Method for rapidly separating sheep serum albumin based on aqueous two-phase system

Abstract

The invention provides a method for rapidly separating sheep serum albumin based on an aqueous two-phase system, the aqueous two-phase system comprises ethanol, sodium dihydrogen phosphate and water, protein aggregation or denaturation can be effectively prevented by using the screened and optimized aqueous two-phase system with specific components and proportions, the quality of sheep serum albumin is guaranteed to a great extent, and the method is suitable for industrial production. The recovery rate and the purity of albumin are remarkably improved, and the sheep blood utilization rate is greatly improved. By virtue of the advantages, the method and the sheep serum albumin extracted by using the method have wide application prospects in various fields such as food and biological materials.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for rapidly separating sheep blood albumin based on a two-aqueous phase system. Background Art

[0002] Sheep blood, as a key by-product of the livestock slaughtering process, is rich in nutrients and bioactive ingredients, such as protein, amino acids, various enzymes, vitamins, hormones, minerals, sugars and lipids, etc. However, the utilization efficiency of sheep blood resources is extremely low, and most slaughtering companies directly discharge the blood, resulting in a large waste of precious protein resources and environmental pollution.

[0003] Sheep blood is rich in protein, amino acids, vitamins and a variety of bioactive substances, among which protein is its main component, with a content of 17% to 19%. Albumin is one of the most important proteins in sheep blood, accounting for 60% of the total plasma protein (Cheng Chi, Cai Yongfeng. Development and Utilization of Edible Animal Blood Resources [J]. Food and Fermentation Industry, 1998, 24(3):7.). Sheep blood albumin is rich in essential amino acids and has a high bioavailability, which makes it show important value in the development of nutritional supplements and health foods. Due to its good nutritional composition, sheep blood albumin has broad application prospects in postoperative repair. It can quickly supplement protein, promote the patient's recovery process, and help accelerate tissue repair and recovery.

[0004] At present, the separation methods of sheep blood albumin mainly include salting-out method, organic solvent precipitation method, ultrafiltration method and chromatographic separation method. However, these traditional methods have certain limitations in practical applications. For example, the salting-out method is widely used because of its simple operation, but it requires the use of a high-concentration salt solution to achieve protein precipitation. This process may damage the purity of the protein and the desalting process is often complicated and time-consuming. Chinese patent CN201710059284.5 uses a saturated ammonium sulfate graded precipitation method to obtain bovine serum albumin, but the preparation process is cumbersome and requires chromatographic column desalination, which increases the cost. The ultrafiltration method uses a selective permeable membrane to remove small molecule impurities, but it is easy to cause membrane contamination and clogging, thereby reducing the recovery efficiency and separation efficiency. Organic solvent precipitation is currently the most commonly used technical means for separating plasma albumin. The separation is based on the precipitation of the protein isoelectric point, but the process may cause the denaturation of sheep blood albumin and affect its functional characteristics. For example, Chinese patent CN200410077667.8 uses cold ethanol to separate albumin from human plasma protein waste components, but this method is limited by strict separation conditions and does not explain the purity and structural characteristics of the obtained albumin. Chromatographic separation technology has shown certain advantages in the field of protein extraction, but the high investment in equipment limits its possibility of large-scale application. Chinese patent CN202410514938.9 discloses a method of using two chromatography to separate canine albumin from canine blood. Although the purity of the protein is improved, the cost of the chromatographic medium is relatively high, which limits the wide application of this method.

[0005] Therefore, how to improve separation purity and reduce costs while maintaining the activity of sheep blood albumin remains a key challenge in current research and application. Summary of the invention

[0006] In order to overcome the shortcomings of the existing technology, improve the separation efficiency of sheep blood albumin and reduce the cost, an efficient and low-cost sheep blood albumin separation technology is developed. The present invention proposes a method for rapid separation of sheep blood albumin based on an ethanol-salt aqueous two-phase system, which significantly improves the separation efficiency by adjusting the system components and operating conditions, realizes the efficient extraction and application of sheep blood albumin, and opens up a new way for the high-value utilization technology of animal blood resources.

[0007] The present invention provides a method for rapidly separating sheep blood albumin based on a two-aqueous phase system, the method comprising the following steps: (1) adding a two-phase aqueous system to sheep plasma, stirring the resulting mixture, letting it stand, centrifuging, and collecting an upper phase solution, wherein the two-phase aqueous system comprises ethanol, sodium dihydrogen phosphate, and water; (2) Remove ethanol from the upper phase solution.

[0008] In some embodiments of the present invention, the two-phase aqueous system consists of ethanol, sodium dihydrogen phosphate and water.

[0009] Specifically, the molecular weight of the sheep blood albumin is about 66-68 kDa, preferably 66 kDa.

[0010] Specifically, the method for obtaining sheep plasma is as follows: an anticoagulant is added to sheep blood, followed by centrifugation, and the supernatant is retained to obtain sheep plasma.

[0011] Specifically, the sheep blood needs to be inspected and quarantined before use and can only be used as a raw material for extracting sheep blood albumin after passing the inspection.

[0012] Preferably, the anticoagulant is selected from ethylenediaminetetraacetic acid, sodium citrate, and heparin, and more preferably 3.8% sodium citrate.

[0013] Preferably, the volume ratio of the anticoagulant to sheep blood is 1:(5-20), such as 1:5, 1:7, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:16, 1:18, or 1:20, etc., and more preferably 1:10.

[0014] Specifically, the obtained sheep plasma can be stored frozen at -20°C. When in use, the frozen sheep plasma can be thawed in a water bath at 37°C before use.

[0015] Preferably, the aqueous two-phase system further includes sodium chloride.

[0016] In a specific embodiment of the present invention, the aqueous two-phase system includes ethanol, sodium dihydrogen phosphate, sodium chloride, and water.

[0017] Preferably, in step (1), the mass percentage of sheep plasma in the mixture is 1-5%, such as 1%, 2%, 3%, 4%, or 5%, etc., and more preferably 2-4%.

[0018] Preferably, in step (1), the mass percentage of ethanol in the mixture is 25-35%, such as 25%, 26%, 27%, 28%, 29%, 30%, 32%, 33%, or 35%, etc., and more preferably 25-30%.

[0019] Preferably, in step (1), the mass percentage of sodium dihydrogen phosphate in the mixture is 16-24%, such as 16%, 17%, 18%, 19%, 20%, 22%, or 24%, etc., and more preferably 16-20%.

[0020] Preferably, in step (1), the mass percentage of sodium chloride in the mixture is 0-10%, such as 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 10%, etc., and more preferably 1-5%.

[0021] In a specific embodiment of the present invention, in the aqueous two-phase system, the mass percentage of the sheep plasma in the mixture is 3%, the mass percentage of the ethanol in the mixture is 30%, and the mass percentage of the sodium dihydrogen phosphate in the mixture is 20%.

[0022] In a specific embodiment of the present invention, in the aqueous two-phase system, the mass percentage of the sheep plasma in the mixture is 4%, the mass percentage of the ethanol in the mixture is 28%, the mass percentage of the sodium dihydrogen phosphate in the mixture is 18%, and the mass percentage of the sodium chloride in the mixture is 3%.

[0023] Preferably, the standing time is 10 to 60 min, for example, 10 min, 15 min, 20 min, 30 min, 35 min, 40 min, 45 min, 50 min or 60 min.

[0024] Preferably, the centrifugation conditions are: the centrifugation temperature is 1~10°C (for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, etc.), the centrifugation speed is 2500~4500rpm (for example, 2500rpm, 3000rpm, 3500rpm, 4000rpm or 4500rpm, etc.), and the centrifugation time is 5~25min (for example, 5min, 10min, 15min, 20min or 25min, etc.).

[0025] In a specific embodiment of the present invention, the centrifugation conditions are: centrifugation temperature is 4°C, centrifugation speed is 3500 rpm, and centrifugation time is 15 min.

[0026] Specifically, the step (1) is carried out at room temperature, and the room temperature is 20-35°C.

[0027] Specifically, the method for removing ethanol in step (2) is dialysis technology, and the dialysis process uses a dialysis bag with a molecular weight cutoff of 10 kDa.

[0028] Specifically, step (2) also includes a freeze-drying operation.

[0029] Preferably, the conditions of the freeze-drying operation are: the freeze-drying temperature is -90 ~ -70°C (for example, -90°C, -85°C, -80°C, -75°C or -70°C, etc.), and the freeze-drying time is 36~60h (for example, 36h, 40h, 44h, 46h, 48h, 50h, 52h, 56h or 60h, etc.).

[0030] In a specific embodiment of the present invention, the method comprises the following steps: (1) After fresh sheep blood has passed the inspection and quarantine, add 10% of the original blood volume of anticoagulant and centrifuge at 4°C 3500rpm for 15 minutes. Then take out the supernatant and store it in a freezer at -20°C; (2) When ready to use, take out the frozen sheep plasma and thaw it in a water bath at 37°C to obtain sheep plasma; (3) successively adding ethanol, sodium dihydrogen phosphate and water to sheep plasma to form a two-phase aqueous system, wherein the sheep plasma accounts for 3% by mass of the mixture, ethanol accounts for 30% by mass of the mixture, sodium dihydrogen phosphate accounts for 20% by mass of the mixture, and the remainder of the two-phase aqueous system is composed of water; stirring and then allowing to stand; (4) After standing, the two phases are separated and the upper phase solution is collected; (5) Remove the ethanol from the upper phase solution and freeze-dry to obtain sheep serum albumin.

[0031] In another aspect of the present invention, there is provided a use of sheep blood albumin prepared by the above method in food or biochemical materials.

[0032] Preferably, the food is a nutritional supplement or a food additive.

[0033] The hydrolyzate of sheep blood albumin is rich in various amino acids, peptides and trace elements, and is suitable as a nutritional supplement to improve the nutritional value of food. At the same time, it has good gelling, emulsifying and moisturizing functional properties, and can be used as a food additive to improve the taste and quality of food.

[0034] Preferably, the biomaterial can be used as a raw material for protein electrophoresis or cell culture, an antigen for immunoassay, a biofilm, a bioglue or a drug carrier.

[0035] In protein electrophoresis, it can be used as an important experimental material. Under the action of the electric field, it can provide a reliable reference standard for the separation and analysis of other proteins, helping researchers to accurately interpret the characteristics and differences of proteins. In the process of cell culture, using sheep blood albumin as an additive to the cell culture medium can provide the necessary nutrients and growth factors for cell growth, creating an ideal microenvironment for cell culture.

[0036] In terms of immunoassay, sheep blood albumin can act as an immunogen and be used to prepare antibodies. These antibodies can accurately recognize and bind to sheep blood albumin or other structurally related proteins, playing a vital role in disease diagnosis and quality testing of biological products.

[0037] In the preparation of biomaterials, sheep blood albumin can be used to manufacture products such as biological glue and biofilm, and plays a role in tissue engineering and wound healing.

[0038] In the field of drug carriers, sheep blood albumin can be used as a drug carrier to accurately deliver drugs to specific tissues or cells, thereby improving the efficacy of drugs and reducing side effects.

[0039] The beneficial effects of the present invention are: (1) The present invention has developed a method for extracting sheep blood albumin through experimental screening, which has the outstanding characteristics of short process, simple operation and high production efficiency. In particular, by using a two-phase aqueous system with specific components and ratios, protein aggregation or denaturation can be effectively prevented during the extraction process, which greatly guarantees the quality of sheep blood albumin. It can also significantly improve the recovery rate and purity of albumin products, greatly improve the utilization rate of sheep blood, and avoid environmental pollution caused by direct discharge of sheep blood. With these advantages, this method and the sheep blood albumin extracted by this method can be widely used in many fields such as food and biomedicine.

[0040] (2) The present invention uses mild conditions to extract proteins. The entire operation process is rigorous and controllable, avoiding damage to the protein structure and allowing the protein to always maintain its original excellent activity.

[0041] (3) The present invention compares the functions of albumin from different species, providing a theoretical basis for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein: Figure 1 : Two-phase aqueous phase diagrams of different two-phase aqueous systems.

[0043] Figure 2 : Result diagram of phase separation process of different aqueous two-phase systems.

[0044] Figure 3 : Electrophoretic diagrams of various layers in the phase separation process of different aqueous two-phase systems, wherein A is the electrophoretic diagram of plasma protein separation by ethanol and sodium dihydrogen phosphate system; B is the electrophoretic diagram of plasma protein separation by ethanol and dipotassium hydrogen phosphate system; C is the electrophoretic diagram of plasma protein separation by ethanol and sodium citrate system; D is the electrophoretic diagram of plasma protein separation by ethanol and sodium carbonate system; M is a protein molecular weight standard; and plasma is extracted sheep plasma.

[0045] Figure 4 : The extraction rate and purity analysis diagram of sheep blood albumin separated by different two-phase aqueous systems, wherein A is the recovery rate diagram of sheep blood albumin separated by two-phase aqueous systems with different types of salts, and B is the purity diagram of sheep blood albumin separated by two-phase aqueous systems with different types of salts.

[0046] Figure 5 : Results of reconstitution of sheep serum albumin after extraction with different aqueous two-phase systems and freeze-dried.

[0047] Figure 6 : SDS-PAGE image of sheep serum albumin separated by ethanol-sodium dihydrogen phosphate two-phase aqueous system, wherein lane 1: M is a protein molecular weight standard; lane 2: plasma is sheep plasma; lane 3: sample A; lane 4: standard product is sheep serum albumin standard product.

[0048] Figure 7 : Infrared spectrum and fluorescence spectrum of sheep blood albumin separated by ethanol-sodium dihydrogen phosphate two-phase system, wherein A is the infrared spectrum and B is the fluorescence spectrum.

[0049] Figure 8 : DSC diagram of separation of sheep serum albumin by ethanol-sodium dihydrogen phosphate aqueous two-phase system.

[0050] Fig. 9 : Comparison of emulsification stability of albumin from different species.

[0051] Fig.10 : Comparison of albumin gel strength from different species.

[0052] Note: a, b, and c in the attached figure represent p <0.05. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0054] Example 1 Screening of Combinations of Two-Aqueous Phase Systems 1. Screening method of two-phase aqueous system: (1) First, weigh a certain amount of inorganic salt (m 1 ) (the inorganic salts are potassium bicarbonate, sodium carbonate, sodium sulfate, sodium citrate, sodium acetate, dipotassium hydrogen phosphate or sodium dihydrogen phosphate) add a certain amount of distilled water (m 2 ) until it is just dissolved; (2) Add ethanol (m 3 ) until the mixed solution just becomes turbid, and then let it stand for equilibrium; (3) The equilibrium process is such that when a drop of water is added, the solution immediately becomes clear, and when a little more ethanol is added, the solution immediately becomes turbid; (4) Repeat the above steps to observe whether the phases can be separated and calculate the mass fraction of each component of the solution at the turbidity point according to the following formula.

[0055] The results are shown in Table 1.

[0056] Table 1 Phase formation of different types of salts

[0057] Calculate the mass fraction of the salts that form a stable phase with ethanol (dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium carbonate) according to the above step (4) and draw a two-phase aqueous phase diagram (such as Figure 1 Determine the phase separation conditions.

[0058] Example 2 Screening of a two-phase aqueous system for separating sheep blood albumin 1. Extraction method of sheep blood albumin: (1) After fresh sheep blood has passed the inspection and quarantine, add 10% of the original blood volume of anticoagulant and centrifuge at 4°C 3500rpm for 15 minutes. Then take out the supernatant and store it in a freezer at -20°C; (2) When ready to use, take out the frozen sheep plasma and thaw it in a water bath at 37°C to obtain sheep plasma; (3) successively adding ethanol, sodium dihydrogen phosphate and water to sheep plasma to form a two-phase aqueous system, wherein the sheep plasma accounts for 3% by mass of the mixture, ethanol accounts for 30% by mass of the mixture, sodium dihydrogen phosphate accounts for 20% by mass of the mixture, and the remainder of the two-phase aqueous system is composed of water; stirring and then allowing to stand; (4) After standing, the two phases are separated and the upper phase solution is collected; (5) Remove the ethanol from the upper phase solution and freeze-dry to obtain sheep serum albumin.

[0059] 2. Method for determining the content of sheep blood albumin: The ethanol solution collected in step (4) was used to determine the total protein content by the Coomassie Brilliant Blue method, and the albumin content by HPLC.

[0060] HPLC conditions were: C8 column (4.6 x 250 mm, 5 μm), column temperature 40 °C, mobile phase A was 0.01% TFA aqueous solution, mobile phase B was 0.01% TFA acetonitrile solution, gradient elution, flow rate was 0.8 mL / min, injection volume was 20 μl, and measurement wavelength was 276 nm.

[0061] The recovery rate and purity of sheep blood albumin were calculated by the following method:

[0062] 3. Results 1) Screening of inorganic salt types The types of inorganic salts in the aqueous two-phase system were screened, and the sodium dihydrogen phosphate in step (3) of the extraction method was replaced by dipotassium hydrogen phosphate, sodium citrate or sodium carbonate, respectively. The other extraction steps were the same as the above-mentioned sheep blood albumin extraction method.

[0063] The results are as follows Figure 2 As shown in the figure, the results of the phase separation process of the two-phase aqueous system of ethanol and dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate and sodium carbonate are shown. Among them, the sheep plasma in the ethanol and sodium dihydrogen phosphate system has completely separated in 30 minutes and remains relatively stable, with only a small amount of interface precipitation; although the sheep plasma in the ethanol and sodium carbonate system has a fast phase separation speed, a large amount of protein denatures and precipitates; the sheep plasma in the ethanol and dipotassium hydrogen phosphate system and the ethanol and sodium citrate system takes 240 minutes to completely separate, and at the same time, the ethanol and dipotassium hydrogen phosphate system is accompanied by a large amount of protein precipitation after complete phase separation, and the ethanol and sodium citrate system has salting out after complete phase separation.

[0064] Electrophoresis was performed on each layer of the system during the phase separation process of different systems using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific test results are as follows: Figure 3 As shown. Figure 3 In the system corresponding to A, the protein with a molecular weight of 66 kD was almost completely extracted into the upper phase, with only a small amount existing in the middle phase. Figure 3 In the system involved in BD, proteins with a molecular weight of 66kD were denatured in the phase separation process in other aqueous two-phase systems and precipitated in the lower phase, with only a small amount being extracted into the upper phase.

[0065] The results of the recovery rate and purity of sheep serum albumin in the aqueous two-phase system composed of ethanol, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate and sodium carbonate are as follows Figure 4 Among them, the two-phase aqueous system composed of ethanol and sodium dihydrogen phosphate has the best separation effect on sheep blood albumin, with a recovery rate of 73.36% and a purity of 89.52%.

[0066] The sheep serum albumin treated with different extraction systems and freeze-dried was reconstituted into an aqueous solution with a concentration of 10 mg / mL. Figure 5 As shown. Figure 5 It can be seen that the sheep blood albumin extracted under the ethanol-sodium dihydrogen phosphate extraction system has the most ideal dissolution effect, and the obtained solution is clear and transparent. In contrast, when the ethanol-sodium citrate and ethanol-sodium carbonate extraction systems are used, the solution is relatively turbid and contains obvious insoluble matter. When the ethanol-dipotassium hydrogen phosphate extraction system is used, the solution is even more turbid.

[0067] Therefore, based on the above results, the ethanol-sodium dihydrogen phosphate system is the most effective for separating and extracting sheep serum albumin from sheep plasma. This system will be selected and its optimal component ratio will be further explored to determine the optimal process conditions.

[0068] 2) Screening of sheep plasma ratio The proportion of sheep plasma added to the aqueous two-phase system was screened, and the mass percentage of sheep plasma in the mixture in step (3) of the extraction method was replaced with 1%, 2%, 3%, 4% and 5%, respectively. Only the proportion of sheep plasma was changed, and the other extraction steps were the same as the above-mentioned extraction method.

[0069] The screening results of the sheep plasma ratio are shown in Table 2. When the mass percentage of ethanol and sodium dihydrogen phosphate in the mixture is constant, the recovery rate of sheep blood albumin is the highest when the mass percentage of the added sheep plasma in the mixture is 4%.

[0070] Table 2 Analysis of the recovery rate of sheep blood albumin in different proportions of sheep plasma

[0071] 3) Screening of ethanol ratio The proportion of ethanol added to the aqueous two-phase system was screened, and the mass percentage of ethanol in the mixture in step (3) of the extraction method was replaced with 25%, 28%, 30%, 32% and 35%, respectively. The mass percentage of sheep plasma in the mixture was set to 4%, and the other extraction steps were the same as the above-mentioned extraction method.

[0072] The screening results of the ethanol ratio are shown in Table 3. When the mass percentage of sheep plasma and sodium dihydrogen phosphate in the mixture is constant, the recovery rate of sheep blood albumin is the highest when the mass percentage of the added ethanol in the mixture is 28%.

[0073] Table 3 Analysis of the recovery rate of sheep blood albumin by different proportions of ethanol

[0074] 4) Screening of sodium dihydrogen phosphate ratio The proportion of sodium dihydrogen phosphate added to the aqueous two-phase system was screened, and the mass percentage of sodium dihydrogen phosphate in the mixture in step (3) of the extraction method was replaced with 16%, 18%, 20%, 22% and 24%, the mass percentage of sheep plasma in the mixture was set to 4%, the mass percentage of ethanol in the mixture was set to 28%, and the other extraction steps were the same as the above-mentioned extraction method.

[0075] The screening results of the sodium dihydrogen phosphate ratio are shown in Table 4. When the mass percentage of sheep plasma and ethanol in the mixture is constant, when the mass percentage of the added sodium dihydrogen phosphate in the mixture is 18%, the recovery rate of sheep blood albumin is the highest.

[0076] Table 4 Analysis of the recovery rate of sheep blood albumin by different proportions of sodium dihydrogen phosphate

[0077] 5) Add a variety of inorganic salts The quantity and quality of inorganic salts added to the aqueous two-phase system were screened. The mass percentage of sheep plasma in the mixture in step (3) of the extraction method was set to 4%, the mass percentage of ethanol in the mixture was set to 28%, and the mass percentage of sodium dihydrogen phosphate in the mixture was set to 18%. Then, different proportions of sodium chloride were added to the aqueous two-phase system, and the mass percentages of sodium chloride in the mixture were 0%, 3%, 5%, 7% and 10%. The other extraction steps were the same as the above-mentioned extraction method.

[0078] The screening results of the sodium chloride ratio are shown in Table 5. When the mass percentage of sheep plasma in the aqueous two-phase system is 4%, the mass percentage of ethanol in the mixture is 28%, the mass percentage of sodium dihydrogen phosphate in the mixture is 18%, and the mass percentage of sodium chloride in the mixture is 3%, the aqueous two-phase system formed at this time has the best separation effect on sheep blood albumin, and the recovery rate can reach 92.05%.

[0079] Table 5 Analysis of the results of sodium chloride on the recovery rate of sheep blood albumin

[0080] Example 3 Structural Identification and Quality Analysis of Sheep Blood Albumin Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to perform comparative detection of protein molecular weight standards on sheep plasma, sheep albumin standard (Solarbio, SP084), and sample A prepared according to the extraction conditions in Example 2 (sheep plasma accounted for 4% of the mass percentage of the mixture, ethanol accounted for 28% of the mass percentage of the mixture, sodium dihydrogen phosphate accounted for 18% of the mass percentage of the mixture, and sodium chloride accounted for 3% of the mass percentage of the mixture).

[0081] Test results such as Figure 6 As shown, the electrophoresis results show that the molecular weight of the albumin obtained from sample A is consistent with that of the standard albumin, which is about 66 kDa, and the electrophoretic purity is higher than 90%.

[0082] The albumin in sample A and the sheep blood albumin standard were analyzed by Fourier transform infrared spectrometer. The spectrum of the sample was 400-4000cm-1 The frequency scanning range is 4cm -1 Scan at a resolution of

[0083] The fluorescence spectra of albumin and sheep blood albumin standard of sample A were plotted using a fluorescence spectrometer, with an excitation wavelength of 280 nm, an emission wavelength of 300-450 nm, and an excitation slit and an emission slit of 3 nm.

[0084] The results are as follows Figure 7 As shown, the albumin structure of sample A is consistent with that of the standard, indicating that the extraction method of Example 2 has less damage to the protein structure.

[0085] Example 4 Stability Determination The thermodynamic stability of sheep blood albumin in sample A was measured by differential scanning calorimetry and compared with standard albumin.

[0086] Weigh about 8 mg of albumin powder and sample A respectively, put them in an aluminum crucible and press them into tablets. Put them into the DSC instrument, set the nitrogen flow rate to 50 ml / min, set the temperature program to 20-120℃, and the heating rate to 10℃ / min.

[0087] The results are as follows Figure 8 As shown in the figure, the albumin molecule in sample A has the same thermodynamic stability as the standard, its secondary structure is intact and has good heat resistance, meeting the stability requirements of biological products. This result further verifies the protective effect of this extraction method on the natural conformation of the protein.

[0088] Example 5 Protein emulsification and emulsion stability Sample A and other pure albumin products of different species were dissolved in deionized water to prepare a 1 mg / mL solution. Other types of pure albumin products included bovine, porcine and chicken albumin. The samples were emulsified with a high-speed shear at a speed of 10,000 r / min. Corn oil was slowly added to the solution while homogenizing. The final ratio of the water phase to the oil phase was 3:1. After homogenization, 50 μl of liquid was drawn from the bottom of the tube at 0 min and 10 min, respectively, and added to 5 mL of SDS solution (0.1%, w / v). The absorbance of the diluted solution was then measured at 500 nm.

[0089] The calculation formula of emulsion stability (ESI) is as follows:

[0090] Among them, A 0 ——Absorbance value at 0 min; A 10 ——Absorbance value at 10 min; The results are as follows Fig. 9As shown, sheep blood albumin is significantly better than bovine, pig and chicken albumin in terms of emulsification stability, which indicates that sheep blood albumin has better emulsification properties and can provide higher performance in food processing and related applications.

[0091] Example 6 Determination of protein gel strength 1. Preparation of albumin gel: (1) Sample A was dissolved in distilled water and magnetically stirred for 30 min to reach a protein concentration of 10% (w / v) and then placed at 4 °C overnight for adequate hydration.

[0092] (2) Heat the protein solution in a 90°C water bath for 30 min and then quickly transfer it to an ice water bath to obtain heat-induced gel.

[0093] 2. Gel strength determination method: The gel sample was cut into equal height cylinders with a diameter of 12 mm and measured by a TA50 spherical probe at 30% deformation, a trigger point load of 5.0 g and a test speed of 0.5 mm / s. The gel strength is equal to the product of the breaking force (g) and the fracture distance (cm).

[0094] 3. Results The results are as follows Fig.10 As shown, sheep blood albumin has obvious significance in gel strength compared with other species and has better gel strength.

[0095] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0096] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for rapidly separating sheep blood albumin based on a two-phase aqueous system, characterized in that: The method comprises the following steps: (1) adding a two-phase aqueous system to sheep plasma, stirring the resulting mixture, letting it stand, centrifuging, and collecting an upper phase solution, wherein the two-phase aqueous system comprises ethanol, sodium dihydrogen phosphate, and water; (2) Removing ethanol from the upper phase solution; Among them, the mass percentage of sheep plasma in the mixture is 1-5%; the mass percentage of ethanol in the mixture is 25-35%; and the mass percentage of sodium dihydrogen phosphate in the mixture is 16-24%.

2. The method according to claim 1, characterized in that The molecular weight of the sheep blood albumin is 66-68 kDa.

3. The method according to claim 1, characterized in that The mass percentage of the sheep plasma in the mixture is 3%, the mass percentage of the ethanol in the mixture is 30%, and the mass percentage of the sodium dihydrogen phosphate in the mixture is 20%.

4. The method according to claim 1, characterized in that: The two-phase aqueous system also includes sodium chloride.

5. The method according to claim 4, characterized in that The mass percentage of the sheep plasma in the mixture is 2-4%, the mass percentage of the ethanol in the mixture is 25-30%, the mass percentage of the sodium dihydrogen phosphate in the mixture is 16-20%, and the mass percentage of the sodium chloride in the mixture is 1-5%.

6. The method according to claim 5, characterized in that The mass percentage of the sheep plasma in the mixture is 4%, the mass percentage of the ethanol in the mixture is 28%, the mass percentage of the sodium dihydrogen phosphate in the mixture is 18%, and the mass percentage of the sodium chloride in the mixture is 3%.

7. The method according to claim 1, characterized in that The standing time is 10 to 60 minutes.

8. The method according to claim 1, characterized in that The centrifugal conditions are as follows: centrifugal temperature is 1-10°C, centrifugal speed is 2500-4500rpm, and centrifugal time is 5-25min.

9. The method according to claim 1, characterized in that: Step (2) also includes a freeze-drying operation.

10. The method according to claim 9, characterized in that The freeze-drying operation conditions are: freeze-drying temperature is -90 ~ -70 ° C, and freeze-drying time is 36 ~ 60h.

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