CN105732794A - Method of recyclable large-scale separation of [beta]-lactoglobulin - Google Patents

Abstract

The invention relates to a recyclable method for large-scale separation of beta-lactoglobulin, which comprises the steps of obtaining whey liquid, extracting two aqueous phases, filtering, drying the retentate, and recycling the filtrate. Separate the whey liquid from fresh Holstein milk and buffalo milk, add PVP/sodium citrate to form a two-phase system, mix well, and centrifuge until the upper and lower phases are separated; extract the lower phase solution obtained in the two-phase extraction step, pass through the membrane Filtration; collect the membrane filtration retentate obtained by filtration, and obtain β-lactoglobulin by spray drying or freeze-drying; recycling of the filtrate Add the filtrate obtained from the filtration step into the two-phase system, which can be recycled for use in β-lactoglobulin Separation of globulins. The advantages are: large-scale and rapid separation of β-lactoglobulin with high recovery rate; the separation system is environmentally friendly and recyclable, which is conducive to reducing production costs and pollution; it is suitable for a variety of raw milk, and the obtained products are suitable for scientific research and functional food In the industrial production and processing.

Description

A recyclable method for large-scale separation of β-lactoglobulin

technical field

The invention relates to a method for separating and purifying β-lactoglobulin, in particular to a method for recyclable and large-scale separation of β-lactoglobulin.

Background technique

β-lactoglobulin exists in the milk of cattle and other ruminants, and is the main protein in whey, and it is also the main substance that dairy products cause allergic reactions in infants and young children. However, with the development of new technology and processing methods, the biological functions and good food processing characteristics of β-lactoglobulin and its peptides have been gradually developed. For example, the special protein spatial structure of β-lactoglobulin can embed fat-soluble vitamins, polyunsaturated fatty acids, and flavonoids, and protect the stability of these substances in the digestive system of the gastrointestinal tract; β-lactoglobulin hydrolyzed peptide It has antibacterial and antioxidative functions; in the food processing of dairy products, meat products and bread production, β-lactoglobulin also exhibits excellent gelling, water holding, emulsifying and foaming properties. Separation and purification of β-lactoglobulin has certain significance for scientific research and industrialization of functional foods.

In the prior art, some technical solutions have been disclosed for the separation of β-lactoglobulin, but none of them can carry out recyclable large-scale separation. For example, the Chinese patent of the existing separation technology CN201410534492.2 discloses the preparation method and product of α-lactalbumin and/or β-lactoglobulin. The whey is ultrafiltered through membranes with a pore size of 100kDa and 30kDa, and the pH is adjusted to 3.5~ 5.0, the purity of β-lactoglobulin powder is greater than 70%. Although this method is simple, it does not remove non-protein substances in whey, and the purity of the obtained β-lactoglobulin powder is low; since the molecular weight of β-lactoglobulin is 18.4kDa and that of α-lactalbumin is 14.2kDa, the two proteins The molecular weight is similar, and the membrane separation method has very high requirements on the membrane; in addition, the method is carried out at a pH of 3.5~5.0, and the isoelectric point PI of β-lactoglobulin powder is 5.1~5.3, which is prone to denaturation and precipitation. Membrane filtration efficiency is low, and high-purity, recyclable and large-scale separation cannot be achieved.

The Chinese patent of existing separation technology CN201210231797.7 discloses a method for isolating β-lactoglobulin from raw milk, using repeated salting out and ultrafiltration to obtain high-purity β-lactoglobulin (purity is 85.2%); The Chinese patent of separation technology CN20140154517.6 discloses separating β-lactoglobulin from desalted whey powder, and obtaining high-purity β-lactoglobulin through the steps of salting out, ultrafiltration, ion exchange chromatography, and gel chromatography ( The purity is not given); although these two methods have obtained β-lactoglobulin with higher purity, the steps are cumbersome and the amount of sample processing is small, so they are suitable for operation in the laboratory.

The Chinese patent of existing separation technology CN200910161153.3 discloses the separation and determination method of whey protein. The β- Lactoglobulin a and b are completely separated. This method has a fast separation speed, but the sample processing volume is 0.02g, which is suitable for the analysis and detection of lactoglobulin.

The two-phase extraction technology is to divide the two phases of the aqueous solution, and the biological substances with different entropy and enthalpy can be separated through the thermodynamic effect. The extraction system has the advantages of mild conditions, fast mass transmission, low cost, easy operation, adjustable and easy to scale up. , Continuous operation and so on. The Chinese patent with patent number CN201310460499.X discloses a method for separating milk-derived whey protein in a PEG/phosphate two-phase aqueous phase. The method uses a PEG/phosphate two-phase aqueous system to combine α-lactalbumin and The globulins were separated into the upper phase PEG solution and the lower phase phosphate solution, and the protein composition of the upper and lower phases was analyzed by SDS-PAGE electrophoresis. However, this method simply separates the α-lactalbumin and β-lactoglobulin in whey into different phases, and does not recover the β-lactoglobulin, nor does it provide the purity of the separated β-lactoglobulin , the amount of sample processing is small, and large-scale separation cannot be carried out.

Contents of the invention

The purpose of the present invention is to provide a recyclable method for large-scale separation of lactoglobulin, which can not only process a large number of samples, but also recycle the separation system; the separated and recovered β-lactoglobulin has high purity. The present invention adopts a two-phase aqueous system formed by polyvinyl pyrrolidone (PVP)/sodium citrate. After phase separation, β-lactoglobulin exists in the lower phase of sodium citrate solution and is filtered out through a membrane. The final sodium citrate solution is still added to the original two-phase system for recycling; in addition, PVP is a synthetic water-soluble polymer compound with good solubility and physiological compatibility, and low toxicity, which can be used in In medicine, food and cosmetics; therefore, the separation method adopted in the present invention is both environmentally friendly and efficient.

The technical scheme adopted in the present invention is as follows:

A recyclable method for large-scale separation of β-lactoglobulin, comprising five steps of 1) sample pretreatment, 2) aqueous two-phase extraction, 3) filtration, 4) drying of the retentate, and 5) recycling of the filtrate , characterized by:

1) Sample pretreatment: Pretreat one of Holstein milk, buffalo milk or commercial whey powder to obtain whey liquid with a protein content of 5% to 50%.

2) Two-phase aqueous extraction: add the whey liquid obtained in step 1) into the two-phase system formed by polyvinyl pyrrolidone (PVP) and sodium citrate according to the volume ratio of 1:20~1:250, Mix and centrifuge until the upper and lower phases are separated; in the two-phase system, the quality of PVP is 9-21% of the total mass of the system, the quality of sodium citrate is 15-25% of the total mass of the system, and the pH is controlled at 5.2-8.2; The above-mentioned mixing means that the stirring speed is 50-70rpm, and the stirring time is 20-40min; the above-mentioned centrifugation means that the speed is 2500-3500rpm, the centrifugation time is 10-30min, and the centrifugation temperature is 4-37°C. The upper phase in which the upper and lower phases are separated refers to a polyvinylpyrrolidone solution containing part of whey protein, and the lower phase refers to a sodium citrate solution containing a large amount of β-lactoglobulin;

3) Filtration: Take out the sodium citrate lower phase solution containing β-lactoglobulin in step 2), and filter it through a 1~10kDa membrane, the membrane filtration temperature is 4~37°C, and the control pressure is ≤0.2MPa to obtain the retentate and filter Out liquid; described retentate refers to the aqueous solution that contains a large amount of β-lactoglobulins intercepted; Described filtrate refers to the sodium citrate solution that filters out;

4) Dry the retentate: collect the aqueous solution containing a large amount of β-lactoglobulin retained in step 3), and obtain β-lactoglobulin by spray drying or freeze drying.

5) Filtrate recycling: the sodium citrate filtrate obtained in step 3) can be added to the two-phase aqueous system in step 3), and can be recycled and applied to the separation of β-lactoglobulin.

Further, the 1) sample pretreatment refers to pasteurization and degreasing of one of Holstein milk and buffalo milk, adjusting the pH to the isoelectric point of casein for precipitation, centrifugation, collecting the upper whey liquid, and passing through 1 ~6kDa membrane filtration, the retentate is concentrated under reduced pressure to a protein concentration of 5%~50%; the commercial whey powder pretreatment refers to the commercial whey powder (WPI80~90) with a purity of 80~90 is dissolved in water, commercial The whey powder mass accounts for 5% to 50% of the total mass of the solution.

Further, in the 2) aqueous two-phase extraction, PVP is one of PVP3500, PVP4000, and PVP5000.

Further, in the above 3) filtration, the material of the membrane is one of polyvinylidene fluoride, cellulose acetate membrane, regenerated cellulose membrane or ceramic membrane.

Further, in the step 4) of drying the retentate, the β-lactoglobulin obtained by spray drying can be applied to the industrial processing of functional foods; the β-lactoglobulin obtained by the freeze-drying step is an active protein and can be used in scientific research.

Further, in the step of 5) recycling the filtrate, adjusting the sodium citrate filtrate to a certain concentration refers to adjusting the concentration of the filtrate to be consistent with the proportion of sodium citrate in the original two-phase system.

The beneficial effects of the present invention are:

1. The present invention is not limited by the source of raw materials, which can be Holstein milk, buffalo milk, or commercial whey powder, and the sample processing volume is large, which increases the resources of β-lactoglobulin.

2. The two-phase aqueous system in the present invention adopts environment-friendly materials PVP and sodium citrate, which can still be recycled after the separation of β-lactoglobulin, which greatly reduces the separation cost.

3. The pH condition adopted by the separation method of the present invention is mild, the temperature is controllable, and the separated and recovered β-lactoglobulin has high purity. Since higher temperature and low pH can lead to denaturation of proteins, the present invention can obtain biologically active β-lactoglobulin suitable for scientific research under freeze-drying conditions according to different separation temperatures, and can obtain biologically active β-lactoglobulin suitable for scientific research under spray-drying conditions. Functional beta-lactoglobulin for food processing.

4. The recyclable large-scale separation method of lactoglobulin provided by the present invention includes five steps of sample pretreatment, two-phase extraction, filtration, retentate drying, and filtrate recycling. The operation process is simple and large-scale , low-cost industrial production.

The raw material that the present invention adopts is described as follows:

The polyvinyl pyrrolidone (polyvinyl pyrrolidone, PVP),

The commercial whey powder pretreatment refers to commercial whey powder with a purity of 80-90 (WPI80-90).

Description of drawings

Fig. 1 is the phase diagram of PVP/3000/sodium citrate

Figure 2 is the effect of two-phase separation of β-lactoglobulin by SDS-PAGE electrophoresis

Among them, Marker is a known molecular weight standard protein, the molecular weight ranges from 14.4kDa to 97kDa; α-la indicates α-lactalbumin in whey; β-lg indicates β-lactoglobulin in whey.

Figure 3-Figure 5 is the high performance liquid chromatogram of β-lactoglobulin

Wherein Fig. 3 is the β-lactoglobulin standard product high-performance liquid chromatogram, Fig. 4 is the whey protein high-performance liquid chromatogram, Fig. 5 is the separated β-lactoglobulin high-performance liquid chromatogram.

Fig. 6 is the standard curve of the β-lactoglobulin established with high performance liquid chromatography,

The standard curve regression equation is:

.

detailed description

The following examples are further illustrations of the present invention, not limitations of the present invention.

Example 1

A recyclable method for large-scale separation of β-lactoglobulin, taking the processing of 50L of Holstein milk to produce β-lactoglobulin for scientific research as an example; the following steps are carried out in sequence:

1) Sample pretreatment: Skim 50L of Holstein milk with a milk fat separator to prepare skim milk with a fat content of ≤0.5wt%; adjust the pH of the skim milk to 4.6~4.7, heat-treat at 60°C for 30min, and take the supernatant for desalination. Concentrate at a low temperature of 30-50°C to obtain 15L of whey liquid, and its whey protein content is 20wt%;

2) Two-phase aqueous phase extraction: add the whey liquid obtained in step 1) into the two-phase aqueous system formed by polyvinyl pyrrolidone (PVP)/sodium citrate at a volume ratio of 1:100, mix well, and centrifuge until The upper and lower phases are separated; for the two-phase system, the mass fraction of PVP and sodium citrate is controlled above the curve of the PVP/sodium citrate phase diagram (Figure 1), so that the mass of PVP is 12% of the total mass of the system, and sodium citrate The mass is 18% of the total mass of the system, the volume ratio of the two phases is 1, and the pH is controlled at 7.0; the whey liquid is added to the above-mentioned two-phase system, and the stirring speed is 60rpm, and the stirring time is 30min. /min Centrifuge for 20min;

3) Separation effect of β-lactoglobulin:

The separation of β-lactoglobulin in whey in two-phase system was detected by polyacrylamide gel electrophoresis.

Electrophoresis conditions: 5% stacking gel, 14% separating gel, loading volume 10 μL, loading sequence is protein standard molecular weight marker, α-lactalbumin standard, β-lactoglobulin standard, whey, upper phase solution, lower phase phase solution. The molecular weight of β-lactoglobulin is 18.2 kDa.

After testing, it was found that β-lactoglobulin was well separated into the lower phase of the two-phase aqueous system, that is, the sodium citrate phase (Figure 2)

4) Filtration: Take out the sodium citrate lower phase solution containing β-lactoglobulin in step 2) from the bottom of the two-phase extraction tank, and filter it through a 10kDa membrane, the membrane filtration temperature is 4°C, and the control pressure is ≤0.2MPa;

5) Dry the retentate: collect the aqueous solution containing a large amount of β-lactoglobulin intercepted in step 3) by membrane filtration, and freeze-dry at -45°C to obtain the active β-lactoglobulin freeze-dried powder (β-lactoglobulin) for scientific research Protein purity ≥95 wt %);

6) Determination of β-lactoglobulin

The content of β-lactoglobulin in whey and β-lactoglobulin freeze-dried powder is determined by high performance liquid chromatography, and the specific measurement conditions are as follows:

Chromatographic column: ZORBAX 300SB-C18 chromatographic column, 4.6×250mm

Mobile phase: Phase A: 0.1% TFA volume concentration ultrapure aqueous solution

Phase B: 0.1% TFA in acetonitrile by volume

Gradient elution: 0-35min 30%-50% phase B

35-40min 50%-30% Phase B

Flow rate: 1mL/min

Detection wavelength: 215nm

Column temperature: 30°C

Injection volume: 10μL

Prepare the commercial β-lactoglobulin standard, the whey protein concentrate obtained in step 1), and the β-lactoglobulin freeze-dried powder obtained in step 5) respectively to a final concentration of 1 mg/mL, and take 10 μL for the above high-efficiency solution β-lactoglobulin was detected by phase chromatography, and the results are shown in Figure 2.

The concentrations of β-lactoglobulin commercially purchased standard products were respectively prepared as follows: 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL, 0.015625mg/mL, to obtain There is a linear relationship between lactoglobulin concentration and high performance liquid chromatography peak area, as shown in Figure 3. The standard curve linear regression equation is:

According to the regression equation, the concentration of β-lactoglobulin in the whey protein concentrate obtained in step 1) is 48.9%, and the concentration of β-lactoglobulin in the β-lactoglobulin freeze-dried powder obtained in step 5) is 98.2%. . Use high-performance liquid chromatography to measure the concentration of β-lactoglobulin in the two-phase system, and calculate the recovery rate Y according to the formula (3):

(1) r=V _up /V _down (1)

(2) K=C _up /C _down (2)

(3) Y=1/(1+r×K)×100% (3)

In the formula: r is the phase; _Vupper and Vlower are the volumes (L) _of the _upper and _lower phases; Cupper and Clower are the mass concentrations (mg/mL) of the target isolate in the upper and lower phases; K is the partition coefficient. When r=1, the calculated recovery of β-lactoglobulin in the sodium citrate phase was 98.2%;

7) Recycling of the filtrate: The sodium citrate filtrate obtained in step 3) is adjusted to 1 according to the upper and lower ratios, adjusted to a final concentration of 18%, and added to the two-phase aqueous system in step 2), which can be recycled for use in β-emulsion Separation of globulins.

Therefore, 150 g of β-lactoglobulin freeze-dried powder can be finally obtained for every 50 L of Holstein's milk, with a purity of 98.2%.

Example 2

Take the processing of 500L Holstein milk to produce β-lactoglobulin for food processing as an example; follow the steps in turn:

1) Sample pretreatment: Skim 500L of Holstein milk with a milk fat separator to prepare skim milk, the fat content of which is ≤0.5wt%. Concentrate at a low temperature of 30~50°C to obtain whey liquid, and its whey protein content is 5wt%;

2) Two-phase aqueous phase extraction: Add the whey liquid obtained in step 1) into the two-phase aqueous system formed by polyvinylpyrrolidone (PVP)/sodium citrate at a volume ratio of 1:25, mix well, and centrifuge up and down phase separation; wherein, the PVP quality is 15% of the total mass of the system in the two-phase aqueous system, and the sodium citrate quality is 22% of the total mass of the system, and the pH is controlled at 7.6; The speed is 60rpm, and the mixing time is 30min. After mixing, centrifuge at 4°C and 3500r/min for 20min;

3) Filtration: Take out the sodium citrate lower phase solution containing β-lactoglobulin in step 2) from the bottom of the two-phase extraction tank, and filter it through a 10kDa membrane, the membrane filtration temperature is 4°C, and the control pressure is ≤0.2MPa;

4) Dry the retentate: collect the aqueous solution containing a large amount of β-lactoglobulin intercepted by the membrane filtration in step 3), and spray dry to obtain β-lactoglobulin for food processing;

5) Filtrate recycling: adjust the sodium citrate filtrate obtained in step 3) to a final concentration of 22%, add the two-phase system in step 2), which can be recycled for the separation of β-lactoglobulin;

Every 500L of Holstein milk can finally get 700g of β-lactoglobulin powder with a purity of 95.3%.

Example 3

To process 50L of buffalo milk, replace the Holstein milk in step 1) with buffalo milk, and the rest is the same as in Example 1.

Every 50L of buffalo milk can finally obtain 197g of β-lactoglobulin powder with a purity of 97%.

Example 4

Process 50kg of commercial whey powder, cancel step 1), prepare the final concentration of commercial whey powder to 20wt%, and the rest is the same as in Example 1.

Every 50kg of commercial whey powder can finally get 18.2kg of β-lactoglobulin powder with a purity of 98%.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (7)

1. A method for recyclable large-scale separation of β-lactoglobulin, comprising five steps of sample pretreatment, aqueous two-phase extraction, filtration, drying retentate, and filtrate recycling, characterized in that: 1) The sample pretreatment step: pretreat one of Holstein milk, buffalo milk or commercial whey powder to obtain whey liquid, the protein content of the whey liquid is 5% to 50%; 2) The two-phase extraction step: add the whey liquid obtained in step 1) into the two-phase system composed of polyvinylpyrrolidone and sodium citrate according to the volume ratio of 1:20~250, mix and centrifuge until The upper and lower phases are separated; the mass of polyvinylpyrrolidone in the two-phase system is 9-21% of the total mass of the system, the mass of sodium citrate is 15-25% of the total mass of the system, and the pH value is controlled in the range of 5.2-8.2 In; the mixing means that the stirring speed is 50~70rpm, and the stirring time is 20~40min; The upper phase separated from the upper and lower phases refers to a polyvinylpyrrolidone solution containing part of whey protein, and the lower phase refers to a sodium citrate solution containing a large amount of β-lactoglobulin; 3) The filtration step: take out the sodium citrate lower phase solution containing β-lactoglobulin in step 2), and filter it through a 1~10kDa membrane, the membrane filtration temperature is 4~37°C, and the control pressure is ≤0.2MPa to obtain the cut-off Liquid and filtrate; Described retentate refers to the aqueous solution that contains a large amount of β-lactoglobulin; Described filtrate refers to sodium citrate solution; 4) The step of drying the retentate: collecting the aqueous solution containing a large amount of β-lactoglobulin retained in step 3) (to obtain β-lactoglobulin after drying; 5) The filtrate recycling step: the sodium citrate filtrate obtained in step 3) can be added to the two-phase aqueous system in step 3), and can be recycled and applied to the separation of β-lactoglobulin. 2. The method of recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, characterized in that: said step 1) sample pretreatment refers to a kind of pasteurized milk in Holstein's milk and buffalo milk. After sterilization and degreasing, adjust the pH to the isoelectric point of casein, precipitate and centrifuge, collect the upper whey liquid, filter it through a 1-6kDa membrane, and concentrate the retentate under reduced pressure to a protein concentration of 5%-50%; the commercial milk Clean powder pretreatment refers to the commercial whey powder with a purity of 80-90 is dissolved in water, and the mass of commercial whey powder accounts for 5%-50% of the total mass of the solution. 3. The method for recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, characterized in that: in the step 2) in the two-phase extraction step, the polyvinylpyrrolidone is PVP3500, One of PVP4000 and PVP5000. 4. The method of recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, characterized in that: in the step 3) in the filtration step, the material of the membrane is polyvinylidene fluoride, acetic acid One of cellulose ester film, regenerated cellulose film or ceramic film. 5. The method for recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, characterized in that: said step 4) in the step of drying the retentate, the β-lactoglobulin obtained by spray drying can be used It is used in the industrial processing of functional foods; the β-lactoglobulin obtained through the freeze-drying step is an active protein and can be used in scientific research. 6. the method for recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, is characterized in that: in the described 5) filtrate recycling step, the sodium citrate filtrate is transferred to a certain concentration of Refers to adjusting the concentration of the filtrate to be consistent with the proportion of sodium citrate in the original two-phase system. 7. The method for recyclable large-scale separation of β-lactoglobulin as claimed in claim 1, characterized in that: in the 4) step of drying the retentate, the drying method of the β-lactoglobulin aqueous solution is spray drying or Freeze dried.