CN111303269B - Method for extracting kappa-casein from milk and product thereof - Google Patents
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Abstract
The invention relates to a method for extracting kappa-casein from milk and a product thereof, which utilizes three casein pairs of alpha-casein, beta-casein and kappa-casein in the milk to carry out Ca separation 2+ And pH sensitivity difference, setting different technological parameters, dissolving urea solution with resolving agent, acid precipitation, and Ca 2+ After operations such as precipitation and the like, three kinds of casein are separated to obtain kappa-casein, and a kappa-casein monomer with high purity is extracted from cow milk with known AA-kappa-casein or BB-kappa-casein types and can be used as an AA-kappa-casein or BB-kappa-casein standardized monomer commodity for high performance liquid chromatography detection.
Description
Technical Field
The invention relates to the technical field of protein separation and extraction from milk, and particularly relates to a method for extracting kappa-casein from milk and a product thereof.
Background
The protein in the milk mainly comprises whey protein and casein, wherein the casein is composed of alpha-casein, beta-casein and kappa-casein, and the three caseins can be further subdivided and can be divided into different configurations. The cow milk can be divided into AA-kappa-casein, BB-kappa-casein and mixed cow milk with two configurations according to kappa-casein types. The literature data at present prove that the milk containing only BB-kappa-casein can improve the yield of cheese, the other two kappa-caseins both have glycosylation structures, the glycosylated kappa-casein is hydrolyzed by pepsin to produce glycomacropeptide, and the glycomacropeptide has physiological activity of improving immunity, so that the identification of the type of the kappa-casein in the milk is necessary for milk production.
The genotype identification of the cow can be carried out by a PCR method, the PCR detection has higher requirements on basic hardware and operators, most dairy enterprises do not have the conditions required by the detection method, and the PCR method is difficult to judge unknown cow milk.
The qualitative and quantitative detection of casein in cow milk by using a high performance liquid chromatograph in the prior art is most convenient and rapid, but high-purity AA-kappa-casein or BB-kappa-casein is required in the detection process, and the kappa-casein which is sold in the market at present is a mixture of AA and BB-kappa-casein and does not belong to a standardized product of monomers, so that a method for extracting a kappa-casein monomer from cow milk is urgently needed, and the method is preferably simple to operate and low in cost.
Disclosure of Invention
The invention aims to: aiming at the technical problems that kappa-casein monomer obtained by extraction in the prior art has low purity and is not suitable for AA-kappa-casein or BB-kappa-casein standardized monomer used for high performance liquid chromatography detection, the method for extracting the kappa-casein monomer from milk and the product thereof are provided.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for extracting kappa-casein from milk comprises the following steps:
step 1, adding a casein crude product extracted from cow milk into a urea solution, adjusting the pH value to 9-10 by using an alkali liquor, heating to 40-45 ℃, stirring for dissolving, then stirring for 2-3h at normal temperature, cooling to 2-6 ℃, and standing for 1-2h.
And 2, under the ice-water bath condition, adjusting the pH value of the material obtained in the step 1 to 5.0 +/-0.2 by using an acid liquor, centrifugally collecting the precipitate, and washing the precipitate with pure water at least once to obtain a first precipitate.
Step 3, taking a proper amount of alkali liquor with the pH value of 11 +/-0.2, heating to 40-45 ℃, adding the first precipitate obtained in the step 2, stirring for dissolving, adjusting the pH value to 10.0 +/-0.2 by using the alkali liquor in the dissolving process, then adjusting the pH value to be neutral, adding soluble calcium salt solution, and enabling Ca in the final solution to be in the form of calcium carbonate 2+ The ion concentration reaches 0.08mol/L to 0.12mol/L, and the supernatant is collected by centrifugation.
And 4, adjusting the pH value of the supernatant obtained in the step 3 to 4.0 +/-0.2 by using acid liquor, centrifuging, collecting precipitate, and washing the precipitate with pure water at least once to obtain the kappa-casein.
The invention utilizes three casein pairs of alpha-casein, beta-casein and kappa-casein in cow milk to carry out Ca 2+ And pH sensitivity difference, setting different technological parameters, dissolving urea solution in a resolving agent, acid precipitation and Ca 2+ After precipitation and the like, three kinds of casein are separated to obtain kappa-casein, and kappa-casein monomer with high purity is extracted from cow milk with known AA-kappa-casein or BB-kappa-casein. The obtained kappa-casein monomer can be used as a standardized monomer required by AA-kappa-casein or BB-kappa-casein types detected by high performance liquid chromatography. The extraction method of the invention has simple operation and low cost.
Further, the concentration of the urea solution in the step 1 is 3.1-3.5 mol/L. Through a large amount of researches of the inventor, the purity of the product is different when different decomposition liquids are tested, the concentration is too low, the urea content in unit volume is too low, the extraction effect on casein is reduced, the concentration is too high, and the decomposition liquids may damage the casein or carry impurities to enter subsequent experiments to influence the concentration of the final product.
Further, the crude casein in the step 1 is prepared by the following method: heating the skim milk to 25-40 ℃, stirring, adjusting the pH value to 4.6 +/-0.2 with acid liquor, collecting the precipitate, washing the precipitate with pure water at least once, and obtaining a crude casein product.
Further, the skim milk is obtained by degreasing raw milk, and the skim milk degreasing method comprises the following steps: centrifuging raw milk at 6000-8000 r/min and 2-4 deg.C for 10-15 min, and filtering to obtain skimmed milk.
Further, the precipitate is washed with pure water in step 2 at least 2 times, such as 2 times, 3 times, 4 times, etc., and increasing the number of washing times can reduce the amount of impurities contained in the precipitate.
Further, the calcium salt solution in step 3 is 0.05mol/L to 0.15mol/L calcium chloride solution, preferably, the calcium salt solution in step 3 is 0.08mol/L to 0.15mol/L calcium chloride solution, such as 0.08mol/L, 0.1mol/L, 0.15mol/L.
Further, ca in the final solution in step 3 2+ The ion concentration is 0.10 mol/L-0.12 mol/L. Preferably, ca is in the final solution in step 3 2+ The ion concentration was 0.10mol/L. The research finds that the purity of the final product can be regulated and controlled by controlling the concentration of calcium ions in the final solution, the three caseins have different sensitivities to the concentration of the calcium ions, and the casein can be further separated by limiting the concentration of the calcium ions.
Further, the precipitate is washed with pure water at least 2 times, such as 2 times, 3 times, 4 times, etc., in step 4, and increasing the number of washing times can reduce the amount of impurities contained in the precipitate.
Further, the acid solution is hydrochloric acid solution, sulfuric acid solution or acetic acid solution, and further, the hydrogen ion concentration of the acid solution is 0.8-1.2 mol/L.
Further, the alkali liquor is sodium hydroxide solution or potassium hydroxide solution, and further, the concentration of the alkali liquor is 0.8 mol/L-1.2 mol/L.
The invention also provides the kappa-casein prepared by the method for extracting the kappa-casein from milk.
The invention utilizes three casein pairs of alpha-casein, beta-casein and kappa-casein in cow milk to carry out Ca 2+ And pH sensitivity difference, setting different technological parameters, dissolving urea solution with resolving agent, acid precipitation, and Ca 2+ After precipitation and the like, three kinds of casein are separated to obtain kappa-casein, and kappa-casein monomer with high purity is extracted from cow milk with known AA-kappa-casein or BB-kappa-casein.
The invention provides application of the prepared kappa-casein as a standard substance for identifying the type of the kappa-casein in raw milk.
The kappa-casein monomer extracted by the method has the purity of over 90 percent, can be used as an AA-kappa-casein or BB-kappa-casein standardized monomer product for high performance liquid chromatography detection, and has simple operation and low cost.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the invention utilizes three casein pairs of alpha-casein, beta-casein and kappa-casein in cow milk to carry out Ca 2+ And pH sensitivity difference, setting different technological parameters, dissolving urea solution with resolving agent, acid precipitation, and Ca 2+ After operations such as precipitation and the like, three kinds of casein are separated to obtain kappa-casein, and kappa-casein monomers with high purity are extracted from the known AA-kappa-casein or BB-kappa-casein type milk, wherein the purity reaches more than 90%.
2. The kappa-casein monomer extracted by the method has high purity, can be used as an AA-kappa-casein or BB-kappa-casein standardized monomer product for high performance liquid chromatography detection, and has simple operation and low cost.
Drawings
FIG. 1 is a high performance liquid chromatography detection profile of AA-kappa-casein extracted in example 1.
FIG. 2 is a high performance liquid chromatography detection profile of BB-kappa-casein extracted in example 2.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Extraction of raw milk containing AA-kappa-casein
Firstly, freezing and centrifuging fresh milk at 7000r/min and 4 deg.C for 10min, and removing fat to obtain skimmed milk. Then heating the skim milk to 40 ℃, adjusting the pH value to 4.6 by using 1mol/L hydrochloric acid under the stirring condition, and collecting precipitate S 1 Washing the precipitate S with pure water 1 The mixture is ready for use after three times. (precipitation S) 1 Casein) next, 10g of the precipitate S were taken 1 Adding the mixture into 300ml of urea solution with the concentration of 3.3mol/L, adjusting the pH value to 9 by using 1mol/L sodium hydroxide, heating to 40 ℃, stirring for dissolving, stirring for 2 hours at normal temperature, cooling to 2 ℃, and standing for 1 hour. Then, the pH value is adjusted to 5.0 by using 1mol/L hydrochloric acid under the condition of ice-water bath, and precipitate S is centrifugally collected 2 Washing the precipitate S with water 2 The mixture is ready for use after three times. (precipitation S) 2 Mainly containing alpha-casein and kappa-casein, and supernatant L 1 Mainly contains beta casein
Finally, taking a proper amount of alkali liquor with the pH value of 11, heating to 40 ℃, and dissolving precipitate S under the stirring condition 2 Adjusting pH to 7.0 with 1mol/L sodium hydroxide, adding 0.8mol/L calcium chloride solution to obtain Ca in the final solution 2+ The ion concentration reaches 0.1mol/L, and supernatant liquid L is collected by centrifugation 2 Centrifuging the resulting precipitate S 3 Mainly contains alpha-casein. Continuously adjusting the supernatant L with acid solution 2 The pH value of the solution is 4.0, and the precipitate is collected by centrifugation to obtain the AA-kappa-casein.
Test 1
Detecting the extracted AA-kappa-casein by high performance liquid chromatography
1.1 Instrument: high performance liquid chromatograph (Agilent 1260)
1.2 chromatographic column:VydacEvrest C18 250*4.6mm 5μm 
1.3 sample introduction amount: 50 μ L
1.4 column temperature: 40 deg.C
1.5 detection wavelength: 241nm and 280nm for assistance
1.6 mobile phase A:0.1wt% aqueous trifluoroacetic acid solution
1.7 mobile phase B:0.1wt% trifluoroacetic acid in acetonitrile
1.8 elution procedure is shown in Table 1.
Table 1 example 1 elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) | Flow rate/ |
| 0 | 75.7 | 24.3 | 1 |
| 2 | 71.2 | 28.8 | 1 |
| 17 | 64.8 | 35.2 | 1 |
| 30 | 61.8 | 38.2 | 1 |
| 45 | 55.0 | 45.0 | 1 |
| 48 | 46.0 | 54.0 | 1 |
| 53 | 46.0 | 54.0 | 1 |
| 55 | 75.7 | 24.3 | 1 |
| 65 | 75.7 | 24.3 | 1 |
1.9 pretreatment of Standard
Weighing 1mg to three 1.5ml centrifugal tubes of sigma brand alpha s-casein, beta-casein and kappa-casein standard products respectively, adding 500 mu L of buffer solution 1 (consisting of 6mol/L guanidine hydrochloride, 0.1mol/LBis-Tris and 5.37mol/L sodium citrate dihydrate), uniformly mixing by vortex, centrifuging at 6000r/min for 15s at room temperature for 1h, then centrifuging at 6000r/min for 10min, adding 490 mu L of 4.5mol/L guanidine hydrochloride and 10 mu L of beta mercaptoethanol after centrifuging, uniformly mixing by vortex, finally filtering by using a 0.45 mu m PTFE filter membrane, and using for liquid phase detection after filtering.
2.0 sample detection
Taking 1mg of AA-kappa-casein obtained in example 1, adding 500. Mu.L of buffer solution 1 (consisting of 6mol/L guanidine hydrochloride, 0.1mol/LBis-Tris and 5.37mol/L sodium citrate dihydrate), uniformly mixing by vortex, centrifuging at 6000r/min for 15s, standing at room temperature for 1h, then centrifuging at 6000r/min for 10min, adding 490. Mu.L of 4.5mol/L guanidine hydrochloride and 10. Mu.L of beta mercaptoethanol after centrifuging, uniformly mixing by vortex, finally filtering by using a 0.45. Mu. MPTE filter membrane, and filtering for liquid phase detection.
Detecting the extracted AA-kappa-casein by high performance liquid chromatography to obtain a spectrogram shown in figure 1, wherein the obtained casein is AA-kappa-casein with high purity, and the purity of the target is measured to be 95% according to peak area ratio after integration processing by chromatographic data acquisition software.
Example 2
Extraction of raw milk containing BB-kappa-casein
Firstly, freezing and centrifuging fresh milk at 2 deg.C for 15min at 6500r/min, removing fat, collecting skim milk, heating to 40 deg.C, adjusting pH to 4.8 with 1.2mol/L hydrochloric acid under stirring, collecting precipitate, washing precipitate with pure water to obtain S 1 The mixture is ready for use after three times. (precipitation S) 1 Casein) next, the precipitate S is taken 1 10g, adding 0.8mol/L sodium hydroxide into 300ml of 3.5mol/L urea solution to adjust the pH value to 9.2, heating to 30 ℃, dissolving under stirring, stirring for 2h at normal temperature, cooling to 2 ℃, and standing for 2h. Then, the pH value is adjusted to 5.1 by using 1.2mol/L hydrochloric acid under the condition of ice-water bath, the precipitate is collected by centrifugation, and the precipitate S is washed by water 2 Three times of the preparation is ready for use. (precipitation S) 2 Mainly containing alpha-casein and kappa-casein, and supernatant L 1 Mainly beta casein
Finally, taking a proper amount of aqueous solution with the pH value of 11.2, heating to 40 ℃, and dissolving the S under the stirring condition 2 1mol/L of hydrogen and oxygenAdjusting pH to 7.0 with sodium chloride, adding 0.8mol/L calcium chloride solution to obtain Ca in the final solution 2+ The ion concentration is 0.12mol/L, and supernatant liquid L is collected by centrifugation 2 . (precipitation S) 3 Containing mainly alpha-casein) to continue conditioning the supernatant L 2 And when the pH value is 4.0, centrifuging and collecting the precipitate to obtain the BB-kappa-casein.
The BB- κ -casein extracted above was detected by high performance liquid chromatography 1.1 instrument: high performance liquid chromatograph (Agilent 1260)
1.2 chromatographic column: vydacEvresst C18 250 x 4.6mm 5 μm
1.3 sample introduction amount: 50 μ L
1.4 column temperature: 40 deg.C
1.5 detection wavelength: 241nm and 280nm for assistance
1.6 mobile phase A:0.1wt% aqueous trifluoroacetic acid solution
1.7 mobile phase B:0.1wt% trifluoroacetic acid acetonitrile solution
1.8 elution procedure is shown in Table 2.
Table 2 example 2 elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) | Flow rate/ |
| 0 | 75.7 | 24.3 | 1 |
| 2 | 71.2 | 28.8 | 1 |
| 17 | 64.8 | 35.2 | 1 |
| 30 | 61.8 | 38.2 | 1 |
| 45 | 55.0 | 45.0 | 1 |
| 48 | 46.0 | 54.0 | 1 |
| 53 | 46.0 | 54.0 | 1 |
| 55 | 75.7 | 24.3 | 1 |
| 65 | 75.7 | 24.3 | 1 |
1.9 pretreatment of Standard
Weighing 1mg to three 1.5ml centrifugal tubes of sigma brand alpha s-casein, beta-casein and kappa-casein standard products respectively, adding 500 mu L of buffer solution 1 (consisting of 6mol/L guanidine hydrochloride, 0.1mol/LBis-Tris and 5.37mol/L sodium citrate dihydrate), uniformly mixing by vortex, centrifuging at 6000r/min for 15s at room temperature for 1h, then centrifuging at 6000r/min for 10min, adding 490 mu L of 4.5mol/L guanidine hydrochloride and 10 mu L of beta mercaptoethanol after centrifuging, uniformly mixing by vortex, finally filtering by using a 0.45 mu m TFE filter membrane, and using for liquid phase detection after filtering.
2.0 sample detection
Adding 500 mu L of buffer solution 1 (consisting of 6mol/L guanidine hydrochloride, 0.1mol/L LBis-Tris and 5.37mol/L sodium citrate dihydrate) into BB-kappa-casein, vortex and mixing uniformly, centrifuging at 6000r/min for 15s, standing at room temperature for 1h, then centrifuging at 6000r/min for 10min, adding 490 mu L of 4.5mol/L guanidine hydrochloride and 10 mu L of beta mercaptoethanol after centrifugation, vortex and mixing uniformly, finally filtering by using a 0.45 mu mPTEF filter membrane, and filtering for liquid phase detection. Detecting the BB-kappa-casein extracted by using a high performance liquid chromatography, wherein the obtained spectrogram is shown in figure 1, the obtained casein is BB-kappa-casein and has high purity, and the purity of the target is measured to be 92% according to the peak area ratio after integration treatment by using software.
Examples 3 to 8
Examples 3 to 8 experiments were conducted using the same batch of raw milk as in example 1, and it was found through extensive experimental studies by the inventors that the concentration of the urea solution directly affects the concentration of the objective product, and examples 3 to 8 were performed while changing the concentration of the urea solution as compared with example 1, and the remaining amount was unchanged, and the extract was measured by the same measurement method as in example 1, and the measurement results are shown in table 3.
TABLE 3 results of urea concentration and purity test of target product in examples 3-8
Examples 9 to 13
Examples 9-13 experiments were carried out using the same batch of raw milk as in example 1, and extensive experimental studies by the inventors have revealed that Ca is present in the final solution 2+ The ion concentration affects the purity of the target product, and examples 9-13 change Ca in the final solution compared to example 1 2+ The ion concentration was the same as that in example 1, and the extract was measured by the same measurement method, and the measurement results are shown in Table 4.
TABLE 4 Ca in final solutions in examples 9-13 2+ Ion concentration and purity detection result of target product
The invention utilizes three casein pairs of alpha-casein, beta-casein and kappa-casein in cow milk to carry out Ca 2+ And pH sensitivity difference, setting different technological parameters, dissolving urea solution in a resolving agent, acid precipitation and Ca 2+ After operations such as precipitation and the like, three kinds of casein are separated to obtain kappa-casein, a kappa-casein monomer with high purity is extracted from cow milk with known AA-kappa-casein or BB-kappa-casein types, the purity can reach more than 90 percent, and the kappa-casein monomer can be used as an AA-kappa-casein or BB-kappa-casein standardized monomer product for high performance liquid chromatography detection.
The descriptions of each patent, patent application, and publication cited in this application are incorporated herein by reference in their entirety. Citation of any reference shall not be construed as an admission that such reference is available as "prior art" to the present application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A method for extracting kappa-casein from milk is characterized by comprising the following steps:
step 1, adding a casein crude product extracted from cow milk into a urea solution with the concentration of 3.1 mol/L-3.5 mol/L, adjusting the pH value to 9-10 by using an alkali liquor, heating to 40-45 ℃, stirring for dissolving, stirring for 2-3h at normal temperature, cooling to 2-6 ℃, and standing for 1-2h;
step 2, under the ice-water bath condition, adjusting the pH value of the material obtained in the step 1 to 5.0 +/-0.2 by using an acid liquor, centrifugally collecting precipitates, and washing the precipitates with pure water at least once to obtain a first precipitate;
step 3, taking a proper amount of alkali liquor with the pH value of 11 +/-0.2, heating to 40-45 ℃, adding the first precipitate obtained in the step 2, stirring to dissolve, adjusting the pH value to 10.0 +/-0.2 by using the alkali liquor in the dissolving process, then adjusting the pH value to be neutral, adding a soluble calcium salt solution to enable Ca in the final solution to be in the range of 2+ The ion concentration reaches 0.08mol/L to 0.12mol/L, and supernatant fluid is collected by centrifugation;
and 4, adjusting the pH value of the supernatant obtained in the step 3 to 4.0 +/-0.2 by using acid liquor, centrifuging, collecting precipitate, and washing the precipitate with pure water at least once to obtain the kappa-casein.
2. The method for extracting kappa-casein from milk according to claim 1, wherein the crude casein in step 1 is prepared by the following steps: heating the skim milk to 25-40 ℃, stirring, adjusting the pH value to 4.6 +/-0.2 with acid liquor, collecting the precipitate, and washing the precipitate with pure water at least once to obtain a crude casein product.
3. The method for extracting kappa-casein from milk according to claim 2, wherein the skim milk is obtained by subjecting raw milk to a degreasing treatment, and the degreasing treatment method of the skim milk comprises the following steps: centrifuging raw milk at 6000-8000 r/min and 2-4 deg.C for 10-15 min, and filtering to obtain skimmed milk.
4. The method for extracting kappa-casein from milk according to claim 1, wherein the calcium salt solution in step 3 is 0.05mol/L to 0.15mol/L calcium chloride solution.
5. The method for extracting kappa-casein from milk as claimed in claim 1, wherein Ca in the final solution in step 3 2+ The ion concentration is 0.10 mol/L-0.12 mol/L.
6. The method for extracting kappa-casein from milk according to claim 1, wherein the acid solution is hydrochloric acid solution, sulfuric acid solution or acetic acid solution.
7. The method for extracting kappa-casein from milk as claimed in claim 1, wherein the alkali solution is sodium hydroxide solution or potassium hydroxide solution.
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| CN113061173B (en) * | 2021-03-18 | 2022-11-04 | 江南大学 | A kind of method for separating αs1-casein |
| CN113214378B (en) * | 2021-06-11 | 2023-03-24 | 新希望乳业股份有限公司 | Method for separating and extracting casein glycomacropeptide |
| CN113461795B (en) * | 2021-07-05 | 2022-08-26 | 湖北百特威生物科技有限公司 | Casein fractional precipitation purification method |
| CN114773448B (en) * | 2022-04-20 | 2023-07-07 | 中国农业科学院生物技术研究所 | Recombinant κ-casein, its preparation method and artificial milk |
| CN114773449B (en) * | 2022-04-20 | 2023-07-07 | 中国农业科学院生物技术研究所 | Artificial optimization and synthesis method of beta-casein and application thereof |
| CN115633726B (en) * | 2022-11-18 | 2023-03-17 | 中国农业大学 | Preparation method and application of casein |
| CN118420742A (en) * | 2024-05-27 | 2024-08-02 | 江南大学 | A method for co-separating kappa- and beta-casein |
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