LU501670B1

LU501670B1 - A Method for as1-Casein Separation

Info

Publication number: LU501670B1
Application number: LU501670A
Authority: LU
Inventors: Peng Zhou; Kai Zhang; Lina Zhang
Original assignee: Univ Jiangnan
Priority date: 2021-03-18
Filing date: 2022-03-16
Publication date: 2022-09-16
Also published as: CN113061173A; CN113061173B

Abstract

The disclosure provides a method for separating as1-casein, which belongs to the technical field of protein separation. The method uses raw cow milk, a cow milk-containing substance, cow milk casein, goat milk, a goat milk-containing substance, and goat milk casein as raw materials, and obtains as1-casein with a purity of 90% or more by steps of defatting, hydrochloric acid precipitation, urea precipitation, dialysis, and calcium ion precipitation. The method can also be used for separation of as1-casein from cow milk powder, which is simple, easy to repeat, and can be accomplished without special instruments. The obtained as1-casein is non-toxic and harmless, and can be directly used in subsequent production or experiments.

Description

A Method for a1-Casein Separation LU501670 Technical Field

[0001] The disclosure relates to a method for asi-casein separation, and belongs to the technical field of protein separation. Background

[0002] Casein is an important component of animal milk, accounting for about 80% of total milk protein, and including às1-Casein, as;-casein, B-casein, and k-casein, with molecular weights in a range of 19-25.2 kDa. Casein, existing in a state of micelles in cow milk, carries a large amount of calcium and phosphates and is rich in amino acids. The às1-casein has the highest content (38%-40% of total casein) in cow milk casein, and is a highly phosphorylated protein containing 8-9 phosphorylated groups. After asi-casein is hydrolyzed, soluble phosphopeptides bound with calcium are obtained, which can promote the absorption of calcium. Hydrolyzed às1-casein is also an important source of active peptides in cow milk. With respect to goat milk, ası-casein accounts for 5.6% of total casein, and it is one of the most important highly phosphorylated proteins, making it more difficult to extract from goat milk.

[0003] With the deepening of dairy science and technology research, the separation and purification technology of milk protein components has increased the requirement in recent years. At present, there are mainly three methods for milk protein separation: an isoelectric point and salting-out method, membrane separation technology, and chromatographic separation. The isoelectric point and salting-out method based on the fact that different components of milk protein have different isoelectric points and different solubilities in different salt concentrations. It has the advantage of simple operation and equipment, but has 1 low purity. The membrane separation technology realizes separation by retaining proteins LU501670 through controlling different pore sizes. It has the advantage that the operating conditions are mild and will not destroy the original properties of proteins, however this method has been currently been used for only separating casein or B-casein. Chromatography separates milk proteins through ion exchange chromatography or high-performance liquid chromatography mainly by virtue of different affinities of different milk proteins to various ions. It has the advantages of high resolution, good repeatability, and high purity, but the disadvantages of requiring specific equipment and low efficiency.

[0004] The methods disclosed at present mainly focus on preparation of as-casein. Xu Mingfang et al. disclosed a method for separating milk-derived as-casein by ion exchange chromatography. Casein was used as a raw material to prepare a 4 mg/mL solution, and the solution was eluted on a DEAE-Sepharose CL-6B chromatographic column to obtain 100% pure as-casein (publication number: CN 102746394 A, published on October 24, 2012). Zhang Liebing et al. disclosed a chemical fractionation method for purifying and separating as-casein. Cow milk casein was used as a raw material to prepare a 2.7% aqueous solution, k-casein is separated by precipitation with 40 mM Ca”, and then, as-casein is separated by virtue of different solubilities between as-casein and B-casein in a urea solution and finally obtained as- casein with a purity of 96.2% (publication number: CN 101824085 A, published on September 8, 2010). Li Quanyang et al. disclosed a method for separating casein components from buffalo milk. This method used buffalo milk as raw material. Casein is separated by isoelectric point acid precipitation, and then as-casein, B-casein and k-casein are separated by Ca?* ions with 2 different concentrations to obtain as-casein with a purity of 91.6% (publication number: CN LU501670 102675448 A, published on September 19, 2012).

[0005] However, the prior art is still limited to the extraction of as-casein, and a real separation and purification technology for ası-casein was still lacking, which limits the development and application of asi-casein.

Summary

[0006] In view of few methods for separating and purifying as1-casein from cow milk or goat milk at present, the disclosure provides a method for separating and purifying asi-casein from raw cow milk or goat milk by using an isoelectric point and salting-out method. The method uses raw cow milk as a raw material, and achieved a high purity of às1-casein by acid precipitation, urea separation, dialysis, and Ca?* precipitation.

[0007] The above objective of the disclosure is achieved by the following technical solutions:

[0008] Cow milk or goat milk is first defatted, and casein is separated by adjusting the pH to an isoelectric point. as-casein can be separated by adjusting the concentration of urea as às-casein, B-casein and k-casein have different solubilitiesin urea,. Since asi-casein and às2-casein have different sensitivities to Ca”* due to different degrees of phosphorylation, they were separated by adjusting the concentration of Ca”.

[0009] The disclosure provides a method for extracting às1-casein by using Ca”.

[0010] In an implementation, the casein is derived from cow milk, a cow milk-containing substance, goat milk, or a goat milk-containing substance.

[0011] In an implementation, when a raw material is cow milk or contains cow milk, the method includes: 3

[0012] (1) extracting casein from the raw material, dissolving casein powder in a 6-7 M urea LU501670 solution, and adjusting the pH to 6.5-7.5;

[0013] (2) adjusting the urea concentration to 4.5-4.8 M, adjusting the pH to 4.5-4.8, and conducting centrifugation to collect a precipitate;

[0014] (3) dissolving the precipitate in a 6-7 M urea solution, and adjusting the pH to 6.5-7.5;

[0015] (4) diluting the urea solution with water until the urea concentration is 3.2-3.5 M, adjusting the pH to 4.5-4.8, and conducting centrifugation to collect a precipitate;

[0016] (5) dissolving the precipitate in an aqueous solution with a pH of 10-11, adjusting the pH to 7.0-7.5, and removing urea by dialysis to obtain a dialysate; and

[0017] (6) collecting the dialysate, adding 1-3 mM CaCl, for centrifugation to collect a supernatant, adding 15-20 mM CaCl; for separation to obtain a precipitate, and drying the precipitate to obtain As1-casein from cow milk.

[0018] In an implementation, in step (2), the urea concentration is 4.6 M, and the pH is 4.6; and in step (4), the urea concentration is 3.3M, and the pH is adjusted to 4.6.

[0019] In an implementation, in step (5), the pH is adjusted to 7.2 after dissolving in an aqueous solution with a pH of 11.

[0020] In an implementation, 3 mM CaCl; is added and centrifuged to collect a supernatant, and then 20 mM CaCl, is added.

[0021] In an implementation, when a raw material is goat milk or contains goat milk, the method includes:

[0022] (1) extracting casein from the raw material, dissolving casein powder in a 6-7 M urea solution, and adjusting the pH to 6.5-7.5; 4

[0023] (2) adjusting the urea concentration to 2.8-3.2 M, adjusting the pH to 4.0-4.2, and LU501670 conducting centrifugation to collect a precipitate;

[0024] (3) dissolving the precipitate in an aqueous solution with a pH of 11, adjusting the pH to

9.0, and removing urea by dialysis to obtain a dialysate; and

[0025] (4) collecting the dialysate, adding 1-3 mM CaCl, for centrifugation to collect a supernatant, adding 50-60 mM CaCl; for separation to obtain a precipitate, and drying the precipitate to obtain às1-casein from goat milk.

[0026] In an implementation, in step (2), the urea concentration is adjusted to 3.0 M, and the pH is adjusted to 4.1; and in step (3), the pH is adjusted to 9.0 after dissolving in an aqueous solution with a pH of 11.

[0027] In an implementation, 3 mM CaCl is added for centrifugation to collect a supernatant, and then 60 mM CaCl, is added for separation to obtain a precipitate.

[0028] In an implementation, first, cow milk or goat milk is defatted, then concentrated hydrochloric acid is added to a pH of 4-5, the mixture is stirred and centrifuged to collect a precipitate, and the precipitate is washed with distilled water and freeze-dried to obtain casein powder.

[0029] Beneficial effects of the disclosure:

[0030] The method is simple in operation. It does not require complex instruments and equipment to obtain às1-casein with a high purity, and does not use toxic and harmful reagents in a purification process. Furthermore, the obtained às1-casein can be directly used in subsequent experiments. The method is suitable for both industrial production and laboratory preparation, and has a good application prospect.

Brief Description of Figures LU501670

[0031] FIG. 1 shows an electrophoretogram of products obtained in different steps; the far left represents a maker; 1 and 2 represent proteins obtained after defatting; 3 and 4 represent precipitated casein; 5 and 6 represent as-casein obtained after precipitation in step (6); and 7 and 8 represent finally extracted às1-casein from cow milk.

[0032] FIG. 2 is a LC-MS identification diagram of the obtained as1-casein from cow milk.

[0033] FIG. 3 shows freeze-dried as1-casein from cow milk after dialysis. 1 mM, 2 mM and 3 mM CaCl, is added respectively after dialysis, and the supernatant is collected after centrifugation at 3000 g for 10 min, then 20 mM CaCl, is added, precipitate is collected after centrifugation at 3000 g for 10 min.and; the far left represents a maker; 1 and 2 represent precipitated casein ; 3 and 4 represent a supernatant obtained after a dialysate is collected and 1 mM CaCl; is added for precipitation; 5 and 6 represent a supernatant obtained after a dialysate is collected and 2 mM CaCl; is added for precipitation; and 7 and 8 represent a supernatant obtained after a dialysate is collected and 3 mM CaCl; is added for precipitation.

[0034] FIG. 4 shows a SDS-PAGE diagram of products obtained in different steps; the far left represents a maker; 1 and 2 represent proteins obtained after defatting in step (1); 3 and 4 represent casein obtained after acid precipitation in step (2); 5 and 6 represent as-casein obtained after precipitation in step (6); and 7 and 8 represent ası-casein from goat milk obtained after precipitation in step (8).

[0035] FIG. 5 is a LC-MS identification diagram of the finally obtained asi-casein from goat milk.

[0036] FIG. 6 shows an SDS-PAGE diagram of proteins extracted without urea being thoroughly removed by dialysis after step (4).

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[0037] FIG. 7 shows that after a dialysate is taken in step (6), 1 mM, 2 mM and 3 mM CaCl; is LU501670 added respectively, a supernatant is collected after centrifugation at 3000 g for 10 min, then 60 mM CaCl, is added, the mixture is centrifuged at 3000 g for 10 min, and a precipitate is collected and freeze-dried to obtain asi-casein from goat milk; the far left represents a maker; 1 and 2 represent casein obtained after acid precipitation in step (2); 3 and 4 represent a supernatant obtained after a dialysate is collected and 1 mM CaCl; is added for precipitation in step (6); 5 and 6 represent a supernatant obtained after a dialysate is collected and 2 mM CaCl, is added for precipitation in step (6); and 7 and 8 represent a supernatant obtained after a dialysate is collected and 3 mM CaCl, is added for precipitation in step (6).

Detailed Description

[0038] Example 1

[0039] (1) Defatting of cow milk: 2 L of fresh raw cow milk was centrifuged at 2500 g, 4°C for 20 min to remove fat.

[0040] (2) Precipitation of casein: Concentrated hydrochloric acid was slowly added and stirred, the pH was adjusted to 4.6, and the mixture was stirred for 30 min and then centrifuged at 3000 g for 10 min to collect a precipitate. After being dispersed, the precipitate was washed with distilled water and centrifuged at 3000 g for 10 min. The operation was repeated twice, and a precipitate was freeze-dried to obtain casein powder.

[0041] (3) The casein powder was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4%, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

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[0042] (4) The solution was diluted with water until the urea concentration was 4.6 M, the pH LU501670 was adjusted to 4.6, and the solution was allowed to stand at 4°C for 30 min and then centrifuged at 4000 g to collect a precipitate.

[0043] (5) The precipitate was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4%, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

[0044] (6) The solution was diluted with water until the urea concentration was 3.3 M, the pH was adjusted to 4.6, and the solution was allowed to stand at 4°C for 30 min and then centrifuged at 4000 g to collect a precipitate.

[0045] (7) The precipitate was dissolved in an aqueous solution with a pH of 11, the pH was adjusted to 7.2, and the solution was dialyzed with distilled water with a pH of 7.2 for 48 h to thoroughly remove urea.

[0046] (8) A dialysate was collected, 3 mM CaCl, was added, and the mixture was centrifuged at 3000 g for 10 min to collect a supernatant. Then 20 mM CaCl, was added, the mixture was centrifuged at 3000 g for 10 min, and a precipitate was collected and freeze-dried to obtain as1- casein, with a purity of 94.25+0.97%, from cow milk.

[0047] Example 2

[0048] (1) Casein powder from cow was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4 g/100 mL, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

[0049] (2) The solution was diluted with water until the urea concentration was 4.6 M, the pH was adjusted to 4.6, and the solution was allowed to stand at 4°C for 30 min and then 8 centrifuged at 4000 g to collect a precipitate. The precipitate was washed twice with distilled LU501670 water and centrifuged at 3000 g for 10 min to obtain freeze-dried casein powder.

[0050] (3) The precipitate was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4 g/100 mL, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

[0051] (4) The solution was diluted with water until the urea concentration was 3.3 M, the pH was adjusted to 4.6, and the solution was allowed to stand at 4°C for 30 min and then centrifuged at 4000 g to collect a precipitate.

[0052] (5) The precipitate was dissolved in an aqueous solution with a pH of 11, the pH was adjusted to 7.2, and the solution was dialyzed with distilled water for 48 h to thoroughly remove urea.

[0053] (6) A dialysate was collected, 3 mM CaCl, was added, and the mixture was centrifuged at 3000 g for 10 min to collect a supernatant. Then 20 mM CaCl, was added, the mixture was centrifuged at 3000 g for 10 min, and a precipitate was collected and freeze-dried to obtain as1- casein, with a purity of 95.57+1.24%, from cow milk.

[0054] Comparative example 1

[0055] For a specific implementation, see Example 1. The difference is that after a dialysate was collected in step (8), 1 mM, 2 mM and 3 mM CaCl, was added respectively. The purities of ds1- casein from cow milk obtained finally by precipitation and freeze-drying were 80.12+3.75 %,

91.34+1.45 % and 94.25+0.97 % respectively.

[0056] Example 3 9

[0057] (1) Defatting of goat milk: 2 L of fresh goat milk was centrifuged at 2500 g and 4°C for 20 LU501670 min to remove fat.

[0058] (2) Precipitation of casein: A hydrochloric acid solution was slowly added and stirred, the pH was adjusted to 4.1, and the mixture was stirred for 30 min and then centrifuged at 3000 g for 10 min to collect a precipitate. After being dispersed, the precipitate was washed with distilled water and centrifuged at 3000 g for 10 min. The operation was repeated twice, and a precipitate was freeze-dried to obtain casein powder.

[0059] (3) The casein powder was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4%, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

[0060] (4) The solution was diluted with water until the urea concentration was 3.0 M, the pH was adjusted to 4.1, and the solution was allowed to stand at 4°C for 30 min and then centrifuged at 4000 g to collect a precipitate.

[0061] (5) The precipitate was dissolved in an aqueous solution with a pH of 11, the pH was adjusted to 9.0, and the solution was dialyzed with distilled water with a pH of 9.0 for 48 h to thoroughly remove urea.

[0062] (6) A dialysate was collected, 3 mM CaCl, was added, and the mixture was centrifuged at 3000 g for 10 min to collect a supernatant. Then 60 mM CaCl, was added, the mixture was centrifuged at 3000 g for 10 min, and a precipitate was collected and freeze-dried to obtain as1- casein, with a purity of 93.8%, from goat milk.

[0063] Example 4

[0064] (1) Defatting of goat milk: 30 g of goat milk powder was dissolved in 200 mL of warm LU501670 water, and centrifuged at 2500 g and 4°C for 20 min to remove fat.

[0065] (2) Precipitation of casein: A hydrochloric acid solution was slowly added and stirred, the pH was adjusted to 4.1, and the mixture was stirred for 30 min and then centrifuged at 3000 g for 10 min to collect a precipitate. After being dispersed, the precipitate was washed with distilled water and centrifuged at 3000 g for 10 min. The operation was repeated twice, and a precipitate was freeze-dried to obtain casein powder.

[0066] (3) The casein powder was dissolved in a 6.6 M urea solution to prepare a solution with a mass-volume percentage concentration of 4%, the pH was adjusted to 7.0, and the solution was allowed to stand at 4°C for 30 min.

[0067] (4) The solution was diluted with water until the urea concentration was 3.0 M, the pH was adjusted to 4.1, and the solution was allowed to stand at 4°C for 30 min and then centrifuged at 4000 g to collect a precipitate.

[0068] (5) The precipitate was dissolved in an aqueous solution with a pH of 11, the pH was adjusted to 9.0, and the solution was dialyzed with distilled water with a pH of 9.0 for 48 h to thoroughly remove urea.

[0069] (6) A dialysate was collected, 3 mM CaCl, was added, and the mixture was centrifuged at 3000 g for 10 min to collect a supernatant. Then 60 mM CaCl, was added, the mixture was centrifuged at 3000 g for 10 min, and a precipitate was collected and freeze-dried to obtain as1- casein, with a purity of 94.2%, from goat milk.

[0070] Comparative example 2 11

[0071] For a specific implementation, see Example 3. The difference is that the urea was not LU501670 removed thoroughly by dialysis after step (4), resulting in a poor separation effect. The results are shown in FIG. 4.

[0072] Comparative example 3

[0073] For a specific implementation, see Example 3. The difference is that after a dialysate was collected in step (6), 1 mM, 2 mM and 3 mM CaCl, was added respectively. The purities of ds1- casein from goat milk obtained finally by precipitation and freeze-drying were 75.33+1.25%,

89.50+0.56% and 93.20+0.44% respectively.

[0074] While the disclosure has been provided in preferred examples above, they are not intended to limit the disclosure. Anyone skilled in the art may conduct various changes and modifications without departing from the spirit and scope of the disclosure, and so the protective scope of the disclosure shall be defined by the claims.

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Claims

What is claimed is: LUS01670

1. A method for extracting asi-casein, wherein às1-casein is separated from casein by means of Ca**.

2. The method of claim 1, wherein the casein is derived from cow milk, a cow milk- containing substance, goat milk, or a goat milk-containing substance.

3. The method of claim 2, wherein when a raw material is cow milk or contains cow milk, the method comprises: (1) extracting casein from the raw material, dissolving casein powder in a 6-7 M urea solution, and adjusting the pH to 6.5-7.5; (2) adjusting the urea concentration to 4.5-4.8 M, adjusting the pH to 4.5-4.8, and conducting centrifugation to collect a precipitate; (3) dissolving the precipitate in a 6-7 M urea solution, and adjusting the pH to 6.5-7.5; (4) diluting the urea solution with water until the urea concentration is 3.2-3.5 M, adjusting the pH to 4.5-4.8, and conducting centrifugation to collect a precipitate; (5) dissolving the precipitate in an aqueous solution with a pH of 10-11, adjusting the pH to 7.0-

7.5, and removing urea by dialysis to obtain a dialysate; and (6) collecting the dialysate, adding 1-3 mM CaCl; for centrifugation to collect a supernatant, adding 15-20 mM CaCl; for separation to obtain a precipitate, and drying the precipitate to obtain As1-casein from cow milk.

4. The method of claim 3, wherein in step (2), the urea concentration is 4.6 M, and the pH is 4.6; and in step (4), the urea concentration is 3.3M, and the pH is adjusted to 4.6.

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5. The method of claim 4, wherein in step (5), the pH is adjusted to 7.2 after dissolving in LU501670 an aqueous solution with a pH of 11.

6. The method of claim 5, wherein 3 mM CaCl, is added and centrifuged to collect a supernatant, and then 20 mM CaCl, is added.

7. The method of claim 2, wherein when a raw material is goat milk or contains goat milk, the method comprises: (1) extracting casein from the raw material, dissolving casein powder in a 6-7 M urea solution, and adjusting the pH to 6.5-7.5; (2) adjusting the urea concentration to 2.8-3.2 M, adjusting the pH to 4.0-4.2, and conducting centrifugation to collect a precipitate; (3) dissolving the precipitate in an aqueous solution with a pH of 11, adjusting the pH to 9.0, and removing urea by dialysis to obtain a dialysate; and (4) collecting the dialysate, adding 1-3 mM CaCl; for centrifugation to collect a supernatant, adding 50-60 mM CaCl, for separation to obtain a precipitate, and drying the precipitate to obtain As1-casein from goat milk.

8. The method of claim 7, wherein in step (2), the urea concentration is adjusted to 3.0 M, and the pH is adjusted to 4.1; and in step (3), the pH is adjusted to 9.0 after dissolving in an aqueous solution with a pH of 11.

9. The method of claim 8, wherein 3 mM CaCl, is added for centrifugation to collect a supernatant, and then 60 mM CaCl, is added for separation to obtain a precipitate.

10. The method of claim 3, wherein first, cow milk or goat milk is defatted, then concentrated hydrochloric acid is added to a pH of 4-5, the mixture is stirred and centrifuged to 14 collect a precipitate, and the precipitate is washed with distilled water and freeze-dried to LU501670 obtain casein powder.