the feed material. For example, 1% w / v casein in a solution means 1 g of casein per 100 ml of solution, or 10 g of casein per 1 liter of solution.

[0028] In connection with the present invention, the terms “casein hydrolysate” or “hydrolyzed protein” mean a composition comprising casein protein subjected to protein hydrolysis. By protein hydrolysis, the protein is cleaved into peptides and free amino acids, and therefore, “hydrolyzed protein” contains peptides and / or free amino acids.

[0029] The term "endogenous," as in "endogenous plasmin," means that the enzyme plasmin is naturally present in the casein protein source used for hydrolysis. Conversely, the term "exogenous" refers to the fact that the enzyme is not naturally present and is added separately.

[0030] In the context of this invention, the term “solid” refers to the molecules that would remain after all water has been removed from milk. The term “solid” includes proteins, enzymes (plasmin or plasminogen), milk fat, carbohydrates, minerals, vitamins, and other small molecules other than water.

[0031] casein: Casein is a milk protein found in milk as a suspension of particles called casein micelles. Within the micelles, casein is bound to calcium ions through hydrophobic interactions.

[0032] In human milk, about 40% of the protein is casein, while in cow's milk, about 80% of the milk protein is casein.

[0033] Casein can be found in different forms, such as alpha-casein and beta-casein. Beta-casein can also be found as mutant A1-beta-casein and A2-beta-casein. A1 and A2-beta-casein are genetic variants of the beta-casein milk protein that differ by one amino acid; proline is present at position 67 of the amino acid chain in A2-beta-casein, while histidine is present at that position in A1-beta-casein. Due to the way beta-casein interacts with enzymes found in the digestive system, A1 and A2 are processed differently by digestive enzymes. However, in this invention, both A1-beta-casein and A2-beta-casein can be used.

[0034] Therefore, in the context of this invention, the term "casein" refers to any type of casein, such as acid casein, beta-casein, beta-concentrated casein, alpha-casein, alpha-concentrated casein, kappa-casein, and kappa-concentrated casein. However, in one embodiment of the present invention, the term "casein" does not apply to casein salts. Casein salts are known to have little or no plasmin activity.

[0035] In a preferred embodiment of the present invention, the casein is alpha-casein and / or beta-casein.

[0036] In one embodiment of the present invention, the solution provided in step i) contains casein in an amount of at least 1% w / v.

[0037] In order to prepare casein hydrolysates, a sufficient amount of casein must be present in the solution. Therefore, in a preferred embodiment of the present invention, the solution contains casein in an amount of at least 3.0% w / v, for example, at least 4% w / v, more preferably at least 5% w / v, and even more preferably at least 6% w / v.

[0038] If the casein content is too high, the solution will gel during heat treatment, becoming viscous and thus difficult to process. Therefore, the casein content must be 20% by weight or less.

[0039] Therefore, in a further embodiment of the present invention, the solution contains casein in an amount ranging from 1% to 20% w / v, for example, 3.0% to 17% w / v casein in solution, preferably 4% to 15% w / v casein in solution. In a preferred embodiment of the present invention, the solution contains casein in an amount ranging from 1% to 15% w / v.

[0040] The casein content in the solution is preferably at least 90% by weight of the total protein content, for example, at least 93% by weight of the total protein content, and more preferably at least 95% by weight of the total protein content.

[0041] A solution containing casein and endogenous plasmin preferably does not contain whey protein. If whey protein is present, it should be in an amount of 10% by weight or less of the total protein content.

[0042] Plasmin has very low activity towards whey protein and therefore cannot hydrolyze / cleave whey protein into peptides and free amino acids. Plasmin is active towards casein. Furthermore, whey protein inactivates plasmin. Unfolded, denatured whey protein (mainly beta-lactoglobulin) binds to plasmin (by disulfide bonds) near its active site, thereby inactivating plasmin. When whey protein is present in large quantities in solution, plasmin activity is significantly affected after extensive heat treatments such as UHT treatment due to interactions with beta-lactoglobulin containing free SH groups, which cause irreversible denaturation of plasmin through SS / SH interactions. Therefore, the whey protein content in the "solution" used in this invention must be low enough to allow an effective level of plasmin activity to remain in the solution after heat treatment.

[0043] The term "whey protein" refers to proteins found in whey. Typical whey proteins include beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin and immunoglobulins, lactoferrin, lactoperoxidase, and milk fat globule membrane proteins. Furthermore, whey proteins found in sweet whey typically also include caseinomacropeptides.

[0044] Preferably, the solution contains whey protein in an amount of 7% by weight or less of the total protein content, for example, 5% or less of the total protein content, and more preferably 3% by weight or less of the total protein content. In a preferred embodiment, the solution containing casein and plasmin is essentially whey protein-free.

[0045] The casein source used in this invention is any casein source, including endogenous plasmin. Preferably, the casein and endogenous plasmin source is a fraction of a dairy product containing casein protein. Preferably, the fraction of dairy milk is obtained by fractionating cow's milk.

[0046] In one embodiment of the present invention, the casein and endogenous plasmin source is a dairy product selected from the group consisting of micellar casein isolate (MCI), micellar casein concentrate (MCC), micellar casein retaining solution (MCR), beta-casein-depleted micellar casein isolate, beta-casein-depleted micellar casein concentrate, milk protein concentrate (MPC), or microfiltration retaining solution of a dairy product. Preferably, the casein and endogenous plasmin source is MCI, MCC, MCR, beta-casein-depleted micellar casein isolate, beta-casein-depleted micellar casein concentrate, or microfiltration retaining solution of a dairy product.

[0047] In a more preferred embodiment, the micellar casein concentrate and isolate will have a high content of micellar casein, so the casein and endogenous plasmin source is selected from the group of micellar casein concentrate and micellar casein isolate. The micellar casein content is higher in the micellar casein isolate than in the micellar casein concentrate, and for this reason, it is preferable to use the micellar casein isolate. Most preferably, the solution containing casein and endogenous plasmin is micellar casein isolate (MCI).

[0048] Micelle casein isolates are produced by a filtration process, preferably by membrane filtration in a microfiltration membrane that separates natural milk casein protein from milk-derived feed in a highly concentrated, undenatured form in a microfiltration holding liquid. Micelle casein isolates (MCIs) contain, for example, 85–95% by weight of casein of the total protein content. In most cases, MCIs contain 93–94% w / w casein of the total protein content.

[0049] MCI can be produced, for example, by providing a milk-derived feed to microfiltration using an MF membrane (or more) typically having a pore size in the range of 0.01 to 1.0 microns. Preferably, the pore size of the MF membrane (or more) is in the range of 0.05 to 0.8 microns. Furthermore, the provided MF membrane (or more) has a molecular weight cutoff in the range of 200 to 2000 kDa. The MF membrane (or more) may be, for example, a polymer membrane or a ceramic membrane.

[0050] For example, MCI can be produced by microfiltration using a ceramic membrane with a pore size of approximately 0.14 microns (Inside Ceram®, Tami Industries, Nyon, France) or a polymer FR membrane with a molecular weight cutoff of approximately 800 kDa (PVDF 800 kDa; Synder Filtration, USA).

[0051] Microfiltration of milk-derived feeds, such as skim milk, to produce micellar casein isolates is well-documented in the literature and is an existing commercial unit operation well-known to those skilled in the art. Microfiltration can be achieved using spiral membranes or ceramic membranes, but is not limited to these, and can be carried out over a wide range of temperatures, such as low-temperature membrane filtration at temperatures below 10°C or heated conditions at temperatures around 50°C, for example, 45–55°C.

[0052] Beta-casein-depleted micellar casein isolate and beta-casein-depleted micellar casein concentrate can be obtained as a second retaining liquid from microfiltration obtained by the patented method, by the method described in International Publication No. 2014 / 114709A2.

[0053] Milk-derived feeds for preparing MCI, MCC, MCR, beta-casein-depleted micellar casein isolate, and beta-casein-depleted micellar casein concentrate may include, or consist of, whole milk, skim milk, non-fat milk, low-fat milk, high-fat milk, and concentrated milk. The milk feed is usually derived from ruminant milk. The term "concentrated milk" refers to milk concentrated by evaporation or by ultrafiltration, nanofiltration, and / or reverse osmosis. It is particularly preferable that the concentrated milk is concentrated non-evaporated milk, i.e., milk concentrated by filtration.

[0054] In one embodiment of the present invention, the solution containing casein and endogenous plasmin may be a solution of organic MCI. When using organic MCI, the milk-derived feed used to prepare the MCI is organic.

[0055] In the context of this invention, the term “organic milk” refers to milk produced by mammals, e.g., cattle, raised in accordance with the following: Mammals must have free access to organically certified pasture throughout the entire grazing season. This period is specific to the geographical climate of the farm, but must be at least 120 days, preferably at least 150 days per year. Depending on the weather, season, or climate, the grazing season may or may not be continuous. Organic feed for cattle must contain at least 30 percent (on average) of dry matter from organically certified pasture. Dry matter intake (DMI) is the amount of feed consumed by the animal per day on an anhydrous basis. The remainder of the feed, including hay, grains, and other agricultural products, must also be organically certified. Livestock must be managed without the use of antibiotics, added growth hormones, mammalian or avian by-products, or other prohibited feed components (e.g., urea or arsenic compounds).

[0056] Endogenous plasmin: Plasmin is a naturally occurring enzyme in milk, and the term "endogenous" means that the plasmin used in the method of the present invention is plasmin that is naturally present in dairy products. Therefore, plasmin is naturally present in the casein source used. For example, micellar casein isolate contains plasmin.

[0057] Plasmin is overwhelmingly active against casein proteins but not against whey proteins. Because these casein proteins contain a lysine-arginine motif recognized by plasmin, plasmin is particularly active against alpha-casein and beta-casein.

[0058] In connection with the present invention, the method does not involve the addition of exogenous enzymes, and in particular, the method does not involve the addition of exogenous plasmin or trypsin. The hydrolysis provided in the present invention is carried out solely by the use of naturally occurring endogenous plasmin in the solution / feed used as the casein source for preparing the casein hydrolysate. Trypsin has a similar effect to plasmin, but trypsin is not endogenously found in milk.

[0059] Therefore, in a preferred embodiment of the present invention, the method excludes the addition of exogenous enzymes, such as the addition of exogenous plasmin or trypsin.

[0060] In relation to the present invention, when a solution is referred to as containing endogenous plasmin, it refers to a solution containing plasmin and related endogenous enzymes, such as midi-plasmin, mini-plasmin, micro-plasmin, and enzymes exhibiting plasmin-like activity derived from the autolysis of plasmin. Therefore, in relation to the present invention, the term "plasmin" refers to plasmin-like enzymes, such as midi-plasmin, mini-plasmin, micro-plasmin, and plasminogen that can be converted to plasmin. Thus, the solution used in the present invention does not necessarily have to contain plasmin from the outset, but may instead contain plasminogen that can be converted to plasmin.

[0061] In one embodiment of the present invention, the term "plasmin" refers to enzymes classified under EC3.4.21.7.

[0062] The plasmin system is highly complex, comprising plasmin (PL), plasmin-like enzymes, plasminogen (PG), plasmin activator (PA), plasmin activator inhibitor (PAI), and plasmin inhibitor (PI). PI and PAI are primarily found in whey, while PG, PL, and PA are bound to casein micelles. Therefore, by isolating casein micelles as MCI, MCC, or MCR by microfiltration, PI and PAI are removed, and PG and PL are concentrated along with PA, thereby promoting the conversion of PG to PL. PI and PAI are also somewhat thermally unstable, and mild heat treatment is expected to inactivate these species, further increasing the conversion of PG to PL. For example, Richardson et al. observed that incubating milk at 37°C for up to 80 hours increased PL and decreased PG.

[0063] The inventors have found that commercial pasteurization conditions (15 seconds at 75°C) inactivate PAI, and in fact, PA is more heat-resistant than PG and PL. This means that skim milk used in the production of MCI also contains inactivated PAI, and once separated from whey protein, it is ready for conversion from PG to PL.

[0064] solution: In connection with the present invention, the term “solution” encompasses compositions comprising a combination of a liquid compound and a solid compound or semi-solid particles, such as protein particles. Therefore, the “solution” may be a suspension or even a slurry. However, the “solution” is preferably pumpable, and the amount of liquid in the solution containing casein and endogenous plasmin is preferably 70-99%, more preferably 80-96%. The liquid used in the solution is typically water.

[0065] As described above, the solution containing casein and endogenous plasmin according to the present invention typically contains casein in an amount of 1% by weight or more of the solution.

[0066] In one embodiment of the present invention, a solution containing casein and endogenous plasmin is a) a fraction of milk-derived feed containing casein and endogenous plasmin, or b) a concentrated fraction of milk-derived feed containing casein and endogenous plasmin, or c) a fraction of milk-derived feed containing casein and endogenous plasmin diluted in a liquid such as water, or d) a solution of a concentrated fraction of milk-derived feed diluted in a liquid such as water.

[0067] For example, a solution containing casein and endogenous plasmin contains a fraction of milk-derived feed having a high casein content among the total protein content.

[0068] Any type of milk fraction containing casein and any of endogenous plasmin, plasminogen, midi-plasmin, mini-plasmin, or microplasmin may be used.

[0069] In the context of this invention, the term "milk fraction" is understood to mean a fraction obtained from a milk-derived feed. Milk fractions can be obtained by membrane filtration of milk-derived feed, but they can also be obtained by other fractionation processes.

[0070] A milk fraction containing casein and endogenous plasmin can be selected from the group consisting of micellar casein isolate (MCI), micellar casein concentrate (MCC), micellar casein retaining solution (MCR), milk protein concentrate (MPC), beta-casein-depleted micellar casein isolate, beta-casein-depleted micellar casein concentrate, or microfiltration retaining solution of dairy products. Preferably, the milk fraction is selected from the group consisting of micellar casein isolate (MCI), micellar casein concentrate (MCC), and micellar casein retaining solution (MCR). Most preferably, the milk fraction is MCI.

[0071] In MCI, MCC, and MCR, plasmin inhibitors and plasminogen activator inhibitors are absent. However, in milk itself (e.g., skim milk), plasmin inhibitors and plasminogen activator inhibitors are present. Plasmin inhibitors inhibit the activity of plasmin and are therefore undesirable in solutions containing casein and plasmin. Plasminogen activator inhibitors inhibit the conversion of plasminogen to plasmin and are therefore also undesirable. Therefore, milk itself is not suitable as a solution containing casein and plasmin. In one embodiment of the present invention, a solution containing casein and plasmin does not contain plasmin inhibitors or plasminogen activator inhibitors, or does not contain at least essential amounts of plasmin inhibitors and / or plasminogen activator inhibitors.

[0072] Micellar casein isolate (MCI) contains, for example, endogenous plasmin, as well as casein, in an amount of 85–95% by weight of the total solids. MCI is produced by a filtration process that separates natural milk casein protein in a highly concentrated, undenatured form.

[0073] A fraction of a milk-derived feed containing casein and plasmin, such as micellar casein or micellar casein-retaining solution or concentrate, is dispersed in water in an amount sufficient to obtain a desired amount of casein.

[0074] The milk-derived feed may be based on the milk of mammals such as cows, buffaloes, goats, sheep, yaks, pigs, camels, horses, ewes, and mare, or mixtures thereof. In a preferred embodiment of the present invention, the solution containing casein and endogenous plasmin contains a milk fraction derived from cow's milk.

[0075] Milk-derived feeds that can be used to obtain fractions with a high casein content may include, for example, whole milk, low-fat milk, low-fat milk, semi-skimmed milk, skim milk, buttermilk, reconstituted milk powder, heat-treated milk (e.g., pasteurized milk, sterilized milk, condensed milk, evaporated milk, and UHT milk), unfiltered raw milk, and homogenized milk.

[0076] The solution of the present invention contains casein in an amount of at least 80% by weight of the total solids, preferably at least 90% by weight of the total solids. Furthermore, the casein content in the solution must be at least 90% by weight of the total protein content, for example, at least 93% by weight of the total protein content. In addition, the amount of whey protein in the solution must be at the lowest possible level. The solution contains whey protein in an amount of up to 10% by weight of the total protein content, preferably up to 7% by weight of the total protein content.

[0077] It was surprising to the inventors that, when using a milk fraction derived from milk that has a higher casein content and a lower whey protein content compared to milk itself, it was possible to prepare a casein hydrolysate by hydrolyzing the casein using endogenous plasmin present in the milk fraction, without adding exogenous enzymes. The inventors believe that one reason why casein could be hydrolyzed using endogenous plasmin was the low whey protein content in the solution used for hydrolysis. While not bound by any theory, the inventors believe that a low content of whey protein (a small amount of whey protein) better protects plasmin from denaturation during the heat treatment process. Whey protein, when present, interacts with plasmin, thus reducing the plasmin efficiency in casein hydrolysis. Therefore, it is desirable to have a small amount of whey protein in the solution to provide efficient hydrolysis of casein.

[0078] Furthermore, although not bound by any theory, the inventors believe that whey protein is the cause of gelation in dairy products, and therefore, for example, the gelation of UHT-treated milk after several months of storage. Therefore, using a solution containing casein and plasmin from which a significant amount of whey protein has been removed compared to the milk itself (including skim milk), the resulting casein hydrolysate does not gel over at least 12 months of storage.

[0079] For example, in milk, the ratio of casein to whey protein is typically 80:20, but in order for plasmin to efficiently hydrolyze casein and prevent the product from gelling, the whey protein content needs to be reduced to less than 10% by weight of the total protein content.

[0080] Since casein hydrolysates cannot be obtained by using milk as is, the inventors have found that dairy products are not suitable as a solution containing casein and plasmin in their raw form. High levels of whey protein inhibit plasmin activity and cause gelation during storage, so milk is not a suitable starting material for preparing casein hydrolysates in its raw form. Another reason why milk is not suitable for preparing casein hydrolysates is that it contains plasmin inhibitors and plasminogen activator inhibitors, which also limit plasmin activity. In the present invention, in which MCI, MCC, MCR, beta-casein-depleted MCI and beta-casein-depleted MCC are typically used for preparing casein hydrolysates, plasmin inhibitors and plasminogen activator inhibitors are not present because they are removed in the serum phase during the preparation of MCI, MCC, MCR, beta-casein-depleted MCI and beta-casein-depleted MCC.

[0081] Therefore, in preferred embodiments, the liquid composition containing casein and endogenous plasmin is essentially free of plasmin inhibitors and plasminogen activator inhibitors. The term "essentially free" in the context of this invention means that the compounds are present only in negligible amounts.

[0082] The inventors also found that in order to efficiently and rapidly obtain casein hydrolysates, other process parameters must preferably be optimized, namely, a pH in the range of 7.2 to 9.0 and a hydrolysis temperature of 25°C to 45°C.

[0083] The temperature of the solution containing casein and endogenous plasmin provided in step i) must be preferably 1°C to 45°C, more preferably 1°C to 20°C, and even more preferably 5°C to 10°C before the start of the hydrolysis process. This is to control microbiology, i.e., to avoid microbial growth. The wide temperature range (1 to 45°C) depends on the storage time of the solution before use. If the storage time before microbial inactivation and enzymatic hydrolysis with endogenous plasmin is short, a higher temperature of the solution before the start of the process is acceptable. However, if the solution containing casein and endogenous plasmin is stored overnight before microbial inactivation and hydrolysis is started, the storage temperature must be low, preferably below 10°C. Therefore, in a preferred embodiment of the present invention, the solution containing casein and endogenous plasmin has a temperature of 1°C to 10°C before use in the hydrolysis process of the present invention.

[0084] PH adjustment In step ii) of the present invention, the solution containing casein and endogenous plasmin is subjected to pH adjustment to adjust the pH of the solution in step i) to a range of 7.2 to 9. Preferably, the pH of the solution is adjusted to a range of 7.5 to 8.7, for example, 7.5 to 8.5, and more preferably to a range of 7.5 to 8.2. The pH of milk is typically 6.7 to 6.8, but the optimal pH for plasmin is about 7.5 to 8.0. The pH must be greater than 7.2. The activity of plasmin at pH 6.7 to 6.8 is much lower than at pH greater than 7.2. At pH lower than 7.2, it takes several weeks for endogenous plasmin to hydrolyze casein, so the pH during hydrolysis must therefore be greater than 7.2. Conversely, using a pH of 7.2 to 9.0 during hydrolysis yields casein hydrolysate in less than 72 hours.

[0085] The solution's pH must be between 7.2 and 9, because the activity of endogenous plasmin is optimal at these pH values. Below a pH lower than 7.2, plasmin activity decreases significantly.

[0086] First heat treatment: In one aspect of the present invention, a pH-adjusted solution is subjected to a first heat treatment step. The first heat treatment is applied to a solution containing casein and endogenous plasmin to reduce the number of microorganisms in the solution, allowing the obtained product to be stored for a longer period before further processing. Therefore, the first heat treatment step is carried out to inactivate and thus control the microorganisms. The first heat treatment step is necessary to avoid undesirable microorganisms in the hydrolysate, and the inventors have surprisingly found that when a solution of casein and endogenous plasmin is subjected to a heat treatment step to inactivate microorganisms, endogenous plasmin is not inactivated. In particular, it was unexpectedly found that plasmin is not inactivated even under broad heat treatment. Broad heat treatment, in the context of the present invention, means heat treatment at a temperature and time such that all microorganisms are inactivated or all microorganisms are unable to grow further.

[0087] In connection with the present invention, the term "microorganism" refers, for example, to bacterial spores, yeasts, molds, and fungal spores.

[0088] The breadth of the first heat treatment process will affect not only the quality of the final product but also its shelf life. Increasing the breadth of the heat treatment, i.e., raising the heat treatment temperature, can reduce the rate of product spoilage. However, this must be balanced with increased chemical, physical, sensory, and nutritional changes in the final product.

[0089] The first heat treatment step used in the method of the present invention may be at a temperature of 85°C to 180°C. The duration of the first heat treatment step depends on the temperature and may therefore be 0.1 to 30 seconds. When the temperature is as low as 85 to 110°C, the heat treatment step should be 15 to 30 seconds. However, when using higher temperatures such as 120 to 180°C, the holding time should be 0.1 to 10 seconds.

[0090] In a preferred embodiment of the present invention, the first heat treatment step is performed at a temperature of 120°C or higher for 0.1 to 10 seconds. In a further embodiment, the first heat treatment step is performed at a temperature of 120°C to 180°C for 1 to 10 seconds.

[0091] Furthermore, in one embodiment, the first heat treatment may be a sequential heat treatment in which the temperature is first raised to 85°C to 100°C for 1 to 15 seconds, and then the temperature is raised to over 120°C for 0.1 to 10 seconds.

[0092] To inactivate all microorganisms, it is preferable to use a temperature of 120°C or higher. The inventors have found that if the temperature of the first heat treatment is lower than 120°C, not all microorganisms are inactivated.

[0093] Preferably, the first heat treatment step includes heating to a temperature exceeding 125°C with a holding time of 0.1 to 10 seconds, and more preferably, the first heat treatment step includes heating to a temperature exceeding 130°C with a holding time of 0.1 to 10 seconds.

[0094] Furthermore, in one embodiment of the present invention, the first heat treatment step includes heating at a temperature of 120°C to 180°C for 0.1 to 10 seconds. For example, the first heat treatment step includes heating at a temperature of 125°C to 170°C for 0.1 to 10 seconds, more preferably at 125°C to 160°C for 0.1 to 10 seconds, and even more preferably at 130°C to 150°C for 0.1 to 10 seconds.

[0095] The holding time in the first heat treatment step can be 0.1 to 10 seconds when temperatures above 120°C are used. However, the inventors have found that when temperatures above 120°C are used, the holding time must preferably be 1 second or longer in order to completely inactivate the microorganisms. Heat treatment at 120°C for 1 to 10 seconds is considered a broad-spectrum heat treatment. Surprisingly, it was found that plasmin is not inactivated in the first heat treatment step with a broad-spectrum heat treatment (1 to 10 seconds at temperatures above 120°C).

[0096] In a further embodiment of the present invention, the first heat treatment step has a holding time of 1 to 5 seconds, more preferably 2 to 4 seconds.

[0097] In a preferred embodiment of the present invention, the first heat treatment step was performed at a temperature of 120°C to 180°C for 1 to 10 seconds, for example, 125°C to 170°C for 1 to 10 seconds, preferably 130°C to 150°C for 1 to 10 seconds.

[0098] In a particularly preferred embodiment of the present invention, the first heat treatment step includes heating to a temperature of 125°C to 160°C for 1 to 5 seconds, preferably using a temperature of 130°C to 145°C for 3 to 5 seconds.

[0099] In one embodiment of the present invention, the first heat treatment of the pH-adjusted solution from step ii) is performed by using ultra-high temperature (UHT) heat treatment, high-pressure sterilization, etc.

[0100] UHT treatment is well known in the art as a process in which a liquid subjected to UHT treatment is heated to a high temperature and rapidly cooled to shorten the holding time. UHT heat treatment may be, for example, direct UHT, infusion-based UHT, or indirect UHT. In the context of this invention, the term "UHT" means heat treatment at an extremely high temperature (e.g., above 120°C) for a short holding time to produce commercially available sterile products that can be stored at room temperature. The aim of this process is to kill all microorganisms, and any remaining microorganisms are unlikely to cause spoilage under normal storage conditions.

[0101] In a direct UHT system, specifically a direct steam injection UHT, a pH-adjusted solution is instantly heated by injecting high-pressure steam at a temperature of approximately 120°C to 180°C into the liquid, and then held in a holding tube for 2 to 5 seconds. The liquid is then immediately cooled to remove condensed steam. Steam injection enables rapid heating and cooling.

[0102] UHT treatment may also be infusion-based, where the solution is pumped into a chamber through a nozzle along with relatively low concentrations of high-pressure steam, providing a large surface contact area. Steam infusion typically uses a temperature of 150°C for 0.1–0.3 seconds. This is not sufficient to inactivate all microorganisms.

[0103] Alternatively, UHT heat treatment may be performed using indirect heat treatment, for example, by using a solid heat exchanger to heat and cool the liquid.

[0104] The first heat treatment in step iii) of the present invention may be performed by using high-pressure sterilization (HPP).

[0105] UHT by steam infusion or injection combined with flash cooling may be used for sterilization. Flash cooling rapidly removes heat by vacuum. UHT with flash cooling has a very short heat treatment holding time, i.e., 0.1 to 0.2 seconds. In one embodiment of the present invention, UHT with a very short holding time is undesirable because a sufficient amount of microorganisms are not removed.

[0106] In a particular embodiment of the present invention, the first heat treatment step is performed by using direct steam injection (UHT) at a temperature of 120°C to 180°C for 0.1 to 5 seconds.

[0107] High-pressure sterilization (HPP) is a food processing method that inactivates microorganisms by exposing food to high pressure, with or without heat. HPP is also known as high-pressure hydrostatic processing (HPP) or ultra-high-pressure processing (UHP). HPP may be a heated or non-heated process. For example, HPP may be a pasteurization technique that applies a pressure of 50-1000 MPa to a solution transmitted by water. The advantage of high-pressure sterilization compared to heat treatment is that it inactivates microorganisms while avoiding protein denaturation and changes in functionality. Furthermore, high pressure can be used to enhance several processing operations, such as freezing, thawing, and extraction, providing new processing options.

[0108] The first heat treatment in the method of the present invention inactivates at least 90% of the microorganisms present in the solution containing casein and endogenous plasmin, but the activity of plasmin must be maintained. Preferably, at least 95% of the microorganisms must be inactivated, and more preferably, at least 99% of the microorganisms must be inactivated.

[0109] In one embodiment of the present invention, pH-adjusted and heat-treated solutions of casein and endogenous plasmin are stored at a temperature of 10°C or lower for at least 10 hours before the hydrolysis step. Plasmin activity increases with low-temperature storage. Furthermore, beta-casein dissociates from casein micelles at low temperatures and associates with casein micelles at high temperatures. Therefore, when the solution of casein and endogenous plasmin is stored at low temperatures, more beta-casein is released from casein micelles into the whey.

[0110] Preferably, the low-temperature storage before the first heat treatment step is 8°C or lower, for example, 5°C or lower. The low-temperature storage may be 1°C to 10°C, for example, 2°C to 8°C. The low-temperature storage is preferably at least 10 hours, preferably at least 20 hours, and more preferably at least 24 hours. Alternatively, the low-temperature storage may be 10 to 72 hours, for example, 20 to 60 hours, most preferably 1 to 2 days. However, the limiting time of low-temperature storage should not be considered a limiting factor of the invention, as low-temperature storage at a temperature of 1 to 10°C for more than 10 hours will result in an increased plasmin effect.

[0111] Hydrolysis / Second heat treatment: In one aspect of the present invention, a casein hydrolysate is obtained by hydrolysis of a pH-adjusted solution containing casein and endogenous plasmin, wherein the solution contains casein at least 80% by weight of the total solids and whey protein at a maximum of 10% by weight of the total protein content. The hydrolysis is obtained by a second heat treatment step of the solution in step iii) at a temperature of 25°C to 45°C for at least 6 hours. This causes the endogenous plasmin to hydrolyze the casein, yielding a casein hydrolysate.

[0112] At temperatures below 25°C, the activity of endogenous plasmin decreases significantly, and the rate of hydrolysis becomes very slow; therefore, it is important that the temperature is higher than 25°C. Accordingly, the temperature during the hydrolysis process must be at least 25°C. Furthermore, in order to obtain the activity of endogenous plasmin, it is desirable that the temperature during hydrolysis be 45°C or lower. In one embodiment of the present invention, the heat treatment in step iv) is at a temperature of 30°C to 42°C, for example, 35°C to 40°C.

[0113] The time of hydrolysis should not be considered a limiting factor; in principle, hydrolysis can be carried out for a sufficient time for endogenous plasmin to hydrolyze casein and thus obtain casein hydrolysates. The degree of hydrolysis depends on the time of hydrolysis; therefore, a shorter hydrolysis time results in a lower degree of hydrolysis, while a longer hydrolysis time results in a higher degree of hydrolysis. The hydrolysis time is preferably at least 6 hours, for example at least 8 hours, and more preferably at least 10 hours. Since plasmin undergoes autolysis (autohydrolysis) after some time, the upper limit of the hydrolysis time is not important in principle. However, an example of a hydrolysis time is 6 hours to 10 days, for example 6 hours to 5 days, and more preferably 6 hours to 72 hours. A hydrolysis time of at least 6 hours provides sufficient hydrolysis to obtain a substantial amount of hydrolyzed casein, but 24 hours and 48 hours also provide acceptable levels of hydrolysis for the proposed applications. Since plasmin is known to autolysis, and without being constrained by theory, it is expected that long incubation times may be undesirable for this reason as well as for capacity limitations and the costs incurred in storage that long processing times impose on the processing line.

[0114] Most preferably, hydrolysis is carried out at a temperature of 30°C to 45°C for 6 hours to 10 days, more preferably at 30°C to 42°C for 6 hours to 10 days, and even more preferably at 35°C to 42°C for 6 hours to 5 days.

[0115] In a preferred embodiment, hydrolysis can also be carried out at 35°C to 42°C for 6 to 72 hours.

[0116] As a result of hydrolysis, endogenous plasmin cleaves beta- and alpha-S1-casein into peptides, leading to the accumulation of peptides from these two proteins. During hydrolysis by endogenous plasmin, kappa-casein was observed to remain essentially intact.

[0117] The present invention, which prepares a casein hydrolysate according to a first aspect of the invention, is an optimized process that focuses on optimizing the hydrolysis of casein. In the most preferred embodiment of the present invention, MCI is stored at 5°C for 24 hours before hydrolysis, and an 8% w / w solution of MCI is hydrolyzed at pH 7.5-8.0 and a temperature of 38°C. After 48 hours of hydrolysis, approximately 85% of the beta-casein was hydrolyzed, meaning that only 15% of the remaining intact beta-casein (based on HPLC peak area) was obtained as a casein hydrolysate. Furthermore, as a result of the above optimized method, 57% of the a-S1 casein was hydrolyzed after 48 hours of hydrolysis. Conversely, if UHT-treated milk is stored, hydrolysis can take several months, for example, and gelling occurs during storage. Gelation of the casein hydrolysate of the present invention is undesirable. When the casein hydrolysate of the present invention is stored for 6 months, the product changes in appearance from milky white to translucent. However, the product does not gel or precipitate during storage. This is different from, for example, UHT-treated milk, which can gel over a shelf life of several months.

[0118] In one embodiment of the present invention, if a casein hydrolysate of a desired degree of hydrolysis is obtained, the activity of endogenous plasmin is inactivated or significantly reduced. Inactivating or reducing the activity of plasmin stops or at least slows down hydrolysis. Endogenous plasmin can be inactivated or reduced in different ways, and the present invention should not be limited to any method of inactivating endogenous plasmin and thus stopping hydrolysis. The plasmin inactivation / activity reduction step preferably requires control of the activity of both plasmin, plasmin-like enzymes, and the proenzyme plasminogen.

[0119] The plasmin inactivation / activity reduction step preferably requires controlling the total activity of plasmin, plasmin-like enzymes, and plasminogen in the treated liquid to at least 80%, preferably at least 90%, of the activity of the untreated liquid.

[0120] However, as an example, endogenous plasmin can be controlled by one or more of the following process steps: i) Adjust the pH to less than 6.0 ii) Lower the temperature to 10°C or below. iii) Dry the obtained casein hydrolysate. iv) Add denatured whey protein. v) Add a plasmin inhibitor and / or a plasmin activity inhibitor. vi) Add cysteine. vii) Raise the temperature to 65°C or higher. Plasmin activity decreases at pH values ​​below 6.8. Adjusting the pH to below 6.8 can significantly slow down hydrolysis.

[0121] Furthermore, plasmin activity can be controlled at temperatures below 20°C and above 55°C. Therefore, when the solution temperature is adjusted, for example, to below 10°C or above 65°C, plasmin activity decreases significantly, and the hydrolysis process is not significant.

[0122] Furthermore, plasmin activity can also be controlled by subjecting the resulting casein hydrolysate to a drying process. The drying process inactivates plasmin and prevents further hydrolysis. Drying may be performed, for example, by spray drying or freeze-drying, but the present invention should not be limited to any drying process.

[0123] Furthermore, the activity of endogenous plasmin may be regulated by adding denatured whey protein to the casein hydrolysate. The whey protein binds to plasmin near its active site, thereby limiting its activity. In addition, the activity of endogenous plasmin may be regulated by adding a plasmin inhibitor (PI) and / or a plasmin activity inhibitor (PAI).

[0124] Inactivation of endogenous plasmin can also be achieved by adding cysteine. Cysteine, like whey protein, binds to plasmin near the active site. Free thiols in cysteine ​​bind to plasmin via thiol disulfide exchange, inactivating the plasmin.

[0125] The hydrolysis of casein can be carried out, for example, by placing the pH-adjusted microbial inactivation solution from step iii) into a sterile bottle. The filled bottle can then be subjected to heat treatment at a temperature of 25°C to 45°C for a sufficient amount of time, for example, 6 hours to 10 days, to hydrolyze the casein and obtain the casein hydrolysate.

[0126] However, the hydrolysis process can also be carried out by transferring a pH-adjusted microbial inactivation solution to a sterile holding tank downstream of the first heat treatment unit for microbial inactivation. Holding tanks are preferred for larger-scale production, while hydrolysis in bottles is used for smaller-scale production.

[0127] The form of the casein hydrolysate obtained by the method according to the present invention should not be considered limiting, as the casein hydrolysate may be in any form, such as a liquid, or it may be a concentrate, powder, or granules. However, in one embodiment, the casein hydrolysate obtained in step iv) of the present invention is dried into a powder. The drying process may be, for example, spray drying or freeze-drying.

[0128] Homogenization: In one embodiment of the present invention, a pH-adjusted solution containing casein and endogenous plasmin (from step ii) is subjected to a homogenization step, and then to a first heat treatment step for inactivating microorganisms.

[0129] Homogenization is a mechanical process that results in a reduction in the size and number of fat globules in milk, as well as an increase in their total surface area. This reduces the milk's tendency to form cream on its surface, enhances its stability when in contact with the container, and improves its mouthfeel for consumers. Furthermore, homogenization ensures a uniform distribution of casein micelles in the solution before the first heat treatment step.

[0130] Casein hydrolysate: One aspect of the present invention relates to a natural casein hydrolysate that can be obtained by the method according to the present invention.

[0131] The natural casein hydrolysate produced by the method according to the present invention has the advantage of being prepared without the use of any added exogenous enzymes and is therefore entirely natural. This is an advantage as it avoids regulatory issues. Furthermore, compared to the use of added exogenous plasmin, the peptide accumulation in the casein hydrolysate obtained by using endogenous plasmin is more similar to that of human milk. Although the amino acid sequence of the peptides present in the casein hydrolysate of the present invention is not similar to the amino acid sequence of the peptides in human milk, the peptide accumulation is similar. The peptides in the casein hydrolysate have up to 50% amino acid sequence similarity to human milk peptides, which is higher than that of casein hydrolysate obtained by hydrolysis with added enzymes. This makes it possible to incorporate a casein peptide source into infant formula products that reflects human milk more accurately than when the casein peptide is obtained using exogenous peptides. Furthermore, in the case of beta-casein, gamma-casein, which is part of the main peptide produced by plasmin, is considered non-allergenic, which is particularly beneficial for infants who have problems with milk protein tolerance.

[0132] Therefore, in one embodiment, the natural casein hydrolysate of the present invention does not contain added exogenous enzymes.

[0133] The casein hydrolysate of the present invention contains at least 80% by weight of casein in total solids. Furthermore, the casein hydrolysate of the present invention contains whey protein in an amount of up to 10% by weight of the total protein content. Further details of the casein hydrolysate disclosed in the section on the method of preparing the casein hydrolysate of this application also apply to the casein hydrolysate.

[0134] The casein hydrolysate of the present invention is a natural product.

[0135] In relation to the present invention, the term "natural" should be understood as a product that does not contain components that are not naturally present in milk, milk fraction, or milk fraction diluted in water. For example, the addition of exogenous enzymes such as plasmin does not fall under the term "natural."

[0136] The casein hydrolysate of the present invention contains a peptide, of which at least 50% has a molecular weight of 1000 to 3000 Da, and less than 1% has a molecular weight of 5000 Da or more. Furthermore, less than 10% of the peptide has a molecular weight of less than 1000 Da.

[0137] The peptides present in the casein hydrolysate of the present invention exhibit functional effects such as immune stimulation that are not observed in commercially available hydrolysates.

[0138] Nutritional composition containing casein hydrolysate of the present invention: In a further embodiment, the present invention relates to the use of casein hydrolysates according to the present invention in nutritional compositions. The nutritional composition may be, for example, a pediatric nutritional product, such as a prepared infant formula (for both full-term and premature infants), a follow-on formula, or a follow-up formula. The nutritional composition may be nutritionally complete for the intended consumer, for example, an infant aged 0–6 months or an infant aged 6–12 months, or it may be a nutritional supplement. The nutritional composition may be in the form of a liquid product, a concentrated liquid product, a paste, or a powder.

[0139] It should be noted that embodiments and features described in one context of the present invention are also applicable to other aspects of the present invention.

[0140] The present invention will be described in further detail by the following non-limiting embodiments. [Examples]

[0141] Example 1 Test Method Example 1.1 HPLC-C18 for analyzing casein in a sample Apparatus and materials Chromatography system: Waters Acquity UPLC system equipped with a UV detector.

[0142] column: First column: Agilent Poroshell 120 SB-C18, 2.1 x 5 mm, 2.7 microns (PN821725~912) Second column: Agilent Poroshell 120 SB-C18, 2.1 x 150 mm, 2.7 microns (PN683775~902) The sample is first passed through the first column to remove impurities, and then passed through the second column for peptide separation.

[0143] Solutions and preparations: ACN 25%: Mix 250 ml of ACN with 750 ml of MQ. Reducing buffer solution: ·6M urea 0.1M sodium citrate 0.02M DTT Eluent A (0.1% trifluoroacetic acid (TFA) in MQ water): Add 1 ml of TFA to 990 ml of MQ water in a 1000 ml volumetric flask (below the surface as it is highly volatile), and fill the flask with water. Mix and transfer to the bottle with the blue cap. • De-air for 20 minutes. Eluent B (0.1% trifluoroacetic acid (TFA) in ACN): Add 1 ml of TFA to 990 ml of ACN in a 1000 ml volume flask (subsurface, as it is highly volatile), and fill with CAN. Mix and transfer to the bottle with the blue cap. Remove air for 20 minutes.

[0144] Eluent A and Eluent B were used in chromatography with the following settings as described in Table 1:

[0145] [Table 1] TIFF0007868024000002.tif25146

[0146] The flow rate was 0.35 ml / min (expected pressure under starting conditions: 4100 psi), and the column temperature was 42°C. The autosampler temperature was 12°C. The injection volume was 5 μl for all samples. UV detection was at 214 nm. After injection of each sample, the HPLC system was washed for 22 minutes (hours 19.5–41.5).

[0147] Example 1.2 Assay for detecting plasmin activity material: 0.4M trisodium citrate buffer, pH 8.9 with NaOH. Assay buffer 1: 0.1M tris-HCl 8 mM EACA (ε-aminocaproic acid) 0.4M NaCl pH 8 with NaOH Substrate S-2251: 4 mM in assay buffer 1

[0148] Commercial plasmin standards: For stabilization, a stock solution of Roche bovine plasmin with reference number 0.5 U in an assay buffer containing 4 mg / ml gelatin. Linear in the range of 15 μU / ml to 1 mU / ml

[0149] procedure: A 1 ml sample solution was mixed with 250 μL of 0.4 M trisodium citrate buffer, pH 8.9, and shaken for 15 minutes to dissociate casein micelles.

[0150] The citrate-treated samples were diluted 1:1 with assay buffer 1 and shaken for 15 minutes to dissociate plasmin and plasminogen from casein.

[0151] Absorbance was measured at 405 nm and 490 nm at 37°C using a plate reader at 5-minute intervals for 120 minutes.

[0152] To correct for turbidity, the background absorbance value at 490 nm was subtracted from the absorbance value at 405 nm.

[0153] ΔA as a function of time 405nm-490nm The increase in absorbance, as shown, was converted to plasmin units using a standard curve.

[0154] Example 1.3: Method for determining peptide distribution in whey protein hydrolysate The molecular weight distribution of peptides in casein hydrolysates was analyzed using size exclusion chromatography (SEC). SEC is used to separate polymer-type molecules by size. A mixture of components of different sizes, in this case peptides, can be separated by SEC. Elution time depends on the size of the molecule; smaller molecules require longer elution times.

[0155] The sample was dissolved in the mobile phase to a concentration of 0.5% w / v. Before injection, the sample was filtered through a 0.45 μm filter. Chromatographic separation was performed using three TSK G2000SWXL (125A, 5 μm, 7.5 mm × 300 mm) columns connected in series. A buffer of 0.0375 M phosphate buffer, 0.375 M ammonium chloride, 0.1% trifluoroacetic acid (TFA), and 25% acetonitrile (CH3CN) was used as the mobile phase at a flow rate of 0.7 mL / min. Peptide detection was performed using a UV detector measuring at 214 nm.

[0156] Based on retention time, the peptide distribution is divided according to size, and the relative amounts are given according to molecular weight.

[0157] Example 2 Plasmin activity in the retaining liquid of microfiltered skim milk (MCI) Micellar casein isolate was prepared by microfiltration of pasteurized skim milk to produce micellar casein isolate.

[0158] The micellar casein isolate was prepared as a retaining liquid for microfiltered skim milk by providing 1200 kg of pasteurized (73°C / 15 seconds) organic concentrated skim milk preheated to 52°C. The pH of the skim milk was 6.7. The skim milk contained 6.4% (w / w) protein, 4.7% (w / w) lactose, 0.1% (w / w) fat, and 12.5% ​​(w / w) total solids.

[0159] Preheated skim milk is subjected to microfiltration (MF) in batch mode on a polymer FR membrane manufactured by Synder Filtration (USA) with a pore size of 800 kDA at 50°C with a transmembrane pressure difference (TMP) of 0.45 bar. After collecting 720 liters of filtration product, dialysfiltration is started by adding reverse osmosis (RO) filtered tap water to the retaining solution at the same flow rate as the MF filtration product. Filtration is completed when 3,000 liters of RO filtered tap water are added. 480 kg of MF retaining solution and 3,720 kg of MF filtration product are collected. 98% lactose and 79% whey protein from the skim milk are collected in the MF filtration product, and 97% micellar casein from the skim milk is collected in the MF retaining solution.

[0160] The residual plasmin in heat-treated MF holding solution (MCI) was analyzed according to the method described in Example 1.2. Samples were prepared using an 8% w / w MCI solution adjusted to pH 7.5 with 10% KOH. Samples were heat-treated at temperatures of 130°C, 135°C, and 140°C for 3-4 seconds, and plasmin activity was analyzed. The plasmin activity is shown in Table 2 below.

[0161] [Table 2]

[0162] Example 3: Effect of incubation temperature on the degree of plasmin-derived hydrolysis of MCI after heat treatment Samples were prepared using an 8% w / w MCI solution adjusted to pH 7.5 with 10% KOH. The samples were heat-treated at 130°C, 135°C, and 140°C for 3-4 seconds and then placed into different bottles by aseptic filling. The bottles were incubated at 20°C and 38°C for 24 hours, respectively. Four batches of MCI were prepared at each temperature (130°C, 135°C, and 140°C). That is, four bottles were heat-treated at 130°C, 135°C, and 140°C, respectively. Then, two of the four bottles were incubated at 38°C and the other two bottles were incubated at 20°C.

[0163] Samples were removed from the incubator after 0, 3, 6, and 24 hours and immediately frozen until HPLC analysis was performed (according to Example 1.1). Samples were prepared by diluting them 8-fold in reducing buffer and left at room temperature for 1 hour. The diluted samples were filtered through a 0.22 μm cellulose acetate filter (VWR#514~0060) and then subjected to HPLC.

[0164] Plasmin activity in the sample before incubation was measured using the method described in Example 1.2. The results are shown in Figures 1 and 2.

[0165] Figure 1 shows the plasmin-derived peptides that elute between 12.00 and 14.00 minutes by RP-HPLC. Figure 2 shows the plasmin-derived peptides that elute between 17.00 and 18.00 minutes by RP-HPLC.

[0166] Figures 1 and 2 show that incubation at 38°C results in increased loss of intact α-S1-casein and β-casein and increased peptide accumulation compared to incubation at 20°C. Both peptides were observed at 13 and 17 minutes by HPLC-RP.

[0167] The loss of intact α-S1-casein and β-casein is approximately 20-25% greater when using 38°C than 20°C, and the peptide increase is approximately four times greater. Therefore, hydrolysis is more efficient at 38°C than at 20°C, which is thought to be due to improved plasmin activity at 38°C compared to 20°C.

[0168] Furthermore, Figures 1 and 2 show that plasmin activity can be maintained after sterilization at temperatures of 130°C, 135°C, and 140°C for 3-4 seconds.

[0169] Example 4: Effects of low-temperature storage of MCI before heat treatment on plasmin activity and casein hydrolysis This example demonstrates the effect of low-temperature storage on plasmin activity and the degree of hydrolysis.

[0170] The samples were prepared using an 8% (w / w) MCI solution adjusted to pH 7.5 with 10% NaOH.

[0171] The heat treatment (first heating step) of MCI was carried out on two different days with a 24-hour time difference, under storage conditions of 4-5°C. This allows the effects on plasmin activity and hydrolysis to be shown after 24 hours of storage at 5°C. Samples from the two different days will be referred to as Day 1 and Day 2 below. Each day, MCI was subjected to heat treatment at temperatures of 130°C, 135°C, and 140°C for 3-4 seconds and aseptically filled into 100 ml bottles.

[0172] Immediately after heat treatment, bottled samples were incubated at 37–40°C for 24 hours. Samples were taken at incubation times of 0, 3, 6, and 24 hours and immediately frozen for analysis by HPLC-RP (HPLC samples were prepared in the same manner as in Example 3). Plasmin activity was measured 5 days after heat treatment according to the method of Example 1.2, and samples were stored at 4°C until analysis.

[0173] The loss of intact α-S1-casein and β-casein was calculated by comparing the peak areas of these proteins obtained by each RP-HPLC chromatogram. The peak areas were normalized to the peak area of ​​a given protein at t=0, i.e., immediately before incubation.

[0174] Figure 3 shows the loss of intact β-casein in different samples.

[0175] Figure 4 shows the loss of intact α-S1-casein in different samples.

[0176] Peptide accumulation in different samples was quantified using the same method and normalized to initial levels of plasmin-derived peptides naturally present in milk and retained in MCI after microfiltration.

[0177] Therefore, the peptide accumulation in the HPLC chromatography region at 12-14 minutes and 17-18 minutes is shown in Figures 5 and 6, respectively.

[0178] From each figure, it is clear that plasmin activity in MCI, and consequently the degree of hydrolysis of intact casein protein, is affected by 24 hours of storage at 4-5°C before heat treatment, and that plasmin is retained in all samples regardless of whether they were heat-treated at 130, 135, or 140°C for 3-4 seconds.

[0179] It is clear that significant accumulation of casein peptides occurs over a 24-hour incubation period. In particular, for samples stored at 5°C for 24 hours before heat treatment, the loss of intact α-S1-casein was greater, resulting in a factor of 1.8–4 compared to t=0, an associated loss of approximately 64% of intact β-casein regardless of heat treatment, and a loss of 34%–54% of α-S1-casein with heat treatment at 130°C for 3–4 seconds. For samples heat-treated on day 1, a loss of 34%–54% of intact β-casein was observed, while a loss of 15–25% of intact α-S1-casein was observed.

[0180] Plasmin activity was measured according to Example 2.1 of heat-treated MCI from day 1 and day 2. See Figure 7. The difference in the degree of casein hydrolysis between the day 1 and day 2 samples can be explained by the increase in plasmin activity after 24 hours of low-temperature storage before heat treatment under all conditions, as shown in Figure 7. During these 24 hours, plasminogen is converted to plasmin, thus increasing plasmin activity.

[0181] In summary, it is clear that substantial hydrolysis of intact α-S1-casein and β-casein, as well as the accumulation of casein peptides as a result of the action of endogenous plasmin, is possible. This is evidenced by the loss peak area (>50%) of these two proteins by RP-HPLC over a 24-hour incubation period. From the superimposed (Figure 8) representative HPLC traces, it is also clear that kappa-casein (retention times 8 and 9.25 minutes) remains intact after the 24-hour incubation period, which is consistent with literature describing the selectivity of plasmin over alpha and beta-casein.

[0182] Example 5: Methodology Examples were provided to demonstrate that microorganisms were inactivated after the heat treatment (first heat treatment step) process.

[0183] Samples were prepared using an 8% w / w MCI solution adjusted to pH 7.5 with 10% KOH. The samples underwent a first heat treatment step using UHT heat treatment at temperatures of 130°C, 135°C, and 140°C for 3–4 seconds, and were aseptically placed into separate bottles. The bottles were stored at 37°C for 10 days before being subjected to microbiological analysis. Analysis was performed for each bottle at all temperatures. The results are shown in Table 3 below.

[0184] The following terms in Table 3 mean the following: BCSP: Bacillus cereus spores CLO.SP: Clostridium spores THF: thermophilic bacteria TPC: Total number of plates CLO.SP-1: Sample diluted 10 times. CLO.SP: Do not dilute the sample. THF-1: Dilute the sample 10-fold. TPC-1: Dilute the sample 10-fold.

[0185] [Table 3]

[0186] As shown in Table 3, all samples stored at 37°C for 10 days had a microbial content of less than 10 cfu / mL. This confirms that the first heat treatment at 130°C–140°C was sterile. Since hydrolysis in this example proceeded for only 24 hours, the results strongly indicate that safe hydrolysis can be performed after UHT treatment in a suitable sterile holding tank.

[0187] Example 6: Determination of LC / MS and proteomic properties of casein hydrolysates High-resolution LC-MS / MS was performed on samples of MCI hydrolyzed according to the method of the present invention to confirm that the accumulation of most of the peptides observed in the hydrolysates is due to the action of plasmin on alpha and beta casein. Furthermore, the peptides were matched with known bioactive peptides using an open-access database.

[0188] To broaden the scope of application and identify as many plasmin-derived peptides as possible, peptides were extracted from hydrolyzed MCI samples using two methods: ultracentrifugation and 10 kDaMWCO column chromatography. These two methods are briefly described below:

[0189] A 100 ml bottle of MCI sample hydrolyzed according to the present invention was removed from -80°C and thawed at room temperature. By ultracentrifugation, approximately 25 ml was taken out, the pH was adjusted to 4.6, and it was centrifuged at 11000 x g for 10 minutes at 4°C. Next, 10 ml of the supernatant was transferred to a 50 ml tube and frozen at -20°C (1 x centrifugation), and another 10 ml of the supernatant was centrifuged at 13200 x g for 10 minutes at 5°C (2 x centrifugation). The supernatant from the second centrifugation, mainly containing casein peptides, was transferred to a new tube. A total of 1 ml of the supernatant from the second centrifugation sample was reduced with 30 μL of 100 mM dithiothreitol (DTT) for 1 hour at 25°C, and then alkylated with 70 μL of 100 mM iodoacetamide (IAA) for 50 minutes at 25°C in the dark.

[0190] In the 10kDaMWCO column method, 1 mL of MCI sample hydrolyzed according to the present invention is diluted with 10 mg / mL of 6 M urea buffer based on a predetermined protein concentration. -1 The solution was diluted to its final concentration, thoroughly mixed, and the protein dissolved in the buffer. Next, 100 μL of the sample solution (1 mg of protein) was reduced with 30 μL of 100 mM dithiothreitol (DTT) for 1 hour at 25°C, alkylated in 70 μL of 100 mM iodoacetamide for 50 minutes at 25°C in the dark, and 800 ml of 50 mM TEAB buffer was added to the sample until the final volume was 1 mL (1 mg / mL). The prepared sample solution was filtered through a 10 kDaMW cutoff column filter (Merk Millipore, Germany) by centrifugation at 14000 g for 20 minutes, and further washed with 0.5 mL of 50 mM TEAB buffer by centrifugation at 14000 RPM for 20 minutes. The filtered peptide was collected in the filtered tube and used for further analysis of endogenous peptides.

[0191] Peptide samples were desalted and purified using hydrophilic-lipophilic balanced solid-phase extraction (OASIS HLB-SPE) (Waters, Bedford, Massachusetts) cartridge column. In short, the column was washed twice with 1 mL of 100% acetonitrile (ACN) and twice with 1 mL of 0.1% trifluoroacetic acid (TFA). Then the peptide sample was loaded onto the column and washed twice with 1 mL of 0.1% TFA. The peptide was then eluted with 1 mL of elution buffer containing 0.1% TFA, 29.9% MQ water, and 70% ACN. The eluted peptide was lyophilized by vacuum centrifugation and then resuspended in 1000 μL of 0.1% formic acid (FA) buffer for further LC-MS analysis.

[0192] Peptide samples were analyzed by LC-MS for protein and peptide identification. Each sample was analyzed with three technically repeatable trials, with 5 μl of peptide sample injected for each analysis. Peptides were automatically injected and loaded onto a Waters ACQUITY UPLC CSH C18 column (130A, 1.7 μm, 2.1 mm × 150 mm) by reverse-phase chromatography performed on a Dionex3000 UPLC system (Thermo Fisher Scientific). The mobile phases were buffer B (99.9% ACN / 0.1% formic acid) and buffer A (0.1% formic acid). Samples were loaded using intelligent flow control at a maximum pressure of 800 bar. An LC gradient was performed at 300 μL / min with the following profile: 18 minutes in 0–30% buffer B, 5 minutes in 30–50% buffer B, 2 minutes and 3 minutes in 50–100% buffer B, then back to buffer A for re-equilibrium. The UPLC was connected online to a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) operating in cation mode and using data-dependent acquisition. The Orbitrap was acquired for a full MS scan with an automated gain control (AGC) target value of 1e6 ions and a maximum filling time of 100 milliseconds. Each MS scan was acquired at high resolution 70,000 full width at half maximum (FWHM) at m / z 200 in the Orbitrap in the range of 400–3000. For high-energy collision dissociation (HCD) MS2 fragmentation (normalized collision energy: 28V) in the Orbitrap, the top 20 positions of the abundant peptide were selected from the MS. Fragmentation was performed at high resolution (17500 FWHM) with a target of 2e4 and a maximum injection time of 150 milliseconds, using a separation window of 1.5 m / z and a dynamic exclusion period of 15 seconds with a tolerance of 10 ppm around the selected precursor. Only precursors with charge states of +2, +3, +4, and +5 were sampled for MS2. Raw data were displayed using Xcalibur v2.0.7 (Thermo Fisher Scientific, USA).

[0193] LC-MS / MS raw data were processed in Proteome Discover 2.1 for peptide and protein identification using the integrated Sequest HT server according to the following criteria: database, UniProt bovine non-redundant proteome database (versions 15-11-2019); enzyme, no specific enzymes were selected; maximum loss cleavage, 2; variable modifications included oxidation (Met), carbamide methylation of cysteine, acetyl (protein N-terminus and lysine (K)), deamidation of Asn and Gln, and phosphorylation (Ser, Thr, and Tyr). MS and MS / MS results were searched with a precursor mass tolerance of 10 ppm and an MS / MS mass tolerance of 0.05 Da. Results were filtered in Proteome Discoverer using the integrated Percolator algorithm to ensure a false discovery rate (FDR) of less than 0.01. Peptides that passed the default score for Sequest HT versus charge state were accepted.

[0194] Peptides identified from each sample were exported to an Excel spreadsheet, further processed manually, and analyzed. The processed data was then visualized, and protein sequence localization and quantity were displayed using the online software Peptigram (http: / / bioware.ucd.ie / peptigram / ).

[0195] The identified peptides were also searched against the Bovine and Human Milk Bioactive Peptide Database (BIOPEP Bioactive Peptide Database) to explore and discover potential endogenous bioactive peptides.

[0196] The results of the LCMS / MS workflow, peptide mix analysis, and database search are shown in Figures 9A, 9B, 10A, 10B, 11A, and 11B.

[0197] Figures 9A and 9B show the identified peptides of α-S1-casein, where the dotted lines represent plasmin cleavage sites.

[0198] Figures 10A and 10B show the identified peptides of α-S2-casein, where the dotted lines again represent plasmin cleavage sites.

[0199] Figures 11A and 11B show the identified peptides of β-casein, where the dotted lines represent plasmin cleavage sites.

[0200] Figures 9A, 9B, 10A, 10B, 11A, and 11B clearly show that the vast majority of peptides are cleaved by plasmin or plasmin-related enzymes on β-casein, α-S1-casein, and α-S2-casein. Furthermore, 33 peptides with demonstrated biological activity were identified by matching them against the aforementioned database. These bioactive peptides are derived from α-S1-casein, α-S2-casein, β-casein, and kappa-casein. In the aforementioned database, 15 of these bioactive peptides were annotated for ACE inhibitory activity, and 16 were annotated for antibacterial activity. Table 4 below lists the peptides identified from α-S1-casein, α-S2-casein, β-casein, and kappa-casein and their biological activities.

[0201] [Table 4] TIFF0007868024000006.tif229157TIFF0007868024000007.tif236157TIFF0007868024000008.tif22155

[0202] Example 7: Determination of molecular weight distribution The molecular weight distribution of the casein hydrolysate prepared according to the present invention was determined by the method described in Example 1.3, and the size of the peptides present was analyzed.

[0203] The results are shown in Figure 12. Figure 12 shows that more than 50% of the identified peptides of the casein hydrolysate of the present invention have molecular weights in the range of 1000 to 3000 Da, and less than 1% of peptides have molecular weights greater than 5000 Da.

[0204] Example 8: Effect of pH The effect of pH on plasmin in heat-treated MF holding solution (MCI) was analyzed.

[0205] Samples were prepared using 8% w / w MCI solutions adjusted to pH 6.8, 7.8, and 8.2 with NaOH and 0.05% NaN3, respectively. The samples were hydrolyzed at 38°C without being subjected to UHT heat treatment. The amounts of beta-casein (beta-CN) and alpha-S1-casein (alpha-S1-CN) were measured by HPLC at the start of hydrolysis (time = 0 hours), and again at 24 hours and 48 hours. The HPLC method used was the same as described in Example 1.1, except that the column used was Waters BioSuite C18 PA-B 3.5 μm, 2.1 × 250 mm (Waters #186002436), and the washing time between injections of each sample was 6.5 minutes.

[0206] Figure 13A shows the effect of pH on the hydrolysis of alpha-S1-casein, and Figure 13B shows the effect on the hydrolysis of beta-casein. Figures 13A and 13B clearly show that the effect of endogenous plasmin on the hydrolysis of casein (both beta-casein and alpha-S1-casein) is optimal at pH 7.5–8.2, and that efficiency is low when the pH is 6.8, which is the pH of natural milk. After 48 hours of hydrolysis, the amount of intact alpha-S1-casein is 40–50% when the pH is 7.8–8.2, compared to approximately 68% when the pH is 6.8. Similarly, the amount of beta-casein after 48 hours of hydrolysis is lower when using pH 7.8–8.2 than when using pH 6.8.

Claims

1. A method for preparing a casein hydrolysate for use in a nutritional composition, the method being: i) A step of providing a solution containing casein and endogenous plasmin, wherein the solution contains casein in an amount of at least 80% by weight of the total solids, and the solution contains whey protein in an amount of up to 10% by weight of the total protein content, and the solution containing casein and endogenous plasmin is a fraction of a milk-derived feed containing casein and endogenous plasmin, selected from the group consisting of micellar casein isolate (MCI), micellar casein concentrate (MCC), micellar casein retaining solution (MCR), beta-casein-depleted micellar casein isolate, beta-casein-depleted micellar casein concentrate, milk protein concentrate (MPC), and microfiltration retaining solution of dairy products. ii) A step of adjusting the pH of the solution in step i) to pH 7.2 to 9, iii) A step of subjecting the pH-adjusted solution to a first heat treatment to inactivate microorganisms, wherein the first heat treatment is performed at a temperature of 85°C to 180°C for a period of 0.1 to 30 seconds, iv) A step of subjecting the solution from step iii) to a second heat treatment at a temperature of 25°C to 45°C for at least 6 hours to obtain a casein hydrolysate. Includes, A method for eliminating the addition of exogenous enzymes.

2. The method according to claim 1, wherein the pH-adjusted solution of step ii) is homogenized.

3. The method according to claim 1 or 2, wherein the first heat treatment in step iii) includes heating to a temperature above 120°C for a period of 0.1 to 10 seconds.

4. The method according to claim 3, wherein the first heat treatment in step iii) includes heating to a temperature of 120°C to 180°C for 0.1 to 10 seconds.

5. The method according to any one of claims 1 to 4, wherein the first heat treatment is selected from ultra-high temperature (UHT) heat treatment and high-pressure sterilization.

6. The method according to any one of claims 1 to 5, wherein the solution comprising casein and endogenous plasmin comprises a milk fraction from milk.

7. The method according to any one of claims 1 to 6, wherein the solution provided in step i) contains casein in an amount of at least 1% w / v.

8. The method according to claim 7, wherein the solution provided in step i) contains casein in an amount of 1% w / v to 20% w / v.

9. The method according to any one of claims 1 to 8, wherein the solution contains casein in an amount of at least 90% of the total protein content.

10. A casein hydrolysate for use in a nutritional composition obtainable by any one of claims 1 to 9, wherein the casein hydrolysate does not contain additional exogenous enzymes, the casein hydrolysate contains at least 80% by weight of hydrolyzed casein of the total solids, and whey protein in an amount of up to 10% by weight of the total protein content.

11. The casein hydrolysate according to claim 10, wherein the casein hydrolysate contains a peptide, at least 50% of the peptide has a molecular weight of 1000 to 3000 Da, and less than 1% of the peptide has a molecular weight of 5000 Da or more.

12. Use of casein hydrolysate according to any one of claims 10 to 11 in a nutritional composition.

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