Functional proteins in milk, including α-lactalbumin (α-La), β-lactoglobulin (β-Lg), lactoferrin (LF), lactoperoxide Enzymes (lactoperoxidase, LP), β-casein (β-casein, β-CN) and milk fat globule membrane protein (milk fat globule membrane protein, MFGMP), etc., have important physiological functions and play a role in human health. It plays an important role and is widely concerned by scientific researchers and production enterprises.
Separation technology of α-La and β-Lg
1.1 Supercritical CO2 (supercritical carbon CO2, scCO2) fractionation technology
Because the isoelectric point difference between α-La and β-Lg is 4.20-4.50 and 5.35-5.49, respectively, α-La is easier to precipitate than β-Lg in acidic medium. The scCO2 fractionation technique has the diffusivity of a gas and the density of a liquid, which increases its water solubility. The solubility of scCO2 depends on the thermodynamic equilibrium, and the thermodynamic equilibrium is regulated by the temperature and pressure of the system. The concentration of CO2 in the aqueous solution of scCO2 will affect the pH value of the medium, and the scCO2 in the aqueous solution can make α-La distillate out and enrich. Therefore, when the CO2 aqueous solution is saturated at atmospheric pressure and the pH value of the solution is reduced to 4.00, the phenomenon of α-La precipitation and β-Lg still exists in the solution. At this point 2 distinct phases are formed: a precipitated phase rich in α-La and low in β-Lg (solid phase) and a soluble phase rich in β-Lg and low in α-La (liquid phase).
At present, many studies have used scCO2 fractionation technology to separate α-La and β-Lg. By improving the separation technology and process parameters, the separation rate and purity of the two proteins have been significantly improved.
1.2 Selective thermal aggregation separation technology
Selective thermal aggregation separation is a method to decompose the α-La aggregates and induce their refolding to the native state by adjusting the pH and temperature of the solution, as well as directional aggregation of β-Lg in a controllable process. In addition to the above conditions, some other influencing factors, including protein concentration, ionic strength, salt type and concentration, chelating agents, the presence and concentration of other proteins (such as casein), etc., can affect the thermal aggregation separation process, so it can be determined by Process parameters were controlled to maximize the yield of α-La and β-Lg in the soluble native form. There are many related studies.
1.3 High Hydrostatic Pressure (HHP) Fractionation Technology
HHP fractionation technology is applied to the separation of α-La and β-Lg. The separation principle is: β-Lg will precipitate and aggregate to different degrees when it changes within a certain pressure range (200-600 MPa). It is easier to make β-Lg aggregate; acidified WP can induce four β-Lg dimers to combine to form β-Lg octamer, while α-La still exists in monomer form. Based on the above, in acidified whey solution at pH 4.6, high-pressure treatment can induce β-Lg aggregation while keeping β-Lg still stably present in the α-La-rich solution, and then centrifuging the solution to obtain α-La-enriched supernatant and β-Lg pellet.
HHP is a multi-cycle continuous treatment technology with relatively high operation and maintenance costs, but at the same time, it has the characteristics of environmental protection and high efficiency. Therefore, it still has development potential for the separation of some high value-added products, and further research is needed.
LF and LP separation technology
2.1 Membrane chromatography technology
Membrane chromatography is a mature protein purification technology, which is a separation technology based on membrane filtration and liquid chromatography. The advantage of membrane chromatography techniques compared to traditional resin chromatography is mainly in the short diffusion time, because the interaction between the molecules and the active sites in the membrane takes place in the convective through pores, rather than static in the pores of the adsorbent particles in liquid. Therefore, when membrane chromatography is applied to separate high flow rate substances and biomacromolecules with small diffusivity, it can effectively reduce the degradation and denaturation of biomolecules. The separation of whey proteins by membrane chromatography has been reported in many studies.
2.2 Multimodal chromatography techniques
Unlike ion exchange chromatography, dye affinity chromatography allows protein adsorption even under low/medium ionic strength conditions. High-efficiency chromatographic supports must have physical properties such as strong binding ability, high stability, and low possibility of being adsorbed by non-specific proteins; functional groups are also required to form cross-linking and immobilization with different ligands (such as triazine dyes). and other structural properties. Therefore, these properties make chitosan an ideal carrier material for the separation of proteins by multimodal chromatography.
Previous studies have used the natural polymer chitosan to separate whey protein, and high-purity LP and LF can be obtained. In addition, multimodal matrices also feature no need for pretreatment of whey, good mechanical resistance, and easier recovery of the resulting proteins after adsorption, washing, and elution steps.
2.3 Simulated moving bed (SMB) technology
While traditional column chromatography is the main technique for separating proteins, which is expensive compared to other separation methods, SMB technology is a way to make the column process more efficient and economical. SMB simulates countercurrent operation between solid and liquid phases by switching valves on the column or moving the column on a rotator. The advantages of countercurrent operation are: more efficient adsorption of liquid streams; higher production efficiency; increased feedstock usage efficiency, production efficiency and product concentration; buffer solution consumption and reduced plant area size.
SMB technology is a continuous chromatography technology, and its application in protein separation and purification is relatively small.
β-CN separation technology
3.1 Membrane separation technology – low temperature microfiltration technology
The separation of casein components mainly includes selective precipitation and low temperature membrane filtration. The principle and characteristics of using microfiltration membrane to separate β-CN mainly include the following aspects: the microfiltration separation process does not change the amount of β-CN in the micellar casein concentrate; CN) and κ-casein (κ-casein, κ-CN) are different in that the dissociation of β-CN from casein micelles is affected by temperature; β-CN in low temperature (<5 °C) milk, The process of separation from the casein micelles to the serum phase is reversible. Therefore, β-CN can be separated from skim milk at low temperature using a microfiltration polymeric membrane.
In the production of fermented protein-rich dairy products such as fresh cheese and “Greek-style” yogurt, centrifugation or membrane filtration is often used to increase dry matter content. And related studies have shown that the content of β-CN in micellar casein concentrate can also be changed by microfiltration technology.
3.2 Selective precipitation separation technology
The selective precipitation method utilizes the calcium sensitivity of β-CN and adds CaCl2 to the alkaline solution to separate the β-CN components from the raw casein micelles. Studies have shown that β-CN is more likely to precipitate when the pH of the solution is greater than 7, and then β-CN can be separated without using high-speed centrifugation, and the separated β-CN can be further purified by filtration and demineralization.
In addition to the influence of the processing scale on the separation results of β-CN, some environmental factors, such as temperature, pH and CaCl2 concentration, have an effect on the purity and yield of β-CN. Therefore, further studies on the impact of environmental factors on the purity and yield of casein fractions are required.