A method for co-separating kappa- and beta-casein

Abstract

The present invention discloses a method for co-separating κ- and β-caseins, belonging to the technical field of dairy product processing. The method uses a micellar casein concentrate (MCC) solution as a raw material, and through fine regulation and combination of key process parameters in steps such as controlling the starting position of the low-temperature step, casein-calcium complexation, low temperature, α s -casein precipitation, κ- and β-casein precipitation, etc., realizes the co-separation of κ- and β-caseins by selective calcium precipitation under alkaline or weakly alkaline environmental conditions. The method of the present invention shortens the separation cycle, simplifies the process flow, and improves production efficiency.

Description

Technical Field

[0001] The invention relates to a method for co-separating kappa- and beta-casein, and belongs to the technical field of dairy product processing. Background Art

[0002] Bovine milk casein is one of the main proteins in infant formula. Its composition is different from breast milk casein, making infant formula difficult to digest and absorb. Bovine milk casein includes α s1 -、α s2 -, κ-, β-casein, α s1 -, β-casein; breast milk casein includes α s1 -, κ-, β-casein, with β- and κ-casein being the main components. Therefore, in order to simulate the composition of breast milk casein, bovine milk casein needs to be separated.

[0003] At present, the separation of casein mainly focuses on single casein, which has problems such as complex separation process, long separation cycle, use of toxic reagents, low yield and purity, making it difficult to achieve industrial scale-up production, and the isolate is not suitable for the production of infant formula milk powder. For example, CN107840883B uses bovine milk casein as raw material, adds urea and β-mercaptoethanol for dissolution, and then performs ion exchange to achieve the separation of three single caseins; CN113461795A uses ethanol dissolution, acid precipitation and other methods to achieve the separation of three single caseins; CN112931616A uses low-temperature membrane filtration method to achieve the co-separation of β-casein and whey protein; CN116355073A uses calcium precipitation method to achieve the separation of κ-casein.

[0004] κ- and β-casein are the two main caseins in breast milk. The one-step method can be used to separate κ- and β-casein from bovine milk casein, which can directly simulate the composition of breast milk casein, shorten the separation process route and separation cycle, and reduce α s -The content of casein, the main allergenic protein. However, κ-casein is a glycosylated protein with strong hydrophilicity and low phosphorylation; β-casein is highly hydrophobic and has a high degree of phosphorylation; α s -Casein has moderate hydrophilicity and high phosphorylation degree; it can be seen that κ-casein, β-casein, α s -The properties of caseins vary greatly, and the ranking of the three in different properties is also different, making the co-separation of κ- and β-caseins a difficult problem in the field, that is, it is necessary to take into account the high extraction rates of κ-casein and β-casein with greatly different properties at the same time. The design of the separation scheme and the selection of conditions are different from those of any single casein, and the requirements for co-separation are higher and more difficult. No relevant reports have been seen so far. 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, which limits its industrial production and application.

[0007] κ-casein, β-casein, α s -The hydrophilicity, hydrophobicity, phosphorylation and other properties of casein are very different, making the co-separation of κ- and β-casein a difficult problem in the field.

[0008] [Technical solution]

[0009] In order to solve the above problems, the present invention uses milk concentrated casein micelle (MCC) solution as raw material, and controls the starting position of the low temperature step, the complexation of casein and calcium, the low temperature, α s -The method of the present invention shortens the separation cycle, simplifies the process flow, and improves production efficiency.

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

[0011] (1) Co-separation:

[0012] Method 1: At 23-27°C, adjust the pH of a casein solution with a mass concentration of 1-7% to 11.0; then add a calcium chloride solution so that the added calcium ion concentration is 5-65mM; balance at 10-40°C for 0-90min to obtain a solution after casein and calcium complexation; then treat the solution after casein and calcium complexation at 0-12°C for 0-60min, adjust the pH to 4.6-5.6 for acid precipitation, and balance at 0-12°C for 0-24h; centrifuge to obtain a solution enriched with α s - precipitation of caseins and supernatant enriched with kappa- and beta-caseins;

[0013] Method 2: At 23-27°C, adjust the pH of a casein solution with a mass concentration of 2-4% to 4.3, heat it to 40-50°C, and balance it for 20-40 minutes; then cool it to 23-27°C and centrifuge it; take the precipitate and add it to ultrapure water, adjust the pH to 6.5-9.0 at 23-27°C, and balance it at 23-27°C for 0-75 minutes to obtain a casein reconstitution solution, wherein the casein mass concentration is 2-4%; add calcium chloride solution to the casein solution until the added calcium ion concentration is 30-70mM, and balance it at 23-27°C for 50-70 minutes; adjust the pH to 5.0 for acid precipitation, balance it at 23-27°C for 0-150 minutes, and then treat it at a low temperature of 0-12°C for 7-9 hours; centrifuge it to obtain a solution enriched with α s precipitation of caseins, and enrichment of the supernatant with κ- and β-caseins;

[0014] Method 3: Adjust the pH of the casein solution with a mass concentration of 2-4% to 5.0 for acid precipitation, balance at 23-27°C for 80-100 minutes, balance at 1-4°C for 7-9 hours, centrifuge, and obtain the enriched α s - precipitation of caseins and supernatant enriched with kappa- and beta-caseins;

[0015] (2) taking the supernatant rich in κ- and β-casein, adjusting the pH to 3.0-5.2, precipitating at 15-55° C. for 0-60 min, and centrifuging to obtain a precipitate rich in 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 1 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 1 is obtained by re-dissolving MCC powder in water or by diluting MCC after re-dissolving it.

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

[0019] In one embodiment of the present invention, the concentration of calcium ions added in step (1) co-separation method 1 is 35 mM.

[0020] In one embodiment of the present invention, the equilibrium temperature after adding calcium chloride in step (1) co-separation method 1 is 10-25°C, further, the temperature is 20°C; the equilibrium time is more than 60 minutes; preferably, the time is 60-90 minutes; further preferably, the time is 60-75 minutes.

[0021] In one embodiment of the present invention, the temperature of the low temperature treatment in the co-separation method 1 of step (1) is 0-4°C, and the time is more than 10 minutes; preferably, the time is 10-50 minutes; more preferably, the time is 10 minutes.

[0022] In one embodiment of the present invention, the pH of 4.6-5.6 in the co-separation method 1 of step (1) is adjusted by using 4M acetic acid solution, 4M hydrochloric acid solution, 4M lactic acid solution or 4M citric acid solution.

[0023] In one embodiment of the present invention, the pH of the acid precipitation in the co-separation method 1 of step (1) is 4.9-5.1.

[0024] In one embodiment of the present invention, the equilibrium time after acid precipitation in the first co-separation method of step (1) is 8-24 hours.

[0025] In one embodiment of the present invention, the centrifugation in the co-separation method 1 of step (1) is performed at 3.5-4.5°C and 2000-10000 g for 5-15 min.

[0026] In one embodiment of the present invention, the balance in the first co-separation method of step (1) is a stirring balance, and the stirring speed is 200-500 rpm.

[0027] In one embodiment of the present invention, the casein solution in the second co-separation method in step (1) is obtained by re-dissolving MCC powder in water or by diluting MCC after re-dissolving it.

[0028] In one embodiment of the present invention, the pH of 4.3 in the second co-separation method of step (1) is adjusted using a 2M HCl solution.

[0029] In one embodiment of the present invention, the centrifugation in the second co-separation method of step (1) is performed at 3.5-4.5°C and 2000-10000 g for 5-15 min.

[0030] In one embodiment of the present invention, the pH value of 6.5-9.0 in the second co-separation method of step (1) is adjusted by using 2M NaOH solution.

[0031] In one embodiment of the present invention, the pH of the casein reconstitution solution in the second co-separation method in step (1) is 7.5.

[0032] In one embodiment of the present invention, the equilibrium time of the casein reconstitution solution in the second co-separation method in step (1) is more than 60 minutes; preferably, the time is 60-75 minutes.

[0033] In one embodiment of the present invention, the pH value of 5.0 in the second co-separation method of step (1) is adjusted by using a 4M acetic acid solution.

[0034] In one embodiment of the present invention, the equilibrium temperature after acid precipitation in the second co-separation method in step (1) is 23-25°C. In one embodiment of the present invention, the equilibrium time after acid precipitation in the second co-separation method in step (1) is more than 90 minutes; preferably, the time is 90-150 minutes; further preferably, the time is 90-120 minutes.

[0035] In one embodiment of the present invention, the temperature of the low temperature treatment in step (1) co-separation method 2 is 0-2°C.

[0036] In one embodiment of the present invention, the centrifugation in the second co-separation method of step (1) is performed at 3.5-4.5°C and 2000-10000 g for 5-15 min.

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

[0038] In one embodiment of the present invention, the casein solution in the third co-separation method in step (1) is obtained by re-dissolving MCC powder in water or by diluting MCC after re-dissolving it.

[0039] In one embodiment of the present invention, the pH of 5.0 in the co-separation method three of step (1) is adjusted by using 4M acetic acid solution, 4M lactic acid solution or 4M citric acid solution.

[0040] In one embodiment of the present invention, the balance in the co-separation mode 3 of step (1) is a stirring balance, and the stirring speed is 200-500 rpm.

[0041] In one embodiment of the present invention, the centrifugation in the co-separation method three of step (1) is performed at 3.5-4.5° C. and 2000-10000 g for 5-15 min.

[0042] 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.

[0043] 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.

[0044] 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 more than 20 min; preferably, the precipitation time is 20-60 min; further preferably, the precipitation time is 20 min.

[0045] In one embodiment of the present invention, the MCC used in preparing the casein solution in step (1) is casein micellar concentrate, or concentrated micellar casein derived from animal milk;

[0046] Among them, MCC comes from animal milk such as cow’s milk;

[0047] MCC can be purchased from the market; or prepared by membrane filtration separation; or obtained by diluting the MCC retentate with water;

[0048] In one embodiment of the present invention, the preparation method of MCC is as follows:

[0049] The skim milk was sterilized with a 1.4 μm pore size ceramic membrane, and the whey protein was removed with a 100 nm pore size ceramic membrane. The volume concentration factor was 4, and the filter was washed with water for 3 times. The retentate was collected and freeze-dried to obtain micellar casein (MCC) powder.

[0050] The second object of the present invention is the kappa- and beta-caseins prepared by the method according to the present invention.

[0051] A third object of the present invention is the method or the use of kappa- and beta-caseins according to the present invention in food.

[0052] In one embodiment of the present invention, the application is for infant food, in particular for preparing infant formula milk powder.

[0053] [Beneficial Effects]

[0054] (1) The first method for co-separating κ- and β-casein of the present invention uses casein micelle powder prepared by membrane filtration as raw material, and controls different low temperature starting positions, casein complexation with calcium, low temperature, α s -Fine regulation and combination of key process parameters of major steps such as casein precipitation; further, by controlling process parameters such as κ- and β-casein precipitation, the yield and purity of κ-casein can reach 82.1% and 15.3%, and the yield and purity of β-casein can reach 96.1% and 73.8%.

[0055] (2) The second method for co-separation of κ- and β-casein described in the present invention uses casein micelle powder as raw material, and controls different low temperature starting positions, weak alkaline re-dissolution conditions, casein and calcium complexation, low temperature, α s-Fine control and combination of key process parameters of main steps such as casein precipitation; further, through κ- and β-casein precipitation, κ-casein with a yield of 73.5% and a purity of 15.2% and β-casein with a yield of 97.7% and a purity of 67.7% were obtained.

[0056] (3) The third method for co-separation of κ- and β-casein described in the present invention uses casein micelle powder as raw material by controlling α s -Control and combination of key process parameters of main steps such as casein precipitation and low temperature; further, through precipitation of κ- and β-casein, κ-casein with a yield of 43.9% and a purity of 14.0% and β-casein with a yield of 76.5% and a purity of 81.4% were obtained.

[0057] (4) The present invention provides a method for co-separating κ- and β-casein with a simple separation process and a short separation cycle. Under the combination of process parameters in each step, the process flow is shortened, simple and efficient, and products with high purity and yield can be co-separated under mild conditions. The method is suitable for large-scale production and has practical industrial application value.

[0058] (5) The separation method of the present invention does not have any food safety issues, and the co-extracted ingredients are closer to breast milk in terms of casein composition, and are expected to replace casein in formula milk powder and be used in formula milk products as an ingredient with a protein composition closer to breast milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the process flow chart of the first method for co-separation of κ- and β-casein.

[0060] Figure 2 This is the process flow chart of the second method for co-separation of κ- and β-casein.

[0061] Figure 3 This is the process flow chart of the third method for co-separation of κ- and β-casein.

[0062] Figure 4 This is the reverse phase high performance liquid chromatography detection spectrum of κ- and β-casein extracted in Example 1. DETAILED DESCRIPTION

[0063] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0064] Test method:

[0065] 1. Reversed-Phase High Performance Liquid Chromatography Analysis

[0066] The 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. Each chromatographic peak was integrated using Empower software.

[0067] 2. Definition of extraction rate, purity, precipitation rate and yield of κ- and β-casein

[0068] Extraction rate: the peak area ratio of κ-casein or β-casein in the supernatant enriched with κ- and β-casein to that in the MCC reconstituted solution at the corresponding protein content;

[0069] Purity: The peak area ratio of κ-casein or β-casein to total casein in the precipitate or supernatant enriched with κ- and β-casein;

[0070] Precipitation rate: 1 minus the percentage of κ-casein or β-casein without precipitation, while the percentage of κ-casein or β-casein without precipitation = the peak area ratio 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;

[0071] Yield: The product of extraction rate and precipitation rate.

[0072] The % involved in the examples without specific meaning is mass percentage; the solution involved without specific solvent is water.

[0073] Example 1 Co-isolation of κ- and β-casein in MCC

[0074] A method for co-separating kappa- and beta-casein comprises the following steps:

[0075] (1) taking MCC powder and re-dissolving it in ultrapure water to obtain a casein reconstituted solution, wherein the mass concentration of casein is 3%;

[0076] (2) Add 2 M NaOH solution to the casein reconstituted solution at 25°C to a pH of 11.0;

[0077] (3) adding 2M calcium chloride solution to a concentration of 35 mM calcium ions, cooling to 20°C and stirring (300 rpm) for 60 min to obtain a solution of casein and calcium complex;

[0078] (4) Cooling to 4°C and stirring at low temperature (300 rpm) for 10 min;

[0079] (5) Add 4 M acetic acid solution to pH 5.0 for acid precipitation, and stir (300 rpm) at 4 °C for 8 h;

[0080] (6) Centrifuge at 4°C and 3000 g for 10 min to obtain the enriched s - precipitation of caseins and supernatant enriched with kappa- and beta-caseins;

[0081] (7) Take the supernatant enriched with κ- and β-casein, add 2 M hydrochloric acid solution to pH 3.6 at 25°C, heat to 50°C and stir (300 rpm) to precipitate for 20 min, cool to 25°C and centrifuge at 10,000 g for 10 min to obtain a precipitate enriched with κ- and β-casein and a supernatant poor in κ- and β-casein.

[0082] Comparative Example 1 Extraction of κ- and β-casein by two-step pH adjustment method

[0083] The two-step pH adjustment method for extracting κ- and β-casein includes the following steps:

[0084] (1) taking MCC powder and re-dissolving it in ultrapure water to obtain a casein reconstituted solution, wherein the mass concentration of casein is 3%;

[0085] (2) Add 2 M NaOH solution to the casein reconstituted solution at 25°C to a pH of 11.0;

[0086] (3) adding 2 M calcium chloride solution at 25° C. to a calcium ion concentration of 35 mM, stirring (300 rpm) for 60 min to obtain a solution of casein and calcium complex;

[0087] (4) Add 2 M acetic acid solution to adjust the pH to 7.0 and equilibrate at 25 °C with stirring (300 rpm) for 60 min;

[0088] (5) After treatment at 4°C with low temperature stirring (300 rpm) for 2 h, the pH was adjusted to 5.0 with 2 M acetic acid solution, and then equilibrated at 4°C with stirring (300 rpm) for 12 h;

[0089] Steps (6) and (7) are consistent with those in Example 1.

[0090] The κ- and β-casein precipitates obtained in Example 1 and Comparative Example 1 were subjected to liquid phase analysis. The results are shown in Table 1.

[0091] As can be seen from Table 1, the treatment of Comparative Example 1 has a great influence on the yield of two caseins during the co-separation of κ- and β-casein. The yield of β-casein obtained in Comparative Example 1 is slightly lower than that of β-casein in Example 1, but the yield of κ-casein is significantly lower than that in Example 1, wherein the process flow of Comparative Example 1 is relatively cumbersome. Therefore, the scheme of Example 1 is preferred for co-separation of κ- and β-casein.

[0092] Table 1 Co-separation results of κ- and β-casein in Example 1 and Comparative Example 1

[0093]

[0094] Example 2 Co-separation method 1: Effect of different low temperature starting positions

[0095] Regulation of different low temperature starting positions:

[0096] (1) Before casein micelle dissociation:

[0097] Step (4) in Example 1 was adjusted to be before step (2), and the temperature of the subsequent steps was adjusted to 4° C., and the rest remained the same as in Example 1.

[0098] (2) Before casein complexes with calcium:

[0099] Step (4) in Example 1 was adjusted to be before step (3), and the temperature of the subsequent steps was adjusted to 4° C., and the rest was kept consistent with Example 1.

[0100] (3) After casein complexes with calcium:

[0101] In step (4) of Example 1, after adding calcium chloride in step (3) to a calcium ion concentration of 35 mM, the temperature was rapidly cooled to 4°C and then stirred at low temperature (300 rpm) for 60 min. The temperature of the subsequent steps was adjusted to 4°C. The rest was kept consistent with Example 1.

[0102] (4)α s -Before casein precipitation:

[0103] Keep the same as in Example 1;

[0104] (5)α s -After casein precipitation:

[0105] Step (4) in Example 1 is adjusted to be after step (6), and the rest remains the same as Example 1.

[0106] The other steps remain the same as in Example 1.

[0107] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under different low temperature position control schemes. The results are shown in Table 2.

[0108] It can be seen from Table 2 that as the starting position of the low-temperature step moves backward, the extraction rates of κ-casein and β-casein show a downward trend overall, but in α s After the precipitation of α-casein, the extraction rate of κ-casein and β-casein decreased significantly. However, the purity of κ-casein and β-casein increased significantly. s The purity of β-casein obtained at low temperature before casein precipitation is significantly greater than that obtained after casein is complexed with calcium. It can be seen that as the starting position of the low temperature step moves backward, the binding ability and affinity of casein monomers with calcium weaken, which is not conducive to the complexation of casein monomers with calcium. Therefore, it is preferred to s -Co-isolation of κ- and β-caseins at low temperature before casein precipitation.

[0109] Table 2 Co-separation results of different low temperature position regulation in Example 2

[0110]

[0111] Example 3 Co-separation method 1: Effects of different added calcium ion concentrations, casein mass concentrations, equilibrium temperatures, and equilibrium times

[0112] (1) Regulation of different added calcium ion concentrations: The calcium ion concentration added in step (3) of Example 1 was adjusted to 5, 15, 25, 45, 55, and 65 mM, and the other steps remained consistent with Example 1.

[0113] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) with respect to MCC and the purity of κ-casein and β-casein were analyzed under the scheme of adding calcium ion concentration. The results are shown in Table 3.

[0114] Table 3 Co-separation results of adding calcium ion concentration control in Example 3

[0115]

[0116] As can be seen from Table 3, the extraction rates of κ-casein and β-casein both decreased significantly as the added calcium ion concentration increased from 5mM to 35mM, and increased slightly from 35mM to 65mM. However, the purity of κ-casein increased significantly as the added calcium ion concentration increased from 5mM to 25mM, remained unchanged at 35mM, and then decreased slightly as the added calcium ion concentration increased between 45mM and 65mM; the purity of β-casein increased significantly within the range of 5mM to 35mM added calcium ion concentration, but as the added calcium ion concentration further increased to 65mM, the purity of β-casein gradually decreased. Therefore, it is preferred that the 35mM added calcium ion concentration be used to co-separate κ- and β-casein by complexing casein with calcium, so that κ- and β-casein with higher purity can be obtained.

[0117] (2) Regulation of different casein concentrations: The casein concentrations in step (1) of Example 1 were adjusted to 1, 2, 4, 5, 6, and 7%, and the other steps remained the same as in Example 1.

[0118] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ-casein and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under the casein quality and concentration control scheme. The results are shown in Table 4.

[0119] As can be seen from Table 4, at the optimal calcium ion concentration (35mM), as the casein mass concentration increases from 1% to 3%, the extraction rates of κ-casein and β-casein both decrease slightly, with little change. As the casein mass concentration further increases from 3% to 7%, the extraction rates of both decrease significantly. As the casein mass concentration increases from 1% to 3%, the purity of κ-casein and β-casein increases slightly. As the casein mass concentration continues to increase to 7%, the purity of κ-casein increases significantly, while the purity of β-casein decreases significantly. This is because when the casein mass concentration is low, the homogenization problem caused by the increase in solution viscosity can be avoided, and the increase in viscosity will reduce the efficiency of the co-separation process. The results of the preliminary experiments show that solutions with a casein mass concentration of ≥8% will form a gel, and subsequent co-separation experiments cannot be performed. Therefore, considering the industrial production efficiency, a casein mass concentration of 3% can be preferably used to co-separate κ- and β-casein by casein and calcium complexation.

[0120] Table 4 Co-separation results of casein quality and concentration control in Example 3

[0121]

[0122] (3) Regulation of different equilibrium temperatures: The temperature of casein complexation with calcium after adding calcium chloride in step (3) of Example 1 was adjusted to 10, 15, 25, 30, 35, and 40° C., and the other steps remained the same as in Example 1.

[0123] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) under the equilibrium temperature control scheme and the purity of κ-casein and β-casein were analyzed relative to MCC. The results are shown in Table 5.

[0124] As can be seen from Table 5, as the temperature increases, the extraction rate of κ-casein decreases steadily between 10-35°C, and when the temperature further increases to 40°C, the extraction rate decreases rapidly. The extraction rate of β-casein does not change significantly between 10-20°C, and then begins to gradually decrease between 20-40°C. As the temperature increases from 10°C to 35°C, the purity of κ-casein and β-casein increases with the increase in temperature. At 15-20°C, the purity of β-casein increases rapidly, and when the temperature increases to 40°C, the purity of both decreases again. Therefore, it is preferred that the calcium equilibrium temperature be 10-25°C for the co-separation of κ- and β-casein, and 20°C is most preferred.

[0125] Table 5 Co-separation results of equilibrium temperature control in Example 3

[0126]

[0127] (4) Regulation of different equilibrium times: The time for casein to complex with calcium after adding calcium chloride in step (3) of Example 1 was adjusted to 0, 15, 30, 45, 75, and 90 min, and the other steps remained the same as in Example 1.

[0128] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) under the equilibrium time control scheme and the purity of κ-casein and β-casein were analyzed relative to MCC, and the results are shown in Table 6.

[0129] As can be seen from Table 6, when the equilibrium time was extended from 0 to 75 min, the extraction rates of κ-casein and β-casein gradually decreased, and remained almost unchanged between 75 and 90 min. With the extension of the equilibrium time after calcium addition, the purity of κ-casein first increased and then stabilized after 60 min, while the purity of β-casein generally showed an increasing trend, but when the equilibrium time was 60 min after calcium addition, the purity of β-casein increased significantly and stabilized within 60-90 min. Therefore, it is preferred to balance for 60-90 min to complex casein with calcium for the co-separation of κ- and β-casein.

[0130] Table 6 Co-separation results of equilibrium time regulation in Example 3

[0131]

[0132] Example 4 Co-separation method 1: Effect of different cryogenic temperatures and cryogenic times

[0133] (1) Regulation of different low temperature: the temperature in step (4) of Example 1 was adjusted to be lowered to 0, 2, 8, and 12° C., and the other steps remained the same as in Example 1.

[0134] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under different low-temperature control schemes. The results are shown in Table 7.

[0135] As can be seen from Table 7, as the temperature increases, the extraction rates of κ-casein and β-casein both show a downward trend. When the temperature is in the range of 0-4°C, the extraction rate of β-casein gradually decreases, and its extraction rate decreases significantly from 4°C to 8°C. When the temperature increases again, the extraction rate decreases slightly. The purity of κ-casein and β-casein increases with the increase of cryogenic temperature. When the cryogenic temperature is between 4-12°C, the purity of β-casein remains almost unchanged. Therefore, good results can be obtained at 0-4°C; among them, the cryogenic temperature of 4°C can be preferably used for the co-separation of κ- and β-casein.

[0136] Table 7 Co-separation results of low temperature temperature control in Example 4

[0137]

[0138] (2) Regulation of different low-temperature times: The low-temperature time after cooling in step (4) of Example 1 was adjusted to 0, 5, 20, 30, 40, 50, and 60 min, and the other steps remained the same as in Example 1.

[0139] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under different low temperature time control schemes. The results are shown in Table 8.

[0140] Table 8 Co-separation results of low temperature time regulation in Example 4

[0141]

[0142] As can be seen from Table 8, the extraction rates of κ-casein and β-casein increased significantly as the low temperature time increased from 0 min to 10 min, and then remained almost unchanged between 10-60 min. It can also be seen that with the increase of low temperature time, the purity of κ-casein and β-casein first increased and then gradually decreased. When the low temperature time was 10 min, the purity of κ-casein was the highest, and the purity of β-casein was relatively high. Therefore, a low temperature time of 10-50 min can achieve better results; among them, 10 min can be preferably used as the low temperature time for the co-separation of κ- and β-casein.

[0143] Example 5 Co-separation method 1: Effects of different acidifiers, acid precipitation pH, and equilibrium time

[0144] (1) Regulation of different acidulants: The acetic acid solution in step (5) of Example 1 is adjusted to hydrochloric acid, citric acid, or lactic acid, and the other steps remain the same as in Example 1.

[0145] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under different acidifier control schemes. The results are shown in Table 9.

[0146] As can be seen from Table 9, the extraction rates of κ-casein and β-casein are in the order of citric acid>lactic acid>acetic acid>hydrochloric acid, and the purity of the two casein monomers follows the order of hydrochloric acid>acetic acid>lactic acid>citric acid. Using acetic acid as an acidulant, the purity of κ-casein and β-casein is close to that when hydrochloric acid is used as an acidulant, and the extraction rates of the two casein monomers obtained are higher than those of hydrochloric acid. Hydrochloric acid, as a strong acid with a relatively high concentration in a casein solution, will locally cause a significant decrease in pH, hydrolyze casein, produce precipitation, and reduce the extraction rate. Acetic acid is a weak acid, and after addition, the solution is more evenly acidified and casein aggregation and precipitation caused by local overacidification are reduced. Therefore, it is most preferred to use acetic acid solution as an acidulant for the co-separation of κ- and β-casein.

[0147] Table 9 Co-separation results of acidifier regulation in Example 5

[0148]

[0149] (2) Regulation of different acid precipitation pH values: The acid precipitation pH values ​​in step (4) of Example 1 were adjusted to 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.4, and 5.6, and the other steps remained the same as in Example 1.

[0150] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) relative to MCC and the purity of κ-casein and β-casein were analyzed under different acid precipitation pH control schemes. The results are shown in Table 10.

[0151] Table 10 Co-separation results of acid precipitation pH control in Example 5

[0152]

[0153] As can be seen from Table 10, the extraction rates of κ-casein and β-casein showed an overall trend of first increasing and then decreasing. When the pH value increased from 4.6 to 4.8, the extraction rates of κ-casein and β-casein increased significantly. When the pH value was between 4.8 and 5.0, the extraction rates of the two casein monomers increased slightly. As the pH value further increased to 5.6, the extraction rates of the two decreased significantly. When the acid precipitation pH value was between 4.6 and 4.9, the purity of κ-casein and β-casein increased significantly. When the pH value increased from 4.9 to 5.2, the purity of the two gradually increased. After the pH continued to increase, the purity of the two slowly decreased. Therefore, better results can be obtained when the acid precipitation pH is in the range of 4.9-5.1, and 5.0 can be preferably used as the acid precipitation pH for the co-separation of κ- and β-casein.

[0154] (3) Regulation of different equilibration times: The stirring equilibration time in step (5) of Example 1 was adjusted to 0, 1, 2, 4, 12, and 24 h, and the other steps remained the same as in Example 1.

[0155] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (6) under different equilibrium time control schemes were analyzed. The results are shown in Table 11.

[0156] As can be seen from Table 11, the extraction rates of κ-casein and β-casein increased significantly as the equilibrium time increased from 0h to 8h, and there was no significant change between 8-24h. The purity of κ-casein gradually decreased within 0-4h as the equilibrium time increased, and its purity increased significantly at 8h, and then stabilized within 8-24h, while the purity of β-casein gradually decreased with the extension of time within 0-24h. Therefore, a better extraction rate and purity can be obtained with an equilibrium of 8h; it can be preferred to use an equilibrium time of 8h for the co-separation of κ- and β-casein.

[0157] Table 11 Co-separation results of equilibrium time regulation in Example 5

[0158]

[0159] Example 6 Co-separation method 1: Effect of different precipitation pH, precipitation temperature, and precipitation time

[0160] (1) Regulation of different precipitation pH values: adjust the pH value of step (7) in Example 1 by adding 2 M hydrochloric acid solution to the supernatant rich in κ- and β-casein to 3.0, 3.2, 3.4, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, and 5.2, and stir at 25° C. for 30 min. The other steps remain the same as in Example 1.

[0161] The results are shown in Table 12.

[0162] As can be seen from Table 12, during the precipitation pH value increasing from 3.0 to 3.6, the precipitation rate of κ-casein has been increasing, reaching a maximum at a pH value of 3.6. When the pH value continues to increase, the precipitation rate of κ-casein begins to decline. At the same time, the precipitation rate of β-casein as a whole shows a trend of first increasing and then decreasing, and reaches a peak value at a pH value of 3.6. Therefore, the pH value of 3.4-3.8 can be preferably used for the precipitation recovery of κ- and β-casein, and the most preferred pH value is 3.6.

[0163] Table 12 Co-separation results of pH regulation of precipitation in Example 6

[0164]

[0165] (2) Regulation of different precipitation temperatures: Adjust the step (7) in Example 1 by adding 2 M hydrochloric acid solution to the supernatant rich in κ- and β-caseins to adjust the pH value to 3.6, and then stir at 15, 25, 35, 45, and 55° C. for 30 min. The other steps remain the same as in Example 1.

[0166] The results are shown in Table 13.

[0167] Table 13 Co-separation results of Example 6 precipitation temperature regulation

[0168]

[0169] As can be seen from Table 13, as the temperature increases from 15°C to 50°C, the precipitation rate of κ-casein also increases, but when the temperature is further increased to 50°C, the precipitation rate remains unchanged. At the same time, the precipitation rate of β-casein increases significantly within 15-25°C, and then increases slightly with the increase in temperature. When the temperature reaches 45°C, the precipitation rate remains almost unchanged. Therefore, the precipitation temperature of 45-55°C can be preferably used for the precipitation recovery of κ- and β-casein, and the most preferred temperature is 50°C.

[0170] (3) Regulation of different precipitation times: After adjusting the pH value to 3.6 by adding 2 M hydrochloric acid solution to the supernatant rich in κ- and β-caseins in step (7) of Example 1, the mixture was stirred at 50° C. for 0, 10, 30, 40, 50, and 60 min, respectively. The other steps remained the same as in Example 1.

[0171] The results are shown in Table 14.

[0172] As can be seen from Table 14, as the precipitation time increases, the precipitation rate of κ-casein increases significantly within 0-20 min, reaches a maximum at 20 min, and as the time further increases, the precipitation rate decreases slightly but has almost no significant change. The precipitation time has no effect on the precipitation rate of β-casein. Therefore, the precipitation time of 20-60 min can be preferably used for the precipitation recovery of κ- and β-casein, and the most preferred time is 20 min.

[0173] Table 14 Co-separation results of Example 6 precipitation time regulation

[0174]

[0175] Example 7 Co-isolation of κ- and β-casein in MCC

[0176] A method for co-separating kappa- and beta-casein comprises the following steps:

[0177] (1) taking MCC powder and re-dissolving it in ultrapure water to obtain a casein reconstituted solution, wherein the casein concentration is 3%;

[0178] (2) adding 2 M HCl solution to the casein reconstituted solution at 25°C to a pH of 4.3, heating to 45°C and stirring (300 rpm) for 30 min; cooling to 25°C and centrifuging at 5000 g for 10 min to obtain a precipitate;

[0179] (3) The precipitate was placed in ultrapure water, 2 M NaOH solution was added to pH 7.5 at 25° C., and the mixture was stirred (300 rpm) at 25° C. for 60 min to obtain a casein reconstitution solution, wherein the casein concentration was 3%;

[0180] (4) adding 2M calcium chloride solution to a concentration of 45 mM calcium ions, stirring (300 rpm) at 25° C. for 60 min to obtain a solution of casein and calcium complex;

[0181] (5) Add 4M acetic acid solution to pH 5.0 for acid precipitation.

[0182] (6) Stirring (300 rpm) at 25°C for 90 min; then stirring (300 rpm) at 2°C for 8 h;

[0183] (7) Centrifuge at 2°C and 3000 g for 10 min to obtain the enriched s - precipitation of caseins and supernatant enriched with kappa- and beta-caseins;

[0184] (8) Take the supernatant enriched with κ- and β-casein, add 2 M hydrochloric acid solution to pH 3.6 at 25°C, heat to 50°C and stir (300 rpm) to precipitate for 20 min, cool to 25°C and centrifuge at 10,000 g for 10 min to obtain a precipitate enriched with κ- and β-casein and a supernatant poor in κ- and β-casein.

[0185] The κ- and β-casein precipitates were taken for liquid phase analysis. The results are shown in Table 15.

[0186] It can be seen from Table 15 that the method of Example 7 can effectively separate κ- and β-casein.

[0187] Table 15 Co-separation results of κ- and β-casein in Example 7

[0188]

[0189] Example 8 Co-separation method 2: Effect of different reconstitution pH and equilibrium time

[0190] (1) Different re-dissolution pH control: The pH value of the alkaline solution in step (3) of Example 7 was adjusted to 6.5, 7.0, 8.0, 8.5, and 9.0, and the other steps remained the same as in Example 7.

[0191] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different re-dissolution pH control schemes were analyzed. The results are shown in Table 16.

[0192] As can be seen from Table 16, as the pH increases from 6.5 to 7.5, the extraction rate of κ-casein and β-casein increases significantly, and the purity of κ-casein and β-casein also shows an upward trend. As the pH further increases to 9.0, the extraction rate of κ-casein and β-casein begins to decrease, and the overall purity also decreases slightly. At a low pH, casein is not completely redissolved and the yield is low. As the pH increases, the net negative charge of casein gradually increases. The better the casein dissolves, the easier it is for β-casein to bind to calcium, and the yield and purity begin to decrease. Taking all factors into consideration, the preferred redissolution pH is 7.5.

[0193] Table 16 Co-separation results of pH control in re-dissolution in Example 8

[0194]

[0195] (2) Regulation of different equilibration times: The equilibration time in step (3) of Example 7 was adjusted to 0, 15, 30, 45, and 75 min, and the other steps remained the same as in Example 7.

[0196] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different equilibrium time control schemes were analyzed. The results are shown in Table 17.

[0197] As can be seen from Table 17, when the equilibrium time is extended from 0 to 60 minutes, the extraction rates of κ-casein and β-casein gradually increase, and remain almost unchanged between 60-75 minutes, indicating that the redissolution is incomplete before 60 minutes, and the acid-precipitated casein is basically completely redissolved after 60 minutes of equilibrium. With the extension of the equilibrium time, the purity of κ-casein has a slight downward trend, but the change is not large, while the purity of β-casein generally shows an upward trend. After 60 minutes of equilibrium, the purity of β-casein tends to be stable. Therefore, preferably 60-75 minutes can obtain better results; among them, the stirring equilibrium 60 minutes of redissolution conditions can be preferably used for the subsequent co-separation of κ- and β-casein.

[0198] Table 17 Co-separation results of equilibrium time regulation in Example 8

[0199]

[0200] Example 9 Co-separation method 2: Effect of different low temperature starting positions

[0201] Regulation of different low temperature starting positions:

[0202] (1) After reconstitution:

[0203] After adjusting step (3) in Example 7, the casein reconstitution solution was quickly cooled to 2°C, and the temperature of the subsequent steps was adjusted to 2°C. The rest was the same as in Example 7.

[0204] (2) After casein complexes with calcium:

[0205] After adjusting step (4) in Example 7, the casein reconstitution solution was quickly cooled to 2°C, and the temperature of the subsequent steps was adjusted to 2°C. The rest was the same as in Example 7.

[0206] (3)α s -After casein precipitation:

[0207] The same as Example 7.

[0208] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different low-temperature position control schemes were analyzed. The results are shown in Table 18.

[0209] It can be seen from Table 18 that as the starting position of the low temperature step moves backward, the extraction rates of κ-casein and β-casein show a downward trend overall, with the α s After the α-casein precipitation, the extraction rate of κ-casein and β-casein decreased significantly. However, the purity of κ-casein and β-casein increased significantly. It can be seen that as the starting position of the low temperature step moves backward, the binding ability and affinity of casein monomers with calcium are enhanced, making monomer casein more selective for calcium and obtaining better purity of β-casein. Therefore, it is preferred to s - After casein precipitation at low temperature, co-isolation of κ- and β-casein was performed.

[0210] Table 18 Co-separation results of different low temperature position regulation in Example 9

[0211]

[0212] Example 10 Co-separation method 2: Effect of different added calcium ion concentrations

[0213] Regulation of different calcium ion concentrations: The calcium ion concentration added in step (4) of Example 7 was adjusted to 30, 35, 40, 50, 55, 60, 65, 70 mM. The other steps were the same as in Example 7.

[0214] The extraction rates of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) relative to MCC and the purity of κ-casein and β-casein were analyzed under different calcium ion concentration control schemes. The results are shown in Table 19.

[0215] As can be seen from Table 19, the extraction rates of κ-casein and β-casein increased with the increase of calcium ion concentration from 30mM to 45mM, while the purity decreased. The extraction rates of κ- and β-casein increased significantly from 45mM to 70mM calcium ion concentration. However, the purity of κ-casein increased significantly with the increase of calcium ion concentration, but the purity of β-casein decreased significantly. Therefore, it is preferred to add calcium ion concentration of 45mM to complex casein with calcium for co-separation of κ- and β-casein, so that κ-casein and β-casein with higher purity can be obtained.

[0216] Table 19 Co-separation results of calcium ion concentration regulation in Example 10

[0217]

[0218] Example 11 Co-separation method 2: Effect of different acid precipitation equilibrium time and low temperature

[0219] (1) Regulation of different acid precipitation equilibrium times: The stirring equilibrium in step (5) of Example 7 was adjusted to 0, 30, 60, 120, and 150 min. The other steps were the same as in Example 7.

[0220] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different acid precipitation equilibrium time control schemes were analyzed. The results are shown in Table 20.

[0221] Table 20 Co-separation results of acid precipitation equilibrium time regulation in Example 11

[0222]

[0223] As can be seen from Table 20, the extraction rate and purity of κ-casein and β-casein increased as the equilibrium time increased from 0 min to 90 min. Between 90 and 150 min, the extraction rate and purity of κ-casein and β-casein fluctuated, but there was no significant change. Therefore, a balance of 90-150 min is preferred to obtain a better extraction rate and purity; and a balance of 90 min can be preferably used as the equilibrium time for the co-separation of κ- and β-casein.

[0224] (2) Control of different low temperature: The low temperature in step (5) of Example 7 is adjusted to 0, 4, 8, 12, and 24° C. The other steps are the same as those of Example 7.

[0225] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different low-temperature control schemes were analyzed. The results are shown in Table 21.

[0226] As can be seen from Table 21, as the temperature increases, the extraction rates of κ-casein and β-casein both show a downward trend. When the temperature is in the range of 0-2°C, the extraction rate of β-casein gradually decreases, and the extraction rate of β-casein decreases significantly when the temperature rises from 4°C to 12°C. The purity of both κ-casein and β-casein increases with the increase of cryogenic temperature. When the cryogenic temperature is between 2-12°C, the purity of β-casein increases slightly, but the fluctuation is not large. Therefore, good results can be obtained at 0-2°C; among them, the cryogenic temperature of 2°C can be preferably used for the co-separation of κ- and β-casein.

[0227] Table 21 Co-separation results of low temperature temperature control in Example 11

[0228]

[0229] Example 12 Co-isolation of κ- and β-casein in MCC

[0230] A method for co-separating kappa- and beta-casein comprises the following steps:

[0231] (1) taking MCC powder and re-dissolving it in ultrapure water to obtain a casein reconstituted solution, wherein the casein concentration is 3%;

[0232] (2) Add 4 M acetic acid solution to pH 5.0, stir (300 rpm) at 25 °C for 90 min, and then stir (300 rpm) at 2 °C for 8 h;

[0233] (3) Centrifuge at 2°C and 3000g for 10 min to obtain the enriched s - precipitation of caseins and supernatant enriched with kappa- and beta-caseins;

[0234] (4) Take the supernatant enriched with κ- and β-casein, add 2 M hydrochloric acid solution to pH 3.6 at 25°C, heat to 50°C and stir for 20 minutes, cool to 25°C and centrifuge at 10,000 g for 10 minutes to obtain a precipitate enriched with κ- and β-casein and a supernatant poor in κ- and β-casein.

[0235] The κ- and β-casein precipitates obtained in step (4) were subjected to liquid phase analysis. The results are shown in Table 22.

[0236] It can be seen from Table 22 that the method of Example 12 can effectively separate κ- and β-casein.

[0237] Table 22 Co-separation results of κ- and β-casein in Example 12

[0238]

[0239] Example 13 Co-separation method 3: Effect of different acidifiers

[0240] Regulation of different acidifiers: the acetic acid solution in step (2) of Example 12 is adjusted to citric acid solution or hydrochloric acid solution; the other steps remain the same as Example 12.

[0241] The extraction rates of κ-casein and β-casein relative to MCC and the purity of κ-casein and β-casein in the supernatant enriched with κ- and β-casein corresponding to step (7) under different acidifier control schemes were analyzed. The results are shown in Table 23.

[0242] As can be seen from Table 23, the extraction rates of κ-casein and β-casein are in the order of acetic acid > citric acid, and the purity of the two casein monomers also follows the order of acetic acid > citric acid. Since citric acid has the ability to complex Ca, the concentration of Ca in the citric acid system is reduced, and the acidification causes the rate and purity to be low. Therefore, it is most preferred to use acetic acid solution as an acidifier for the co-separation of κ- and β-casein.

[0243] Table 23 Co-separation results of different acidifiers in Example 13

[0244]

[0245] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for co-separating κ- and β-casein from casein micelles, characterized in that: The steps include: (1) Co-separation: Method 1: At 23-27°C, adjust the pH of a casein solution with a mass concentration of 2-4% to 11.0; then add a calcium chloride solution to make the calcium ion concentration 5-65mM; balance at 10-25°C for 60-90 min to obtain a solution of casein and calcium complex; then treat the solution of casein and calcium complex at 0-12°C for 0-60 min, adjust the pH to 4.9-5.1 for acid precipitation, and balance at 0-12°C for 8-24 h; Centrifugation to obtain the enriched s Precipitation of casein and supernatant enriched with kappa- and beta-casein; wherein the pH of 4.9-5.1 is adjusted by using 4 M acetic acid solution, 4 M lactic acid solution or 4 M citric acid solution; Method 2: At 23-27 °C, adjust the pH of a casein solution with a mass concentration of 2-4% to 4.3, heat it to 40-50 °C, and balance it for 20-40 min; then cool it to 23-27 °C and centrifuge it; take the precipitate and add it to ultrapure water, adjust the pH to 7.5 at 23-27 °C, and balance it at 23-27 °C for 60-75 min to obtain a casein reconstitution solution, in which the casein mass concentration is 2-4%; add calcium chloride solution to the casein reconstitution solution until the added calcium ion concentration is 45-70 mM, and balance it at 23-27 °C for 50-70 min; adjust the pH to 5.0 for acid precipitation, balance it at 23-27 °C for 60-150 min, and then treat it at low temperature at 0-12 °C for 7-9 h; centrifuge it to obtain a solution enriched with α s Precipitation of casein and supernatant enriched with κ- and β-casein; wherein the pH is adjusted to 5.0 by 4 M acetic acid solution; (2) Take the supernatant rich in κ- and β-casein, adjust the pH to 3.4-3.8, precipitate at 45-55 °C for 20-60 min, and centrifuge to obtain a precipitate rich in κ- and β-casein.

2. The method according to claim 1, characterized in that The equilibrium temperature after acid precipitation in step (1) co-separation method 2 is 23-25°C.

3. The method according to claim 1, characterized in that The balance in the co-separation method 1 of step (1) is a stirring balance, and the stirring speed is 200-500 rpm.

4. The method according to claim 1, characterized in that: The mass concentration of the casein solution in the co-separation method 1 of step (1) is 3%.

5. The method according to claim 1, characterized in that In step (1) of the co-separation method 1, the casein solution is obtained by re-dissolving MCC powder in water or by diluting MCC after re-dissolving it.

6. The method according to claim 1, characterized in that In step (1) of the co-separation method 1, the concentration of calcium ions added was 35 mM.

7. The method according to claim 1, characterized in that In step (1) of the second co-separation method, the centrifugation is performed at 3.5-4.5°C and 2000-10000 g for 5-15 min.

8. The method according to claim 1, characterized in that In step (1) of the co-separation method 1, the pH value of 11.0 is adjusted using a 2 M NaOH solution.

9. The method according to claim 1, characterized in that In step (1), the centrifugation in the first co-separation method is performed at 3.5-4.5°C and 2000-10000 g for 5-15 min.

10. The method according to claim 1, characterized in that The temperature of the low temperature treatment in the second co-separation method of step (1) is 0-2°C.

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