Method for co-separating κ-casein and β-casein to simulate composition of human milk casein

Abstract

A method for co-separating κ-casein and β-casein to simulate the composition of human milk casein, relating to the technical field of dairy product processing. A micellar casein concentrate (MCC) solution is used as a raw material, selective calcium precipitation technology under alkaline environment or weak alkaline environment conditions is used, and the starting position of a low-temperature step and key process parameters in steps such as complexation of casein and calcium, low-temperature, precipitation of αs-casein, and precipitation of κ-casein and β-casein are finely adjusted and combined, to achieve co-separation of κ-casein and β-casein.

Description

A method for co-separating κ- and β-casein to mimic the composition of human milk casein Technical Field

[0001] This invention relates to a method for co-separating κ- and β-casein to mimic the composition of human milk casein, belonging to the field of dairy processing technology. Background Technology

[0002] Bovine and ovine casein are among the main proteins in infant formula. Their composition differs from that of breast milk casein, making them less easily digestible and absorbed. Bovine casein includes α-lactalbumin... s1 -、α s2 -, β-, κ-casein, with α s1 The main components are β-casein, with a ratio of approximately 40:10:36:14; sheep milk casein includes α-casein. s1 -、α s2 α, β, and κ-casein, in a ratio of approximately 24:18:46:12; breast milk casein includes α... s1 It contains β- and κ-casein, mainly β- and κ-casein, and does not contain α-casein. s2 The ratio of bovine and ovine casein is 9-12:68-70:18-21. Therefore, to mimic the casein composition of breast milk and improve digestibility, bovine and ovine casein need to be separated.

[0003] Currently, the separation of casein mainly focuses on the separation of single casein proteins. This process suffers from problems such as complex separation techniques, long separation cycles, use of toxic reagents, low yields and purity, and the use of strongly alkaline conditions, making it difficult to achieve industrial-scale production. The separated products are unsuitable for infant formula production. Under strongly alkaline conditions, casein undergoes dephosphorylation and deamidation reactions, leading to changes in its molecular structure and the generation of harmful byproducts such as lysine, which can pose safety risks after consumption. For example, CN107840883B uses bovine milk casein as raw material, adding urea and β-mercaptoethanol for dissolution, followed by ion exchange to achieve the sequential separation of three single casein proteins; CN113461795A uses ethanol dissolution and acid precipitation to achieve the sequential separation of three single casein proteins; CN112931616A uses low-temperature membrane filtration to achieve the co-separation of β-casein and whey protein; and CN116355073A uses calcium precipitation at pH 12 to achieve the separation of κ-casein. When these single casein proteins are used in the production of infant formula, they need to be compounded to mimic the composition of breast milk casein, making the overall process complex and costly.

[0004] κ- and β-casein are the two most abundant caseins in breast milk. A one-step method for separating κ- and β-casein from bovine and ovine milk caseins directly simulates the composition of breast milk caseins, shortening the separation process and cycle time, reducing costs, and also lowering α-casein levels. s - Casein, a major allergenic protein, does not need to be combined with other caseins when used in the production of infant formula. However, κ-casein is a glycosylated protein with strong hydrophilicity and low phosphorylation; β-casein is strongly hydrophobic and has a higher degree of phosphorylation; α-casein... s Casein has moderate hydrophilicity and hydrophobicity, and is highly phosphorylated; it can be seen that κ-casein, β-casein, and α-casein... s Casein exhibits significant differences in properties, and the order of the three proteins varies depending on their properties. This makes the co-separation of κ- and β-casein a challenging problem in the field. Achieving both high extraction rates and high purity for κ- and β-casein, which have vastly different properties, requires a different separation scheme and conditions compared to the separation of any single casein. The co-separation of κ- and β-casein presents even greater requirements and difficulties; no relevant reports have been found to date. Summary of the Invention

[0005] [Technical Issues]

[0006] The separation of single casein from animal milk has problems such as complex separation process, long separation cycle, use of toxic reagents, low yield and purity, and the generation of harmful substances due to the use of strong alkaline conditions. When used in the production of infant formula, it also needs to be compounded with other proteins, which limits industrial production and application.

[0007] κ-casein, β-casein and α s The significant differences in hydrophilicity, phosphorylation, and glycosylation of caseins make the co-separation of κ- and β-caseins a challenging problem in the field.

[0008] [Technical Solution]

[0009] To address the aforementioned problems, this invention uses micellar casein concentrate (MCC) solution as raw material and employs selective calcium precipitation technology under alkaline or weakly alkaline conditions. Furthermore, it focuses on the starting point of the low-temperature step, the complexation of casein with calcium, low temperature, and α-precipitation. s The method of this invention achieves the co-separation of κ- and β-casein by precisely controlling and combining key process parameters in steps such as casein precipitation, κ- and β-casein precipitation. This simplifies the process flow, shortens the separation cycle, improves production efficiency, and is gentle and safe. The resulting co-separate has a composition similar to that of breast milk casein.

[0010] The first objective of this invention is to provide a method for co-separating κ- and β-casein from casein micelles, comprising the following steps:

[0011] (1) Co-separation:

[0012] Method 1: Adjust the pH of a 1-7% casein solution to 11.0 at 23-27℃; then add calcium chloride solution to achieve a calcium ion concentration of 5-65 mM; equilibrate at 10-40℃ for 0-90 min to obtain a casein-calcium complexed solution; then treat the casein-calcium complexed solution at 0-12℃ for 0-60 min, adjust the pH to 4.6-5.6 for acid precipitation, and equilibrate at 0-12℃ for 0-24 h; centrifuge to obtain a solution enriched with α- s -Casein precipitation, and supernatant enriched with κ- and β-casein;

[0013] Method 2: Adjust the pH of a 2-4% casein solution to 4.3 at 23-27℃, raise the temperature to 40-50℃, and equilibrate for 20-40 minutes; then cool to 23-27℃ and centrifuge; collect the precipitate in ultrapure water, adjust the pH to 6.5-9.0 at 23-27℃, and equilibrate at 23-27℃ for 0-75 minutes to obtain a casein reconstituted solution with a casein mass concentration of 2-4%; add calcium chloride solution to the casein solution until the added calcium ion concentration is 30-70 mM, and equilibrate at 23-27℃ for 50-70 minutes; adjust the pH to 5.0 for acid precipitation, equilibrate at 23-27℃ for 0-150 minutes, and then treat at 0-12℃ for 7-9 hours; centrifuge to obtain a solution enriched with α- s Precipitation of casein, and supernatant enriched with κ- and β-casein;

[0014] Method 3: Adjust the pH of a 2-4% casein solution to 5.0 for acid precipitation, equilibrate at 23-27℃ for 80-100 min, then at 1-4℃ for 7-9 h, and centrifuge to obtain a solution enriched with α-protein. s -Casein precipitation, and supernatant enriched with κ- and β-casein;

[0015] (2) Take the supernatant enriched with κ- and β-casein, adjust the pH to 3.0-5.2, precipitate at 15-55℃ for 0-60 min, centrifuge to obtain the precipitate enriched with κ- and β-casein.

[0016] In one embodiment of the present invention, the mass concentration of the casein solution in step (1) co-separation method one is preferably 2-4%, and more preferably 3%.

[0017] In one embodiment of the present invention, the casein solution in step (1) co-separation method one is obtained by reconstituted MCC powder with water or by diluting MCC after reconstitution.

[0018] In one embodiment of the present invention, in step (1) co-separation method one, the pH is adjusted to 11.0 using a 2M NaOH solution.

[0019] In one embodiment of the present invention, in step (1) co-separation method one, the concentration of calcium ions added is 35mM; the equilibrium temperature after adding calcium chloride is 10-25℃, further, the temperature is 20℃; the equilibrium time is more than 60min; preferably, the time is 60-90min; more preferably, the time is 60-75min.

[0020] In one embodiment of the present invention, the temperature of the low-temperature treatment in step (1) co-separation method one is 0-4°C and the time is more than 10 min; preferably, the temperature is 4°C and the time is 10-50 min; more preferably, the time is 10 min.

[0021] In one embodiment of the present invention, in step (1) co-separation method one, the pH of 4.6-5.6 is adjusted by using a 4M acetic acid solution, a 4M hydrochloric acid solution, a 4M lactic acid solution or a 4M citric acid solution. Preferably, a 4M acetic acid solution is used for adjustment.

[0022] In one embodiment of the present invention, the pH of acid precipitation in step (1) co-separation method one is 4.9-5.1, preferably pH 5.0; the equilibration time after acid precipitation is 8-24h, preferably 8h.

[0023] In one embodiment of the present invention, in step (1) co-separation method one, centrifugation is performed at 3.5-4.5℃ and 2000-10000g for 5-15 minutes; equilibration is performed under stirring at a speed of 200-500rpm.

[0024] In one embodiment of the present invention, the casein solution in step (1) co-separation method two is obtained by reconstituted MCC powder with water or by diluting MCC after reconstitution.

[0025] In one embodiment of the present invention, in step (1) co-separation method two, the pH is adjusted to 4.3 using a 2M HCl solution.

[0026] In one embodiment of the present invention, in step (1) co-separation method two, the centrifugation after cooling to 23-27°C is performed at 2000-10000g for 5-15 minutes.

[0027] In one embodiment of the present invention, in step (1) co-separation method two, the pH is adjusted to 6.5-9.0 using 2M NaOH solution.

[0028] In one embodiment of the present invention, the pH of the casein reconstitution solution in step (1) co-separation method two is 7.5; the equilibration time of the casein reconstitution solution is more than 60 min; preferably, the time is 60-75 min; more preferably, the time is 60 min.

[0029] In one embodiment of the present invention, the concentration of calcium ions added in step (1) co-separation method two is 45mM; the equilibrium temperature after adding calcium chloride is 25°C.

[0030] In one embodiment of the present invention, in step (1) co-separation method two, the pH is adjusted to 5.0 using a 4M acetic acid solution; the equilibrium temperature after acid precipitation is 23-27℃, preferably 27℃.

[0031] In one embodiment of the present invention, the equilibration time after acid precipitation in step (1) co-separation method two is more than 90 min; preferably, the time is 90-150 min; more preferably, the time is 90-120 min.

[0032] In one embodiment of the present invention, the temperature of the low-temperature treatment in step (1) co-separation method two is 0-2℃, preferably 2℃; the time of low-temperature treatment is 7-9h, preferably 9h; the centrifugation after low-temperature treatment at 0-12℃ for 7-9h is centrifugation at 0-4℃ and 2000-10000g for 5-15min.

[0033] In one embodiment of the present invention, the equilibrium in step (1) co-separation method two is equilibrium under stirring, and the stirring speed is 200-500 rpm.

[0034] In one embodiment of the present invention, the mass concentration of the casein solution in step (1) co-separation method two is 2-4%, preferably 4%.

[0035] In one embodiment of the present invention, the casein solution in step (1) co-separation method three is obtained by reconstituted MCC powder with water or by diluting MCC after reconstitution.

[0036] In one embodiment of the present invention, in step (1) co-separation method three, the pH of 5.0 is adjusted by using a 4M acetic acid solution, a 4M lactic acid solution or a 4M citric acid solution, preferably by using a 4M acetic acid solution.

[0037] In one embodiment of the present invention, the equilibrium in step (1) co-separation method three is stirring equilibrium, and the stirring speed is 200-500 rpm.

[0038] In one embodiment of the present invention, the centrifugation in step (1) co-separation method three is centrifugation at 0-4℃ and 2000-10000g for 5-15 minutes.

[0039] In one embodiment of the present invention, the pH value in step (2) is 3.4-3.8; preferably, the pH value is 3.6.

[0040] In one embodiment of the present invention, the pH in step (2) is adjusted to 3.0-5.2 using a 2M hydrochloric acid solution.

[0041] In one embodiment of the present invention, the precipitation temperature in step (2) is 45-55°C; preferably, the temperature is 50°C; the precipitation time is 20 min or more; preferably, the precipitation time is 20-60 min; and more preferably, the precipitation time is 20 min.

[0042] In one embodiment of the present invention, the MCC used in step (1) to prepare the casein solution is a micellar casein concentrate, or a casein micellar concentrate derived from animal milk.

[0043] MCC is derived from animal milk such as cow's milk; MCC can be purchased commercially; or it can be prepared by membrane filtration separation; or it can be obtained by diluting the MCC retentate obtained by membrane filtration with water.

[0044] In one embodiment of the present invention, the method for preparing MCC is as follows:

[0045] Skim milk was sterilized using a 1.4 μm pore size ceramic membrane, whey protein was removed using a 100 nm pore size ceramic membrane, the volume concentration factor was 4, water was added and the mixture was washed and filtered 3 times, the retentate was collected and freeze-dried to obtain micellar casein concentrate (MCC) powder.

[0046] A second objective of this invention is to prepare κ- and β-casein using the method described herein.

[0047] A third objective of this invention is the application of the method described herein or κ- and β-casein in food.

[0048] In one embodiment of the invention, the application is for infant food, particularly for the preparation of infant formula milk powder.

[0049] [Beneficial Effects]

[0050] (1) The first method for co-separating κ- and β-casein described in this invention uses micellar casein concentrate prepared by membrane filtration as raw material, and controls different low-temperature initiation positions, casein complexation with calcium, low temperature, and α-casein complexation.s - By finely controlling and combining key process parameters in major steps such as casein precipitation, and further, by controlling the process parameters in steps such as κ- and β-casein precipitation, the yield and purity of κ-casein can reach 82.1% and 15.3%, respectively, while the yield and purity of β-casein can reach 96.1% and 73.8%, respectively.

[0051] (2) The second method for co-separating κ- and β-casein described in this invention uses micellar casein concentrate as raw material. This is achieved by controlling weakly alkaline reconstitution conditions, different low-temperature initiation positions, and the complexation of casein with calcium, low temperature, and α-casein. s - By precisely controlling and combining key process parameters in major steps such as casein precipitation, the yield and purity of κ-casein can reach 73.5% and 15.2%, respectively, while the yield and purity of β-casein can reach 97.7% and 67.7%, respectively. Furthermore, by controlling the equilibration temperature, low-temperature equilibration time, and casein mass concentration after acid precipitation, the yield and purity of κ-casein can reach 85.7% and 18.3%, respectively, while the yield and purity of β-casein can reach 98.7% and 69.6%, respectively.

[0052] (3) The third method for co-separating κ- and β-casein described in this invention uses micellar casein concentrate as raw material, and controls α... s - By regulating and combining key process parameters in the main steps such as casein precipitation and low temperature, and further, by controlling the process parameters in the κ- and β-casein precipitation steps, the yield and purity of κ-casein can reach 43.9% and 14.0%, respectively, while the yield and purity of β-casein can reach 76.5% and 81.4%, respectively.

[0053] (4) This invention provides a one-step separation method for co-separating κ- and β-casein. Compared with the multi-step separation method that sequentially separates single casein, it simplifies the process flow, shortens the separation cycle, and achieves high yield and purity of κ- and β-casein through the specific combination and synergistic effect of key process parameters in each step. It also realizes the co-separation of κ- and β-casein at a high casein concentration, which is suitable for large-scale industrial production and improves production efficiency.

[0054] (5) The purity of κ- and β-casein in the co-isolated fraction obtained in this invention is very close to the purity of the two caseins in breast milk, and α-casein... s - The low content of casein, a major allergenic protein, allows it to be directly used in the production of infant formula, mimicking the composition of breast milk casein. This eliminates the need to adjust the proportions of various caseins by blending them with other caseins, resulting in lower production costs. It can serve as a novel casein ingredient, promoting the iterative upgrading of infant formula.

[0055] (6) Compared with traditional strong alkaline process conditions, the yield and purity of κ-casein and β-casein in the co-separates obtained by the weak alkaline process conditions (pH 7.5) of this invention are significantly higher. The purity of κ-casein and β-casein is closer to that of the two caseins in breast milk, and α-casein... s2 - The casein content is extremely low, making the casein composition closer to that of breast milk.

[0056] (7) The separation method of the present invention does not use toxic reagents, and compared with the strong alkali process, the degree of dephosphorylation and deamidation reaction during the weak alkali process is extremely low, and the content of the harmful substance lysine generated is also extremely low, which shows the mildness and safety of the weak alkali process. The obtained co-separate is suitable for the production of foods such as infant formula milk powder that have high requirements for raw materials. Attached Figure Description

[0057] Figure 1 is a process flow diagram of the first method for co-separating κ- and β-casein.

[0058] Figure 2 is a process flow diagram of the second method for co-separating κ- and β-casein.

[0059] Figure 3 is a process flow diagram of the third method for co-separating κ- and β-casein.

[0060] Figure 4 shows the reversed-phase high-performance liquid chromatograms of MCC (a) and co-separated κ- and β-casein (b) in Example 1.

[0061] Figure 5 is a reversed-phase high-performance liquid chromatogram of the co-separated κ- and β-casein proteins in Example 14. Detailed Implementation

[0062] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0063] Test method:

[0064] 1. Reversed-phase high-performance liquid chromatography analysis

[0065] Casein composition was determined using an e2695 high-performance liquid chromatograph (Waters Corp., Milford, MA, USA) with an XBridge BEH C18 column (250 mm × 4.6 mm) and a detection wavelength of 220 nm. Empower software was used to integrate each chromatographic peak.

[0066] 2. Definitions of extraction rate, purity, precipitation rate, and yield of κ- and β-casein.

[0067] Extraction rate: The ratio of the peak area of ​​κ-casein or β-casein in the supernatant enriched with κ- and β-casein to that in the initial MCC reconstitution solution;

[0068] Purity: The ratio of the peak area of ​​κ-casein or β-casein to the total casein in a precipitate or supernatant enriched with κ- and β-casein.

[0069] Precipitation rate: 1 minus the proportion of κ-casein or β-casein without precipitation, and the proportion of κ-casein or β-casein without precipitation = the ratio of the peak area of ​​κ-casein or β-casein in the supernatant lacking κ- and β-casein to that in the supernatant enriched with κ- and β-casein.

[0070] Yield: The product of extraction rate and precipitation rate.

[0071] 3. Analysis of phosphate group removal, amide group removal, and lysine content.

[0072] Before centrifugation to separate κ- and β-casein, the solution was mixed with 30% trichloroacetic acid solution, and the supernatant was collected. The phosphate content was determined using the phosphomolybdic acid colorimetric method, and the free ammonia content was determined using the phenol-sodium hypochlorite colorimetric method. Casein was hydrolyzed with 6M HCl, and the lysine content was determined using liquid chromatography-mass spectrometry (LC-MS / MS).

[0073] Unless otherwise specified, the percentages (%) in the examples refer to mass percentages; and unless otherwise specified, the solvent used in the solutions is water.

[0074] Example 1: Co-segregation of κ- and β-casein in MCC - Method 1

[0075] A method for co-separating κ- and β-casein includes the following steps:

[0076] (1) MCC powder was redissolved in ultrapure water to obtain a casein redissolved solution, wherein the mass concentration of casein was 3%;

[0077] (2) Add 2M NaOH solution to the casein reconstituted solution at 25℃ until the pH reaches 11.0;

[0078] (3) Add 2M calcium chloride solution until the concentration of added calcium ions is 35mM, cool to 20℃ and stir (300rpm) for 60min to obtain a solution after casein and calcium complexation.

[0079] (4) After cooling to 4℃, stir at low temperature (300 rpm) for 10 min;

[0080] (5) Add 4M acetic acid solution to pH 5.0 for acid precipitation, and equilibrate at 4°C with stirring (300 rpm) for 8 hours;

[0081] (6) Centrifuge at 4℃ and 3000g for 10 min to obtain α-enriched product. s -Casein precipitation, and supernatant enriched with κ- and β-casein;

[0082] (7) Take the supernatant enriched with κ- and β-casein, add 2M hydrochloric acid solution to pH 3.6 at 25℃, heat to 50℃ and stir (300rpm) for equilibration for 20min, cool to 25℃ and centrifuge at 10000g for 10min to obtain the precipitate enriched with κ- and β-casein and the supernatant depleted with κ- and β-casein.

[0083] Comparative Example 1: Two-step pH-lowering method for extracting κ- and β-casein

[0084] Two-step pH-lowering extraction of κ- and β-casein includes the following steps:

[0085] (1) MCC powder was redissolved in ultrapure water to obtain a casein redissolved solution, wherein the mass concentration of casein was 3%;

[0086] (2) Add 2M NaOH solution to the casein reconstituted solution at 25℃ until the pH reaches 11.0;

[0087] (3) Add 2M calcium chloride solution at 25℃ until the calcium ion concentration is 35mM, stir (300rpm) for 60min to obtain a solution after casein and calcium complexation.

[0088] (4) Add 2M acetic acid solution to adjust the pH to 7.0, and stir (300 rpm) at 25°C for 60 min to equilibrate;

[0089] (5) After being treated at 4℃ with low-temperature stirring (300 rpm) for 2 h, the pH was adjusted to 5.0 with 2M acetic acid solution, and then stirred again at 4℃ (300 rpm) for equilibration for 12 h.

[0090] Steps (6) and (7) are the same as steps (6) and (7) in Example 1.

[0091] The yields and purities of κ-casein and β-casein obtained in step (7) of Example 1 and Comparative Example 1 are shown in Table 1. As can be seen from Table 1, the method in Comparative Example 1 has a significant impact on the yields of the two caseins during the co-separation of κ- and β-casein. The yield of β-casein in Comparative Example 1 is lower than that in Example 1, and the yield of κ-casein in Comparative Example 1 is significantly lower than that in Example 1. Furthermore, the process flow in Comparative Example 1 is relatively cumbersome. Therefore, the scheme of Example 1 is preferred for the co-separation of κ- and β-casein.

[0092] Table 1. Results of co-separation of κ- and β-casein in Example 1 and Comparative Example 1

[0093] Example 2: Co-separation Method 1: The Influence of Different Low Temperature Starting Positions

[0094] Control of different low-temperature initiation positions:

[0095] (1) Before casein micelle dissociation:

[0096] Adjust step (4) in Example 1 to step (2), and adjust the temperature in subsequent steps to 4°C. Keep everything else the same as in Example 1.

[0097] (2) Before casein complexes with calcium:

[0098] Adjust steps (4) to (3) in Example 1, and adjust the temperature in subsequent steps to 4°C, while keeping everything else the same as in Example 1.

[0099] (3) Casein complexes with calcium:

[0100] In Example 1, after adjusting the addition of calcium chloride to step (3) to step (4) to the concentration of added calcium ions to 35mM, the subsequent treatment was to cool down to 4°C and then stir (300rpm) for 60min to equilibrate. The temperature in subsequent steps was also adjusted to 4°C. Everything else remained the same as in Example 1.

[0101] (4)α s -Before casein precipitation:

[0102] Consistent with Example 1.

[0103] (5)α s - After casein precipitation:

[0104] After adjusting steps (4) to (5) in Example 1 to add 4M acetic acid solution to pH 5.0 for acid precipitation, the rest remains the same as in Example 1.

[0105] In the low-temperature initiation position control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 2. Table 2 shows that as the initiation position of the low-temperature step shifts later, the extraction rates of both κ-casein and β-casein decrease. Specifically, when the initiation position of the low-temperature step is at α... s - After casein precipitation, the extraction rates of both types of casein decreased significantly; with the later shift of the low-temperature step initiation position, the purity of both κ-casein and β-casein increased, especially when the low-temperature step initiation position was at α s- After casein precipitation, the purity of the two types of casein showed little change. As the starting point of the low-temperature step shifts further, the binding affinity of casein to calcium increases, which is beneficial for the selective separation of casein. Therefore, the preferred starting point for the low-temperature step is α. s - Before casein precipitation, co-separation of κ- and β-casein is performed.

[0106] Table 2. Co-separation results in the low-temperature initiation position control scheme of Example 2

[0107] Example 3: Co-separation Method 1: Effects of Different Added Calcium Ion Concentrations, Casein Protein Mass Concentration, Equilibrium Temperature, and Equilibrium Time

[0108] (1) Control of different calcium ion concentrations: The calcium ion concentration in step (3) of Example 1 was adjusted to 5, 15, 25, 45, 55 and 65 mM, while the other steps remained the same as in Example 1.

[0109] In the calcium ion concentration control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 3. Table 3 shows that the extraction rates of both κ-casein and β-casein decrease between 5-35 mM calcium ion concentrations, and increase between 35-65 mM. The purity of κ-casein and β-casein increases between 5-35 mM calcium ion concentrations and decreases between 35-65 mM. Therefore, a calcium ion concentration of 35 mM is preferred for co-separation of κ- and β-casein to obtain κ-casein and β-casein with higher purity.

[0110] Table 3. Coseparation results in the calcium ion concentration control scheme of Example 3.

[0111] (2) Regulation of casein mass concentration: The casein mass concentration in step (1) of Example 1 was adjusted to 1, 2, 4, 5, 6, 7%, while the other steps remained the same as in Example 1.

[0112] In the casein mass concentration control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 4. Table 4 shows that as the casein mass concentration increases from 1% to 3%, the extraction rates of both κ-casein and β-casein decrease slightly; as the casein mass concentration further increases to 7%, the extraction rates of both decrease significantly. As the casein mass concentration increases from 1% to 3%, the purities of both κ-casein and β-casein increase slightly; as the casein mass concentration further increases to 7%, the purity of κ-casein further increases, while the purity of β-casein decreases significantly. Higher casein mass concentrations result in higher solution viscosity, making it difficult for added calcium ions and acidifying agents to be evenly dispersed, leading to localized casein aggregation. Higher viscosity also hinders casein precipitation, all of which negatively impact casein separation. The experiment also found that when the casein mass concentration is ≥8%, the solution forms a gel during separation, making subsequent processing impossible. Therefore, a casein concentration of 3% is preferred for the co-separation of κ- and β-casein.

[0113] Table 4. Co-segregation results in the casein mass concentration control scheme of Example 3.

[0114] (3) Adjustment of different equilibrium temperatures: The temperature of the solution after adding calcium chloride in step (3) of Example 1 is adjusted to 10, 15, 25, 30, 35 and 40℃, while the other steps are the same as in Example 1.

[0115] In the equilibrium temperature control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 5. Table 5 shows that the extraction rates of both κ-casein and β-casein decrease slightly between 10-20℃ and significantly between 20-40℃. Between 10-35℃, the purities of both κ-casein and β-casein show an increasing trend; between 15-20℃, the purity of β-casein increases significantly; and between 35-40℃, the purity of both κ-casein and β-casein decreases. Therefore, an equilibrium temperature of 10-25℃ is preferred for the co-separation of κ- and β-casein, with 20℃ being the most preferred.

[0116] Table 5. Co-separation results in different equilibrium temperature control schemes in Example 3.

[0117] (4) Adjustment of different equilibrium times: Adjust the stirring equilibrium time in step (3) of Example 1 to 0, 15, 30, 45, 75 and 90 min, and keep the other steps the same as in Example 1.

[0118] In the equilibration time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 6. Table 6 shows that the extraction rates of both κ-casein and β-casein gradually decreased between 0 and 75 min, and remained almost unchanged between 75 and 90 min. The purity of κ-casein initially increased gradually with time, then stabilized after 60 min; the purity of β-casein showed an increasing trend with time, significantly increasing between 45 and 60 min, and then stabilizing after 60 min. Therefore, an equilibration time of 60-90 min is optimal for the co-separation of κ- and β-casein.

[0119] Table 6 shows the co-separation results in the equilibration time control scheme of Example 3.

[0120] Example 4: Co-separation Method 1: The Influence of Different Low Temperatures and Low Temperature Times

[0121] (1) Control of different low temperatures: Adjust the temperature in step (4) of Example 1 to 0, 2, 8 and 12°C, and keep the other steps the same as in Example 1.

[0122] In the low-temperature control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 7. Table 7 shows that the extraction rates of both κ-casein and β-casein decrease with increasing temperature; the extraction rate of β-casein decreases slightly between 0-4℃, significantly between 4-8℃, and slightly between 8-12℃. The purity of both κ-casein and β-casein increases with increasing temperature; the purity of β-casein remains almost unchanged between 4-12℃. Therefore, good results can be obtained at 0-4℃; among these, 4℃ is the preferred low-temperature temperature for the co-separation of κ- and β-casein.

[0123] Table 7. Co-separation results in the low-temperature control scheme of Example 4.

[0124] (2) Adjustment of different low temperature time: The stirring time in step (4) of Example 1 is adjusted to 0, 5, 20, 30, 40, 50, 60 min, and the other steps are consistent with Example 1.

[0125] In the low-temperature time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 8. Table 8 shows that the extraction rate of κ-casein increased significantly between 0-10 min and remained almost unchanged between 10-60 min; the extraction rate of β-casein increased significantly between 0-10 min, remained almost unchanged between 10-50 min, and decreased between 50-60 min; the purity of κ-casein increased significantly between 0-10 min, decreased slightly between 10-20 min, and remained almost unchanged between 20-60 min; the purity of β-casein increased between 0-10 min, remained almost unchanged between 10-50 min, and increased slightly between 50-60 min. Therefore, good results can be obtained within the range of 10-50 min; among these, a low-temperature time of 10 min is preferred for the co-separation of κ- and β-casein.

[0126] Table 8. Co-separation results in the low-temperature time control scheme of Example 4.

[0127] Example 5: Co-separation Method 1: Effects of Different Acidifying Agents, Acid Precipitation pH, and Equilibrium Time

[0128] (1) Regulation of different acidifying agents: The acetic acid solution in step (5) of Example 1 was adjusted to hydrochloric acid, citric acid and lactic acid solution, while the other steps remained the same as in Example 1.

[0129] In the acidifying agent control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 9. Table 9 shows that the extraction rates of κ-casein and β-casein are ranked as follows: citric acid > lactic acid > acetic acid > hydrochloric acid, and the purity of the two caseins is ranked as follows: hydrochloric acid > acetic acid > lactic acid > citric acid. When acetic acid is used as the acidifying agent, the purity of both κ-casein and β-casein is close to that when hydrochloric acid is used as the acidifying agent, and the extraction rates of both caseins are higher than those when hydrochloric acid is used. Hydrochloric acid is a strong acid; adding a high concentration of hydrochloric acid to the casein solution will significantly lower the local pH, causing casein aggregation and reducing its extraction rate. Acetic acid is a weak acid; its addition can produce more uniform acidification of the casein solution, reducing casein aggregation caused by excessively low local pH. Therefore, acetic acid solution is the optimal acidifying agent for the co-separation of κ- and β-casein.

[0130] Table 9. Co-separation results in the acidifier control scheme of Example 5.

[0131] (2) Adjustment of pH of different acid precipitation: The pH value of acid precipitation in step (5) of Example 1 was adjusted to 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.4 and 5.6, while the other steps were the same as in Example 1.

[0132] In the pH-controlled acid precipitation scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 10. Table 10 shows that with increasing pH, the extraction rates of both κ-casein and β-casein initially increased and then decreased. The extraction rate of κ-casein reached its maximum at pH 4.9, while that of β-casein reached its maximum at pH 5.0. Between pH 4.6 and 4.9, the purities of both κ-casein and β-casein significantly increased. Between pH 4.9 and 5.2, their purities increased slightly. Between pH 5.2 and 5.6, the purity of κ-casein increased slightly, while the purity of β-casein decreased. Therefore, a better effect can be obtained between pH 4.9 and 5.1; among these, a pH of 5.0 is preferred for the co-separation of κ- and β-casein.

[0133] Table 10 shows the co-separation results in the pH adjustment scheme for acid precipitation in Example 5.

[0134] (3) Adjustment of different equilibrium times: The stirring equilibrium time in step (5) of Example 1 is adjusted to 0, 1, 2, 4, 12, 24h, while other steps remain the same as in Example 1.

[0135] In the equilibration time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (6) are shown in Table 11. Table 11 shows that the extraction rates of both κ-casein and β-casein increased significantly between 0 and 8 h, and remained almost constant between 8 and 24 h. The purity of κ-casein gradually decreased between 0 and 4 h, increased between 4 and 8 h, and remained almost constant between 8 and 24 h; the purity of β-casein gradually decreased between 0 and 24 h. Therefore, a better extraction rate and purity can be obtained between 8 and 24 h; an optimal equilibration time of 8 h is recommended for the co-separation of κ- and β-casein.

[0136] Table 11 shows the co-separation results in the equilibration time control scheme of Example 5.

[0137] Example 6: Co-separation Method 1: Effects of Different Precipitation pH, Precipitation Temperature, and Precipitation Time

[0138] (1) Adjustment of pH for different precipitation: The pH value after adding hydrochloric acid solution in step (7) of Example 1 was adjusted to 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, and 5.2, and the subsequent equilibration treatment was adjusted to stirring at 25°C for 30 min. Other steps were the same as in Example 1.

[0139] In the precipitation pH control scheme, the precipitation rates of κ-casein and β-casein obtained in step (7) are shown in Table 12. Table 12 shows that the precipitation rates of both κ-casein and β-casein increase between pH 3.0 and 3.6, and decrease between pH 3.6 and 5.2. Therefore, a pH of 3.4-3.8 is preferred for the precipitation and recovery of κ- and β-casein, with pH 3.6 being the most preferred.

[0140] Table 12 shows the co-separation results in the precipitation pH control scheme of Example 6.

[0141] (2) Control of different precipitation temperatures: The equilibration treatment in step (7) of Example 1 was adjusted to be stirred for 30 min at 15, 25, 35, 45, 50, and 55 °C, while the other steps remained the same as in Example 1.

[0142] In the precipitation temperature control scheme, the precipitation rates of κ-casein and β-casein obtained in step (7) are shown in Table 13. Table 13 shows that the precipitation rate of κ-casein gradually increases between 15-50℃ and remains almost constant between 50-55℃. The precipitation rate of β-casein gradually increases between 15-45℃ and remains almost constant between 45-55℃. Therefore, a temperature of 45-55℃ is preferred for the precipitation and recovery of κ- and β-casein, with 50℃ being the most preferred temperature.

[0143] Table 13 shows the co-separation results in the precipitation temperature control scheme of Example 6.

[0144] (3) Adjustment of different sedimentation times: Adjust the equilibrium time in step (7) of Example 1 to 0, 10, 30, 40, 50 and 60 min, and keep the other steps the same as in Example 1.

[0145] In the precipitation time control scheme, the precipitation rates of κ-casein and β-casein obtained in step (7) are shown in Table 14. Table 14 shows that the precipitation rate of κ-casein increases significantly between 0 and 20 min, and decreases slightly between 20 and 60 min. The precipitation rate of β-casein remains unchanged with increasing time. Therefore, a precipitation time of 20-60 min is preferred for the precipitation and recovery of κ- and β-casein, with 20 min being the most optimal time.

[0146] Table 14 shows the co-separation results in the sedimentation time control scheme of Example 6.

[0147] Example 7: Co-segregation of κ- and β-casein in MCC (Method 2)

[0148] A method for co-separating κ- and β-casein includes the following steps:

[0149] (1) MCC powder was redissolved in ultrapure water to obtain a casein redissolved solution with a casein mass concentration of 3%;

[0150] (2) Add 2M HCl solution to the casein reconstituted solution at 25℃ to pH 4.3, heat to 45℃ and stir (300rpm) for equilibration for 30min, cool to 25℃ and centrifuge at 5000g for 10min to obtain the precipitate;

[0151] (3) Take the precipitate into ultrapure water, add 2M NaOH solution at 25℃ to pH 7.5, stir at 25℃ (300rpm) for 60min to obtain casein reconstituted solution, in which casein mass concentration is 3%;

[0152] (4) Add 2M calcium chloride solution until the concentration of added calcium ions is 45mM, stir at 25℃ (300rpm) for 60min to obtain a solution after casein and calcium complexation.

[0153] (5) Add 4M acetic acid solution to pH 5.0 for acid precipitation, and stir (300 rpm) at 25°C for 90 min to equilibrate;

[0154] (6) After cooling to 2℃, stir at low temperature (300rpm) for 8 hours to equilibrate;

[0155] (7) Centrifuge at 2℃ and 3000g for 10 min to obtain α-enriched product. s -Casein precipitation, and supernatant enriched with κ- and β-casein;

[0156] (8) Take the supernatant enriched with κ- and β-casein, add 2M hydrochloric acid solution to pH 3.6 at 25℃, heat to 50℃ and stir (300rpm) for equilibration for 20min, cool to 25℃ and centrifuge at 10000g for 10min to obtain the precipitate enriched with κ- and β-casein and the supernatant depleted with κ- and β-casein.

[0157] The yields and purities of κ-casein and β-casein obtained in step (8) are shown in Table 15. As can be seen from Table 15, the method of Example 7 can effectively co-separate κ- and β-casein, with both yields and purities being high.

[0158] Table 15 shows the co-separation results of κ- and β-casein in Example 7.

[0159] Example 8: Co-separation Method 2: Effects of Different Reconstitution pH and Equilibration Time

[0160] (1) pH adjustment for different reconstitution: The pH value in step (3) of Example 7 was adjusted to 6.5, 7.0, 8.0, 8.5 and 9.0, while the other steps remained the same as in Example 7.

[0161] In the reconstitution pH control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 16. Table 16 shows that between pH 6.5 and 7.5, the extraction rates of both κ-casein and β-casein significantly increased, while the purity of the former decreased and the purity of the latter increased. Between pH 7.5 and 9.0, the extraction rates of both κ-casein and β-casein gradually decreased, with the purity of the former slightly increasing and the purity of the latter significantly decreasing. At low pH, casein reconstitution is incomplete, resulting in a lower extraction rate. As pH increases, the net negative charge of casein gradually increases, increasing its binding to calcium and thus reducing the extraction rate. Therefore, a reconstitution pH of 7.5 is preferred.

[0162] Table 16 shows the co-separation results in the reconstitution pH adjustment scheme of Example 8.

[0163] (2) Adjustment of different equilibration times: Adjust the equilibration time in step (3) of Example 7 to 0, 15, 30, 45 and 75 min, while keeping other steps consistent with Example 7.

[0164] In the equilibration time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 17. Table 17 shows that the extraction rates of both κ-casein and β-casein gradually increased between 0 and 60 min, and remained almost constant between 60 and 75 min, indicating that extending the extraction time promotes the resolution of acid-precipitated casein, thereby increasing the casein extraction rate. Between 0 and 60 min, the purity of κ-casein decreased, while the purity of β-casein increased. Between 60 and 75 min, the purity of both remained almost constant. Therefore, a good effect can be obtained between 60 and 75 min; among these, a equilibration time of 60 min is preferred for the co-separation of κ- and β-casein.

[0165] Table 17 shows the co-separation results in the equilibration time control scheme of Example 8.

[0166] Example 9: Co-separation Method Two: The Influence of Different Low Temperature Starting Positions

[0167] Control of different low-temperature initiation positions:

[0168] (1) After reconstitution:

[0169] The low-temperature treatment in step (6) of Example 7 is adjusted to be after step (3), that is, the casein reconstituted solution is cooled to 2°C, and the temperature in subsequent steps is adjusted to 2°C. Everything else is the same as in Example 7.

[0170] (2) Casein complexes with calcium:

[0171] The low-temperature treatment in step (6) of Example 7 is adjusted to be after step (4), that is, the solution after casein and calcium complexation is cooled to 2°C, and the temperature in subsequent steps is adjusted to 2°C, while the rest remains the same as in Example 7.

[0172] (3)α s - After casein precipitation:

[0173] Consistent with Example 7.

[0174] In the low-temperature initiation position control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 18. Table 18 shows that the extraction rate of κ-casein decreases as the initiation position of the low-temperature step is shifted further; specifically, when the initiation position of the low-temperature step is at α... s - After casein precipitation, the purity of β-casein significantly increases. With a later initiation of the low-temperature step, the binding affinity of casein to calcium increases, leading to enhanced selective precipitation of casein by calcium ions and thus increased purity of the co-separated β-casein. Therefore, the preferred low-temperature initiation position is α... s- After casein precipitation, κ- and β-casein are co-separated.

[0175] Table 18 shows the co-separation results in the low-temperature initiation position control scheme of Example 9.

[0176] Example 10: Co-separation Method Two: Effects of Different Added Calcium Ion Concentrations and Equilibrium Temperature

[0177] (1) Control of different calcium ion concentrations: The calcium ion concentrations added in step (4) of Example 7 were adjusted to 30, 35, 40, 50, 55, 60, 65, and 70 mM, while the other steps remained the same as in Example 7.

[0178] In the calcium ion concentration control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 19. Table 19 shows that between 30-45 mM of calcium ion concentration, the extraction rates of both κ-casein and β-casein increased, while the purity of the former increased and the purity of the latter decreased. Between 45-70 mM of calcium ion concentration, the extraction rate of κ-casein further increased, while the extraction rate of β-casein remained almost unchanged. The purity of κ-casein further increased, while the purity of β-casein significantly decreased. Therefore, a calcium ion concentration of 45 mM is preferred for the co-separation of κ- and β-casein.

[0179] Table 19 shows the co-separation results in the calcium ion concentration control scheme of Example 10.

[0180] (2) Adjustment of different equilibrium temperatures: Adjust the equilibrium temperature in step (4) of Example 7 to 10, 15, 20, 30, 35, 40℃, and keep the other steps the same as in Example 7.

[0181] In the equilibrium temperature control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 20. Table 20 shows that with increasing temperature, the extraction rates of both κ-casein and β-casein decrease, while their purities first increase and then decrease, reaching their maximum values ​​at 25℃. Therefore, an equilibrium temperature of 25℃ is preferred for the co-separation of κ- and β-casein.

[0182] Table 20 shows the co-separation results in the equilibrium temperature control scheme of Example 10.

[0183] Example 11: Co-separation Method Two: Effects of Different Acid Precipitation Equilibrium Times and Low Temperature

[0184] (1) Adjustment of different acid precipitation equilibrium time: The stirring equilibrium time in step (5) of Example 7 was adjusted to 0, 30, 60, 120 and 150 min, while other steps remained the same as in Example 7.

[0185] In the acid precipitation equilibration time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 20. Table 20 shows that the extraction rates and purities of both κ-casein and β-casein gradually increased between 0 and 90 min, but showed no significant changes between 90 and 150 min. Therefore, high extraction rates and purities can be obtained between 90 and 150 min; among these, a equilibration time of 90 min is preferred for the co-separation of κ- and β-casein.

[0186] Table 21 shows the co-separation results in the acid precipitation equilibrium time control scheme of Example 11.

[0187] (2) Control of different low temperatures: Adjust the temperature in step (6) of Example 7 to 0, 4, 8, 12 and 24°C, and keep the other steps the same as in Example 7.

[0188] In the low-temperature control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 21. Table 21 shows that the extraction rates of both κ-casein and β-casein decrease with increasing temperature. Specifically, the extraction rate of β-casein decreases slightly between 0-2℃ and significantly between 2-12℃. The purities of both κ-casein and β-casein increase slightly with increasing temperature. Therefore, good results can be obtained at 0-2℃; among these, 2℃ is the preferred low-temperature temperature for the co-separation of κ- and β-casein.

[0189] Table 22 shows the co-separation results in the low-temperature control scheme of Example 11.

[0190] Example 12 Further optimization of co-separation method II of κ- and β-casein in MCC: Effect of different acid precipitation equilibrium temperatures

[0191] The temperature in step (5) of Example 7 was adjusted to 19, 21, 23, 27, 29, and 31°C, while the other steps remained the same as in Example 7.

[0192] In the acid precipitation equilibrium temperature control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 23. Table 23 shows that with increasing temperature, the extraction rates and purities of both κ-casein and β-casein exhibit a trend of first increasing and then decreasing, significantly decreasing at 29℃ and reaching a relatively high value at 23-27℃. Therefore, the optimal acid precipitation equilibrium temperature is 23-27℃, with 27℃ being the most preferred, for the co-separation of κ- and β-casein.

[0193] Table 23 shows the co-separation results in the acid precipitation equilibrium temperature control scheme of Example 12.

[0194] Example 13 Further optimization of co-segregation method II of κ- and β-casein in MCC: the effect of different low-temperature equilibration times

[0195] The time in step (6) of Example 12 is adjusted to 5, 6, 7, 9, 10, 11 hours, and the temperature in step (5) is 27°C. Other steps are kept the same as in Example 12.

[0196] In the low-temperature equilibration time control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (7) are shown in Table 23. Table 23 shows that the extraction rates and purities of both κ-casein and β-casein increase with time, showing a significant increase between 5-7 h, a slight increase between 7-9 h, and remaining unchanged between 9-11 h. Therefore, a low-temperature equilibration time of 7-9 h, with 9 h being the most preferred, is optimal for the co-separation of κ- and β-casein.

[0197] Table 23 shows the co-separation results in the low-temperature equilibrium time control scheme of Example 13.

[0198] Example 14 Further optimization of co-segregation method II of κ- and β-casein in MCC: Effect of different casein mass concentrations

[0199] The casein protein concentration in step (1) of Example 13 was adjusted to 2, 4, 5, 6, and 7%, and the time in step (6) was 9 hours. Other steps remained the same as in Example 13.

[0200] In the casein mass concentration control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (1) are shown in Table 24. Table 24 shows that with increasing casein mass concentration, the extraction rates and purities of both κ-casein and β-casein exhibit a trend of first increasing and then decreasing, reaching a relatively high value in the 2-4% range. Therefore, a casein mass concentration of 2-4%, and most preferably 4%, is optimal for the co-separation of κ- and β-casein.

[0201] Table 24 shows the co-segregation results in the casein mass concentration control scheme of Example 14.

[0202] Comparative Example 2: Extraction of κ- and β-casein under strongly alkaline conditions

[0203] Extraction of κ- and β-casein under strongly alkaline conditions includes the following steps:

[0204] The casein protein concentration in step (1) of Example 14 is selected as 4%, and the pH in step (3) of Example 14 is adjusted to 11. Other steps are consistent with those in Example 14.

[0205] The yields and purities of κ-casein and β-casein obtained in step (8) of Example 14 and Comparative Example 2 are shown in Table 25. As can be seen from Table 23, compared to the strong alkali process in Comparative Example 2, the yields and purities of κ-casein and β-casein obtained by the weak alkali process in Example 14 are significantly higher, and the purity of κ-casein and β-casein is closer to that of the two caseins in breast milk. s2 - The casein content is extremely low, indicating that the casein composition extracted with weak alkali is closer to that of breast milk. Furthermore, compared to the strong alkali process, the degree of dephosphorylation and deamidation reactions during the weak alkali process is extremely low, and the content of the harmful substance lysine generated is also extremely low, demonstrating the mildness and safety of the weak alkali process.

[0206] Table 25 shows the co-separation results of κ- and β-casein in Example 14 and Comparative Example 2.

[0207] Example 15: Co-segregation of κ- and β-casein in MCC (Method 3)

[0208] A method for co-separating κ- and β-casein includes the following steps:

[0209] (1) MCC powder was redissolved in ultrapure water to obtain a casein redissolved solution with a casein mass concentration of 3%;

[0210] (2) Add 4M acetic acid solution to pH 5.0, stir at 25°C (300 rpm) for 90 min to equilibrate, and then stir at 2°C (300 rpm) for 8 h to equilibrate.

[0211] (3) Centrifuge at 2℃ and 3000g for 10 min to obtain α-enriched products. s -Casein precipitation, and supernatant enriched with κ- and β-casein;

[0212] (4) Take the supernatant enriched with κ- and β-casein, add 2M hydrochloric acid solution to pH 3.6 at 25℃, heat to 50℃ and stir for equilibration for 20 min, cool to 25℃ and centrifuge at 10000g for 10 min to obtain the precipitate enriched with κ- and β-casein and the supernatant depleted with κ- and β-casein.

[0213] The yields and purities of κ-casein and β-casein obtained in step (4) are shown in Table 26. As can be seen from Table 26, the method of Example 12 can effectively co-separate κ- and β-casein.

[0214] Table 26 shows the co-separation results of κ- and β-casein in Example 15.

[0215] Example 16: Co-separation Method 3: The Effect of Different Acidifying Agents

[0216] Regulation of different acidifiers:

[0217] The acetic acid solution in step (2) of Example 15 was changed to citric acid solution and hydrochloric acid solution, while the other steps remained the same as in Example 15.

[0218] In the acidifying agent control scheme, the extraction rates and purities of κ-casein and β-casein obtained in step (3) are shown in Table 27.

[0219] Table 27 shows the co-separation results in the acidifier control scheme of Example 16.

[0220] As shown in Table 27, the extraction rates of both κ-casein and β-casein are ranked as follows: acetic acid > hydrochloric acid > citric acid. Similarly, the purity of both types of casein is ranked as follows: acetic acid > hydrochloric acid > citric acid. Therefore, acetic acid solution is the optimal acidifying agent for the co-separation of κ- and β-casein.

Claims

1. A method for co-separating κ- and β-casein from casein micelles, characterized in that, Includes the following steps: (1) Co-separation: Method 1: Adjust the pH of a 1-7% casein solution to 11.0 at 23-27℃; then add calcium chloride solution to make the calcium ion concentration 5-65M; equilibrate at 10-40℃ for 0-90 min to obtain a solution complexed with calcium; then treat the casein-calcium complexed solution at 0-12℃ for 0-60 min, adjust the pH to 4.6-5.6 for acid precipitation, and equilibrate at 0-12℃ for 0-24 h. Centrifugation yields α-enriched products. s Precipitation of casein, and supernatant enriched with κ- and β-casein; Method 2: Adjust the pH of a 2-4% casein solution to 4.3 at 23-27℃, raise the temperature to 40-50℃, and equilibrate for 20-40 minutes; then cool to 23-27℃ and centrifuge; collect the precipitate in ultrapure water, adjust the pH to 6.5-9.0 at 23-27℃, and equilibrate at 23-27℃ for 0-75 minutes to obtain a casein reconstituted solution with a casein mass concentration of 2-4%; add calcium chloride solution to the casein reconstituted solution until the added calcium ion concentration is 30-70 mM, and equilibrate at 23-27℃ for 50-70 minutes; adjust the pH to 5.0 for acid precipitation, equilibrate at 23-27℃ for 0-150 minutes, and then treat at 0-12℃ for 7-9 hours. Centrifugation yields α-enriched products. s Precipitation of casein, and supernatant enriched with κ- and β-casein; Method 3: Adjust the pH of a 2-4% casein solution to 5.0 for acid precipitation, equilibrate at 23-27℃ for 80-100 min, then at 1-4℃ for 7-9 h, and centrifuge to obtain a solution enriched with α-protein. s -Casein precipitation, and supernatant enriched with κ- and β-casein; (2) Take the supernatant enriched with κ- and β-casein, adjust the pH to 3.0-5.2, precipitate at 15-55℃ for 0-60 min, centrifuge to obtain the precipitate enriched with κ- and β-casein.

2. The method according to claim 1, characterized in that, In step (1), after adding calcium chloride in co-separation method one, the equilibrium temperature is 10-25℃ and the time is 60-90min.

3. The method according to claim 1, characterized in that, In step (1), the equilibration time after acid precipitation in co-separation method one is 8-24 hours.

4. The method according to claim 1, characterized in that, In step (1), the mass concentration of the casein solution in co-separation method one is 2-4%.

5. The method according to claim 1, characterized in that, In step (1), the pH of the co-separation method 1 is 4.6-5.6, which is adjusted by using 4M acetic acid solution, 4M hydrochloric acid solution, 4M lactic acid solution or 4M citric acid solution.

6. The method according to claim 1, characterized in that, In step (1), the pH of the casein reconstitution solution in co-separation method two is 7.

5.

7. The method according to claim 1, characterized in that, In step (1), the equilibration time of the casein reconstituted solution in co-separation method two is more than 60 minutes.

8. The method according to claim 1, characterized in that, In step (1), the pH of the co-separation method 2 is adjusted to 5.0 using a 4M acetic acid solution.

9. The method according to claim 1, characterized in that, In step (2), the pH value is 3.4-3.

8.

10. The method according to claim 1, characterized in that, In step (2), the precipitation temperature is 45-55℃ and the precipitation time is more than 20 minutes.

11. The method according to claim 1, characterized in that, In step (1), the temperature of the low-temperature treatment in the co-separation method two is 0-2℃.

12. The method according to claim 1, characterized in that, In step (1), the equilibration time after acid precipitation in co-separation method two is more than 90 minutes.

13. The method according to any one of claims 1-12 is used to prepare κ- and β-casein.

14. The method according to any one of claims 1-12 or the use of the κ- and β-casein according to claim 13 in food.

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