CN102675448B - Method for isolating casein components in milk - Google Patents

Abstract

A method for separating casein components in buffalo milk, using buffalo milk as raw material, adopting isoelectric point slow acid precipitation, combined with the method of graded calcium precipitation for αs-casein, β-casein and κ-casein in buffalo milk Casein Three casein fractions were separated. The total recovery rate of each component of fractionated calcium precipitated casein reached 88%, and the mass fractions of separated αs-casein and β-casein reached 91.6% and 67.8%, respectively, and the mass fraction of κ-casein also increased from 13.8 % increased to 43.3%, which laid the foundation for further purification. At the same time, in theory, the mass fraction of β-casein can be increased to 100% if the technical route of secondary graded calcium precipitation is adopted. The method is simple to operate, has obvious separation effect, and is suitable for industrial production.

Description

A method for separating casein components in buffalo milk

technical field

The invention relates to a method for separating casein components in buffalo milk, in particular to a method for simultaneously separating αs-casein, β-casein and κ-casein through a simple process.

Background technique

The milk protein in buffalo milk is mainly composed of casein and whey protein. Taking the hybrid high generation as an example, α _s1 -casein, α _s2 -casein, β-casein and κ-casein account for 35.30% of the casein respectively , 16.97%, 33.88%, 13.85%, α-lactalbumin, β-lactoglobulin accounted for 30.95%, 48.49% of whey protein respectively. Casein exists in the form of a complex formed by casein micelles combined with calcium phosphate, which is called "Ca-phosphocaseinate or Ca-caseinate-phosphate-complex".

In recent decades, in order to study the stability mechanism of the casein micellar system and the structural and functional properties of each casein component, the separation and purification of casein components is the basis for the above research. The efficiency and quality of separation and purification are directly related to the follow-up. The success or failure of this work has become a hot research topic. At present, the separation methods of casein components mainly include precipitation separation, chromatographic separation, membrane separation and enzymatic separation, etc. Among them, chromatographic separation is mainly used on a laboratory scale, while membrane separation and enzymatic separation generally need to be combined with other methods Together to complete the separation steps, the most widely used is precipitation separation, which mainly uses the difference in solubility of each casein component in solutions of different temperatures, ionic strengths, and calcium concentrations to separate. The separation process mainly focuses on the yield of the target product and purity, but there are few systematic studies on its separation process.

Contents of the invention

The purpose of the present invention is to provide a method for separating casein components in buffalo milk, which can simultaneously separate αs-casein, β-casein and κ-casein, has simple operation, obvious separation effect and is suitable for industrial production.

The object of the present invention is achieved through the following technical solutions: a method for separating casein components in buffalo milk, comprising the following steps:

①Fresh buffalo milk, after centrifugation at 8000g, 4°C for 20-30min, use the method of isoelectric point slow acid precipitation, slowly adjust the pH of the skimmed milk to 4.5-4.7 with acid solution, and centrifuge at 8000g, 4°C Keep the precipitate for 20-30 minutes, wash it twice with distilled water to obtain crude casein powder, and freeze-dry it for later use;

②The isoelectric point is slowly acid-precipitated, and the casein powder obtained after freeze-drying is prepared into a 2% casein solution, and the pH of the casein solution is slowly adjusted to 7.0-7.5 with an alkaline solution;

③Add divalent soluble calcium salt to the casein solution in step ② according to the Ca ²⁺ concentration of 0.06mol/L, centrifuge at 8000g for 20-30min, keep the supernatant and precipitate, dissolve the precipitate in ethylenediaminetetraacetic acid di In sodium solution, α _s -casein with a mass fraction of 91.6% can be obtained after dialysis and freeze-drying;

④ Continue to add divalent soluble calcium salts to the supernatant retained by centrifugation in step ③, so that the final concentration of Ca2 ⁺ reaches 0.3mol/L, centrifuge again, retain the supernatant and precipitate, and obtain the supernatant after dialysis and freeze-drying The κ-casein with a mass fraction of 43.3% was dissolved in disodium edetate solution, dialyzed and freeze-dried to obtain β-casein with a mass fraction of 100%.

The casein is the casein of buffalo milk and buffalo milk products.

The buffalo milk is purebred buffalo milk of Mora, first-generation hybrid buffalo milk of Mora hybrid, and high-generation buffalo milk of Mora hybrid; the buffalo milk products are buffalo milk yoghurt, buffalo milk cheese, and buffalo milk powder.

The divalent soluble calcium salt is calcium chloride.

The disodium edetate solution is a 0.06-0.3 mol/L solution prepared by disodium edetate.

The acid solution is prepared as a 0.05-0.1 mol/L solution with glacial acetic acid or hydrochloric acid.

The alkaline solution is prepared by sodium hydroxide or ammonia water to form a 0.1-0.2 mol/L solution.

The present invention has the following advantages:

1. The present invention uses graded calcium precipitation to simultaneously separate the three casein components of αs-casein, β-casein and κ-casein. The operation process is simple, and it provides a method for separating casein components on a large scale. The simple and feasible method, on the one hand, provides a reference for the preparation of samples in the laboratory, and at the same time, it also has certain reference significance for large-scale industrial preparation, and provides convenience for subsequent industrial applications.

2. The αs-casein, β-casein and κ-casein produced by the present invention have high purity, which provides a method for the production of reagent type αs-casein and β-casein, and the reagents used in this process are few , which saves the preparation cost.

3. The yields of αs-casein, β-casein and κ-casein produced by the present invention are all high, which can effectively utilize resources.

Description of drawings

Figure 1 is the electrophoresis diagram of casein obtained after slow acid precipitation at the isoelectric point.

1-Low molecular weight protein standard 2-Buffalo milk whole protein after defatting 3-Whey protein after isoelectric point slow acid removal of casein 4-Casein obtained by isoelectric point slow acid precipitation, the loading concentration was diluted to 1000μg/mL, sample volume 10μL.

Figure 2 is the casein electrophoresis image obtained after grading calcium precipitation.

1- Low molecular weight protein standard, 2-, 3-, 4-, 5-, 6-, 7- respectively 0.30mol/L, 0.25mol/L, 0.20mol/L, 0.15mol/L, 0.10mol/L L, 0.06mol/LCa ²⁺ precipitate solution after treating casein, 8-, 9-, 10-, 11-, 12-, 13- respectively 0.30mol/L, 0.25mol/L, 0.20mol/L, 0.15mol/L, 0.10mol/L, 0.06mol/L Ca ²⁺ treated casein supernatant, the sample concentration was diluted to 1000μg/mL, and the sample volume was 10μL.

Figure 3 is the standard curve of protein content obtained by Coomassie brilliant blue method.

Detailed ways

The separation effect of the casein component is evaluated through experiments below.

1. Evaluation of the effect of isoelectric point slow acid precipitation for crude separation of casein

In order to evaluate the purity of casein after isoelectric point slow acid precipitation, we used SDS-PAGE to compare and analyze the protein composition in skimmed milk and the protein composition in the precipitate and supernatant after isoelectric point slow acid precipitation. The results are shown in Figure 1.

It can be seen from Figure 1 that after the whole protein in lane 2 in Figure 1 is slowly acid-precipitated through the isoelectric point, the bovine serum albumin and whey protein in buffalo milk can basically be completely removed, and the crude cheese in lane 4 in Figure 1 can be obtained albumen, while impurities such as bovine serum albumin and whey protein remain in the acid whey of swimming lane 3 in Figure 1 after acid precipitation of casein. The method is simple to operate, has good separation effect and is suitable for large-scale production.

2. Evaluation of the effect of grading calcium-precipitated casein components

In order to evaluate the separation of casein components under various calcium salt concentrations, we used SDS-PAGE and Coomassie The purity and yield of casein components treated with L and 0.30mol/LCa ²⁺ were analyzed. The results are shown in Figure 2, Figure 3 and Table 1.

It can be seen from Figure 2 that when the Ca ²⁺ concentration reaches 0.06 mol/L, the α _s -casein in the casein has completely precipitated, and β-casein also precipitates at the same time, and in the precipitate except the above two In addition to the casein fraction, bovine serum albumin also completely precipitated. At the same time, in the electrophoresis bands of casein precipitation lysates treated with 0.06mol/L-0.30mol/L Ca ²⁺ concentration ^{, a weak κ-casein band appeared at 0.06mol/L Ca 2+ concentration} . As the concentration increased, the κ-casein band weakened, especially when the Ca ²⁺ concentration reached 0.20mol/L, the κ-casein band almost disappeared. In the supernatant of casein treated at 0.06mol/L-0.30mol/LCa ²⁺ concentration, there are mainly two bands of β-casein and κ-casein, and with the gradual increase of Ca ²⁺ concentration, β -The casein band gradually weakened, which was consistent with the change trend of the protein content in the supernatant determined by Coomassie Brilliant Blue. According to the above phenomena, it can be judged that the sequence of the three components of casein precipitated by Ca ²⁺ is: αs-casein is the first to precipitate calcium, followed by β-casein, and finally κ-casein. It can also be seen from Figure 2 that κ-casein begins to precipitate in the presence of an extremely low 0.06 mol/L Ca ²⁺ concentration, and with the continuous increase of Ca ²⁺ concentration, κ-casein dissolves instead and does not Re-precipitate out. This is related to the cross-linking and precipitation of κ-casein caused by α _s -casein encountering calcium precipitation. All bands of the casein supernatant after Ca ²⁺ treatment do not contain bovine serum albumin, but all contain β-lactoglobulin and α-lactalbumin with very weak bands, indicating that bovine serum albumin has a strong effect on Ca ²⁺ Very sensitive, precipitation occurs at very low concentrations, while β-lactoglobulin and α-lactalbumin have a certain tolerance to Ca ²⁺ .

Table 1 The quality of casein in various grades of calcium precipitation components

According to Table 1, it can be clearly seen that in the supernatant of casein treated at a concentration of 0.06mol/L-0.30mol/L Ca ²⁺ , the protein quality gradually decreases. They are β-casein and κ-casein, and the precipitation of κ-casein in the precipitation is small, indicating that the precipitation reaction of β-casein mainly occurs in this process. Based on this, a simple method for separating β-casein - the secondary calcium precipitation method can be proposed: firstly, the casein is treated with a concentration of 0.06mol/L Ca ²⁺ , and the precipitate is discarded by centrifugation, that is, the α _s -casein is discarded. Keep the supernatant, add divalent soluble calcium salt to make the final concentration of Ca ²⁺ reach 0.3mol/L, centrifuge again, keep the precipitate, dissolve the precipitate in disodium edetate solution, stir to fully dissolve, After dialysis, β-casein with a mass fraction of 100% can be obtained.

In order to further evaluate the mass fractions of αs-casein, β-casein and κ-casein in each casein fraction after the casein was treated with various calcium salt concentrations, we used the following calculation method to estimate them:

If bovine serum albumin and κ-casein in the pellet and whey protein in the supernatant are ignored, the ratio of α _s -casein, β-casein and κ-casein in buffalo milk is 3.77:2.45:1 , combined with the total recovery rate of graded calcium-precipitated casein, the mass fraction ratio of various casein components in the precipitate and supernatant can be estimated, taking the mass fraction of αs-casein in the precipitate as an example:

A ₁ =B ₁ ×B ₂ ×B ₃

In the formula: A ₁ is the mass of αs-casein in the precipitate; B ₁ is the total mass of casein; B ₂ is the total recovery rate of casein; B ₃ is the theoretical mass fraction of αs-casein in casein.

$\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; /</span><span\; class="notranslate">\; \%</span><span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="2"\; label="A"\; filenames="RE-HDA00001741262200011.png,RE-HDA00001741262200012.png"\; state="\{\{state\}\}">A</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}$

In the formula: A is the mass fraction of αs-casein in the precipitate; A ₁ is the mass of αs-casein in the precipitate; A ₂ is the mass of the precipitate. The calculation results are shown in Table 2:

Table 2 Mass fractions of α _s -casein, β-casein and κ-casein in calcium precipitate components of each grade

According to Table 2, within the concentration range of 0.06mol/L-0.30mol/L Ca ²⁺ , the mass fraction of α _s -casein in the precipitate decreased from 91.6% to 76.8%, and the mass fraction of β-casein in the supernatant The mass fraction of κ-casein increased from 32.2% to 43.3% from 67.8% to 56.7%.

Example

A method for separating casein components in buffalo milk, comprising the steps of:

Take 1.00kg of Morrah hybrid high-generation buffalo milk, centrifuge at 8000g, 4°C for 20 minutes, and slowly adjust the pH of the defatted milk to 4.5-4.7 with 0.1mol/L glacial acetic acid, and centrifuge at 8000g, 4°C for 30 minutes to retain the precipitate , freeze-dried to obtain crude casein freeze-dried powder, the casein sample was prepared into a 2% protein solution, and the pH of the casein solution was slowly adjusted to 7.0-7.5 with 0.2mol/L ammonia solution, according to the ratio of 0.06mol/L Add divalent soluble calcium salt, centrifuge to retain the supernatant and precipitate, dissolve the precipitate in 0.06mol/L EDTA disodium solution, dialyze and freeze-dry to obtain αs-casein with a mass fraction of 91.6%; Continue to add divalent soluble calcium salt to the supernatant to make the concentration reach 0.3mol/L, centrifuge again, keep the supernatant and precipitate, and dissolve the precipitate in 0.20mol/L EDTA disodium solution , 100% β-casein was obtained by lyophilization after dialysis; 43.3% κ-casein was obtained by lyophilization after dialysis of the retained supernatant.

Claims (1)

1. a method for separating casein components in buffalo milk, characterized in that, the method may further comprise the steps: Take 1.00kg of Morrah hybrid high-generation buffalo milk, centrifuge at 8000g, 4°C for 20 minutes, and slowly adjust the pH of the defatted milk to 4.5-4.7 with 0.1mol/L glacial acetic acid, and centrifuge at 8000g, 4°C for 30 minutes to retain the precipitate , freeze-dried to obtain crude casein freeze-dried powder, the casein sample was prepared into a 2% protein solution, and the pH of the casein solution was slowly adjusted to 7.0-7.5 with 0.2mol/L ammonia solution, according to the ratio of 0.06mol/L Add divalent soluble calcium salt, centrifuge to retain the supernatant and precipitate, dissolve the precipitate in 0.06mol/L EDTA disodium solution, dialyze and freeze-dry to obtain αs-casein with a mass fraction of 91.6%; Continue to add divalent soluble calcium salt to the supernatant to make the concentration reach 0.3mol/L, centrifuge again, keep the supernatant and precipitate, and dissolve the precipitate in 0.20mol/L EDTA disodium solution , 100% β-casein was obtained by lyophilization after dialysis; 43.3% κ-casein was obtained by lyophilization after dialysis of the retained supernatant.

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