A method for rapid separation of albumin from sheep blood based on an aqueous two-phase system

Abstract

The present invention provides a method for rapidly separating ovine blood albumin based on an aqueous two-phase system, and the aqueous two-phase system includes ethanol, sodium dihydrogen phosphate and water. By using an aqueous two-phase system with specific components and ratios screened and optimized, protein aggregation or denaturation can be effectively prevented, the quality of ovine blood albumin is guaranteed to a great extent, the recovery rate and purity of albumin are significantly improved, and the utilization rate of ovine blood is greatly increased. With these advantages, this method and the ovine blood albumin extracted by using this method show broad application prospects in many fields such as food and biomaterials.

Description

Technical Field

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

[0002] Sheep blood, as a key by-product in the livestock slaughtering process, is rich in various nutritional elements and bioactive components, such as proteins, amino acids, various enzymes, vitamins, hormones, minerals, carbohydrates, and lipids. However, the utilization efficiency of sheep blood resources is extremely low, and most slaughter enterprises directly discharge the blood, resulting in a large waste of valuable protein resources and environmental pollution.

[0003] Sheep blood is rich in proteins, amino acids, vitamins, and various bioactive substances, among which proteins are the main components, with a content of 17% - 19%. Albumin is one of the most important proteins in sheep blood, accounting for 60% of the total plasma proteins (Cheng Chi, Cai Yongfeng. Development and utilization of edible animal blood resources [J]. Food and Fermentation Industries, 1998, 24(3):7.). Sheep blood albumin contains rich essential amino acids and has a high biological utilization rate, which makes it show important value in the development of nutritional supplements and health foods. Due to its good nutritional components, sheep blood albumin has broad application prospects in postoperative repair, can quickly supplement proteins, promote the recovery process of patients, 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 chromatography separation method, etc. However, these traditional methods have certain limitations in practical applications. For example, the salting-out method is widely used due to its simple operation. However, it needs to use a high-concentration salt solution to achieve protein precipitation, which may damage the purity of the protein and the desalting process is often complex and time-consuming. Chinese Patent CN201710059284.5 used the saturated ammonium sulfate fractional precipitation method to obtain bovine serum albumin, but the preparation process is cumbersome and requires column chromatography for desalting, increasing the cost. The ultrafiltration method uses a selective permeable membrane to remove small molecule impurities, but it is prone to membrane fouling and blockage, thus reducing the recovery efficiency and separation efficiency. The organic solvent precipitation method is the most commonly used technical means for separating plasma albumin at present. The separation is based on the isoelectric point of the protein for precipitation, but the sheep blood albumin may be denatured during the treatment process, affecting its functional characteristics. For example, Chinese Patent CN200410077667.8 used the cold ethanol method to separate albumin from the waste components of human plasma protein, but this method is limited by strict separation conditions and does not describe the purity and structural characteristics of the obtained albumin. Chromatography separation technology shows certain advantages in the field of protein extraction, but the high equipment investment limits the possibility of its large-scale application. Chinese Patent CN202410514938.9 discloses a method for separating canine albumin from canine blood by using two chromatographies. Although the purity of the protein is improved, the cost of the chromatography medium is relatively high, restricting the wide application of this method.

[0005] Therefore, how to improve the separation purity and reduce the cost 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 deficiencies of the prior art, improve the separation efficiency of sheep blood albumin and reduce the cost, a high-efficiency and low-cost sheep blood albumin separation technology is developed. The present invention proposes a rapid separation method of sheep blood albumin based on an ethanol-salt aqueous two-phase system. By adjusting the system components and operating conditions, the separation efficiency is significantly improved, realizing the efficient extraction and application of sheep blood albumin, and opening 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 an aqueous two-phase system, which includes the following steps:

[0008] (1) Add an aqueous two-phase system to sheep plasma, stir the obtained mixture, let it stand, centrifuge, and collect the upper-phase solution. The aqueous two-phase system includes ethanol, sodium dihydrogen phosphate and water;

[0009] (2) Remove the ethanol in the upper-phase solution.

[0010] In some embodiments of the present invention, the aqueous two-phase system is composed of ethanol, sodium dihydrogen phosphate and water.

[0011] Specifically, the molecular weight of the sheep blood albumin is about 66 - 68 kDa, preferably 66 kDa.

[0012] Specifically, the method for obtaining sheep plasma is as follows: an anticoagulant is added to sheep blood, centrifuged, and the supernatant is retained to obtain sheep plasma.

[0013] 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.

[0014] Preferably, the anticoagulant is selected from ethylenediaminetetraacetic acid, sodium citrate, heparin, and further preferably 3.8% sodium citrate.

[0015] 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 further preferably 1:10.

[0016] 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.

[0017] Preferably, the aqueous two-phase system further includes sodium chloride.

[0018] In a specific embodiment of the present invention, the aqueous two-phase system includes ethanol, sodium dihydrogen phosphate, sodium chloride and water.

[0019] 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 further preferably 2 - 4%.

[0020] 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 further preferably 25 - 30%.

[0021] 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 further preferably 16 - 20%.

[0022] 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 further preferably 1 - 5%.

[0023] 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 ethanol in the mixture is 30%, and the mass percentage of sodium dihydrogen phosphate in the mixture is 20%.

[0024] 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 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%.

[0025] Preferably, the standing time is 10 - 60 min, such as 10 min, 15 min, 20 min, 30 min, 35 min, 40 min, 45 min, 50 min or 60 min, etc.

[0026] Preferably, the centrifugation conditions are: the centrifugation temperature is 1 - 10 °C (such as 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 - 4500 rpm (such as 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm or 4500 rpm, etc.), and the centrifugation time is 5 - 25 min (such as 5 min, 10 min, 15 min, 20 min or 25 min, etc.).

[0027] In a specific embodiment of the present invention, the centrifugation conditions are: the centrifugation temperature is 4 °C, the centrifugation speed is 3500 rpm, and the centrifugation time is 15 min.

[0028] Specifically, step (1) is carried out at room temperature, and the room temperature is 20 - 35 °C.

[0029] Specifically, in step (2), the method for removing ethanol is dialysis technology, and a dialysis bag with a molecular weight cut-off of 10 kDa is used during the dialysis process.

[0030] Specifically, step (2) further includes a lyophilization operation.

[0031] Preferably, the conditions for the lyophilization operation are: the lyophilization temperature is -90 ~ -70 °C (such as -90 °C, -85 °C, -80 °C, -75 °C or -70 °C, etc.), and the lyophilization time is 36 - 60 h (such as 36 h, 40 h, 44 h, 46 h, 48 h, 50 h, 52 h, 56 h or 60 h, etc.).

[0032] In a specific embodiment of the present invention, the method includes the following steps:

[0033] (1) After the fresh sheep blood passes the inspection and quarantine, add an anticoagulant accounting for 10% of the original blood volume, and centrifuge at 4°C and 3500 rpm for 15 min. Then take out the supernatant and store it frozen at -20°C;

[0034] (2) When in use, take out the frozen sheep plasma and melt it in a water bath at 37°C to obtain sheep plasma;

[0035] (3) Successively add ethanol, sodium dihydrogen phosphate and water to the sheep plasma to form an aqueous two-phase system. 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 remaining part of the aqueous two-phase system consists of water. Stir and then let it stand;

[0036] (4) After standing, separate the two phases and collect the upper phase solution;

[0037] (5) Remove the ethanol from the upper phase solution and freeze-dry to obtain sheep blood albumin.

[0038] In another aspect of the present invention, there is provided an application of the sheep blood albumin prepared by the above method in food or biochemical materials.

[0039] Preferably, the food is a nutritional supplement or a food additive.

[0040] The hydrolysis products of sheep blood albumin are rich in various amino acids, polypeptides and trace elements, and are suitable as nutritional supplements to improve the nutritional value of food; at the same time, it has good functional properties such as gelation, emulsification and moisturizing, and can be used as a food additive to improve the taste and quality of food.

[0041] Preferably, the biological material can be used as a raw material for protein electrophoresis or cell culture, an antigen for immunoassay, a biomembrane, a bioadhesive or a drug carrier.

[0042] In protein electrophoresis, it can be used as an important experimental material, and under the action of an electric field, it can provide a reliable reference standard for the separation and analysis of other proteins, helping researchers accurately interpret the characteristics and differences of proteins. During cell culture, using sheep blood albumin as an additive to the cell culture medium can provide necessary nutrients and growth factors for cell growth and create an ideal microenvironment for cell culture.

[0043] In immunoassay, sheep blood albumin can act as an immunogen for the preparation of antibodies, and these antibodies can accurately recognize and bind to sheep blood albumin or other proteins with a related structure, playing a crucial role in disease diagnosis, quality detection of biological products, etc.

[0044] In the preparation of biomaterials, sheep blood albumin can be used to manufacture products such as bio - glue and bio - film, playing a role in tissue engineering and wound healing, etc.

[0045] In the field of drug carriers, sheep blood albumin can serve as a drug carrier, accurately delivering drugs to specific tissues or cells, thereby improving the efficacy of drugs and reducing side effects.

[0046] The beneficial effects of this invention are as follows:

[0047] (1) Through experimental screening, this invention has developed a method for extracting sheep blood albumin, which has the outstanding characteristics of a short process, simple operation, and high production efficiency. In particular, by using an aqueous two - phase system with specific components and ratios, during the extraction process, it can effectively prevent protein aggregation or denaturation, greatly guaranteeing the quality of sheep blood albumin. It can also significantly improve the recovery rate and purity of albumin products, greatly increasing the utilization rate of sheep blood, and avoiding environmental pollution caused by direct discharge of sheep blood. With these advantages, this method and the sheep blood albumin extracted by this method have been widely applied in many fields such as food, biopharmaceuticals, etc.

[0048] (2) This invention uses mild conditions for protein extraction. The entire operation process is rigorous and controllable, avoiding damage to the protein structure and enabling the protein to always maintain its original excellent activity.

[0049] (3) This invention has compared the functions of albumin from different species, providing a theoretical basis for future applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Hereinafter, the embodiments of this invention will be described in detail with reference to the drawings, where:

[0051] Figure 1 : The phase diagrams of aqueous two - phase systems with different compositions.

[0052] Figure 2 : The result diagrams of the phase - separation process of different aqueous two - phase systems.

[0053] Figure 3 : The electrophoresis diagrams of each layer system during the phase - separation process of different aqueous two - phase systems. Among them, A is the electrophoresis diagram of separating plasma proteins with an ethanol and sodium dihydrogen phosphate system; B is the electrophoresis diagram of separating plasma proteins with an ethanol and dipotassium hydrogen phosphate system; C is the electrophoresis diagram of separating plasma proteins with an ethanol and sodium citrate system; D is the electrophoresis diagram of separating plasma proteins with an ethanol and sodium carbonate system; M is the protein molecular weight standard; Plasma is the extracted sheep plasma.

[0054] Figure 4: Diagram of extraction rate and purity analysis of separating sheep blood albumin by different aqueous two-phase systems. Among them, A is the recovery rate diagram of separating sheep blood albumin by aqueous two-phase systems with different salts, and B is the purity diagram of separating sheep blood albumin by aqueous two-phase systems with different salts.

[0055] Figure 5 : Diagram of the reconstitution results of freeze-dried sheep blood albumin after extraction by different aqueous two-phase systems.

[0056] Figure 6 : SDS-PAGE diagram of separating sheep blood albumin by ethanol-sodium dihydrogen phosphate aqueous two-phase system. Among them, Lane 1: M is the protein molecular weight standard; Lane 2: Plasma is sheep plasma; Lane 3: Sample A; Lane 4: Standard is the sheep blood albumin standard.

[0057] Figure 7 : Infrared spectrum and fluorescence spectrum diagrams of separating sheep blood albumin by ethanol-sodium dihydrogen phosphate aqueous two-phase system. Among them, A is the infrared spectrum diagram, and B is the fluorescence spectrum diagram.

[0058] Figure 8 : DSC diagram of separating sheep blood albumin by ethanol-sodium dihydrogen phosphate aqueous two-phase system.

[0059] Figure 9 : Diagram of comparing the emulsification stability of albumin from different species.

[0060] Figure 10 : Diagram of comparing the gel strength of albumin from different species.

[0061] Note: In the attached drawings, a, b, and c all represent p <0.05. Specific implementation manners

[0062] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0063] Example 1 Screening of combinations of aqueous two-phase systems

[0064] 1. Screening method of aqueous two-phase system:

[0065] (1) First, weigh a certain amount of inorganic salt (m1) (the inorganic salts are potassium bicarbonate, sodium carbonate, sodium sulfate, sodium citrate, sodium acetate, dipotassium hydrogen phosphate or sodium dihydrogen phosphate) and add a certain amount of distilled water (m2) to just dissolve it;

[0066] (2) Slowly add ethanol (m3) drop by drop until the mixed solution just becomes turbid, then let it stand to reach equilibrium;

[0067] (3) Keep the equilibrium process in a state where adding a drop of water makes the solution immediately clear, and adding a little more ethanol makes the solution immediately turbid again;

[0068] (4) Repeat the above operations, observe whether phase separation can occur, and calculate the mass fractions of each component in the solution at the cloud point according to the following formula.

[0069] The results are shown in Table 1.

[0070] Table 1 Phase separation situations of different kinds of salts

[0071]

[0072] Calculate the mass fractions of the salts (dipotassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium carbonate) that can stably form a phase with ethanol according to the method in step (4) above, and draw a phase diagram of the aqueous two-phase system (as shown in Figure 1 ) to determine the phase separation conditions.

[0073] Example 2 Aqueous two-phase system combination for screening and separating sheep blood albumin

[0074] 1. Extraction method of sheep blood albumin:

[0075] (1) After the fresh sheep blood passes the inspection and quarantine, add an anticoagulant accounting for 10% of the original blood volume, and centrifuge at 3500 rpm for 15 minutes at 4°C. Then take out the supernatant and store it frozen at -20°C;

[0076] (2) When in use, take out the frozen sheep plasma and thaw it in a water bath at 37°C to obtain sheep plasma;

[0077] (3) Successively add ethanol, sodium dihydrogen phosphate and water to the sheep plasma to form an aqueous two-phase system. The mass percentage of sheep plasma in the mixture is 3%, the mass percentage of ethanol in the mixture is 30%, the mass percentage of sodium dihydrogen phosphate in the mixture is 20%, and the remaining part of the aqueous two-phase system is composed of water. Stir and then let it stand;

[0078] (4) After standing, separate the two phases and collect the upper-phase solution;

[0079] (5) Remove the ethanol in the upper-phase solution, and freeze-dry to obtain sheep blood albumin.

[0080] 2. Content determination method of sheep blood albumin:

[0081] Use the Coomassie Brilliant Blue method to determine the total protein content and HPLC to determine the albumin content for the ethanol solution collected in step (4).

[0082] The HPLC conditions were as follows: C8 column (4.6 x 250 mm, 5 μm), column temperature 40 °C, mobile phase A was 0.01% aqueous TFA 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 the detection wavelength was 276 nm.

[0083] The recovery rate and purity of sheep blood albumin were calculated by the following method:

[0084]

[0085] 3. Results

[0086] 1) Screening of inorganic salt types

[0087] The types of inorganic salts in the aqueous two-phase system were screened. Sodium dihydrogen phosphate in step (3) of the extraction method was replaced with potassium hydrogen phosphate, sodium citrate or sodium carbonate respectively, and the other extraction steps were the same as the extraction method of sheep blood albumin above.

[0088] The results were as Figure 2 shown, demonstrating the phase separation process results of the aqueous two-phase systems of ethanol with potassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate and sodium carbonate. Among them, the sheep plasma in the ethanol-sodium dihydrogen phosphate system was completely phase-separated in 30 minutes and remained relatively stable, with only a small amount of interfacial precipitation; although the phase separation rate of sheep plasma in the ethanol-sodium carbonate system was fast, a large amount of protein was denatured and precipitated; the sheep plasma in the ethanol-potassium hydrogen phosphate system and the ethanol-sodium citrate system took 240 minutes to be completely phase-separated. At the same time, a large amount of protein precipitation occurred after the ethanol-potassium hydrogen phosphate system was completely phase-separated, and salting-out phenomenon occurred after the ethanol-sodium citrate system was completely phase-separated.

[0089] Electrophoresis detection was carried out on each layer system during the phase separation process of different systems. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used. The specific detection results were as Figure 3 shown. In the system corresponding to Figure 3 A, the protein with a molecular weight of 66 kD was almost all extracted into the upper phase, and only a small amount was present in the middle phase. In the systems involved in Figure 3 B - D, for the protein with a molecular weight of 66 kD, other aqueous two-phase systems were denatured during the phase separation process and precipitated in the lower phase, and only a small amount was extracted into the upper phase.

[0090] The results of the recovery rate and purity of sheep blood albumin for the aqueous two-phase systems composed of ethanol with potassium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate and sodium carbonate were as Figure 4As shown in the figure. Among them, the aqueous two-phase system composed of ethanol and sodium dihydrogen phosphate has the best separation effect on sheep blood albumin, with a recovery rate of up to 73.36% and a purity of up to 89.52%.

[0091] The freeze-dried sheep blood albumin treated with different extraction systems was reconstituted into an aqueous solution with a concentration of 10 mg / mL, and the results are as Figure 5 shown. From 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 resulting solution is clear and transparent. In contrast, when using the ethanol-sodium citrate and ethanol-sodium carbonate extraction systems, the solution is relatively turbid and contains obvious insoluble substances. When using the ethanol-potassium hydrogen phosphate extraction system, the turbidity of the solution is even greater.

[0092] Therefore, from the above results, it can be seen that choosing the ethanol-sodium dihydrogen phosphate system has the best effect on separating and extracting sheep blood albumin from sheep plasma. Subsequently, this system is selected and its optimal component ratio is further explored to determine the optimal process conditions.

[0093] 2) Screening of the proportion of sheep plasma

[0094] The proportion of sheep plasma added to the aqueous two-phase system was screened. 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 other extraction steps were the same as the above extraction method.

[0095] The screening results of the proportion of sheep plasma are shown in Table 2. When the mass percentages of ethanol and sodium dihydrogen phosphate in the mixture are certain, when the mass percentage of added sheep plasma in the mixture is 4%, the recovery rate of sheep blood albumin is the highest.

[0096] Table 2 Analysis of the results of the recovery rate of sheep blood albumin with different proportions of sheep plasma

[0097]

[0098] 3) Screening of the proportion of ethanol

[0099] The proportion of ethanol added to the aqueous two-phase system was screened. 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 other extraction steps were the same as the above extraction method.

[0100] The screening results of the proportion of ethanol are shown in Table 3. When the mass percentages of sheep plasma and sodium dihydrogen phosphate in the mixture are certain, when the mass percentage of added ethanol in the mixture is 28%, the recovery rate of sheep blood albumin is the highest.

[0101] Table 3 Results Analysis of the Recovery Rate of Sheep Serum Albumin with Different Proportions of Ethanol

[0102]

[0103] 4) Screening of the Proportion of Sodium Dihydrogen Phosphate

[0104] The proportion of sodium dihydrogen phosphate added to the aqueous two-phase system was screened. 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 at 4%, the mass percentage of ethanol in the mixture was set at 28%, and the other extraction steps were the same as the above extraction method.

[0105] The screening results of the proportion of sodium dihydrogen phosphate are shown in Table 4. When the mass percentages of sheep plasma and ethanol in the mixture are constant, the recovery rate of sheep serum albumin is the highest when the mass percentage of added sodium dihydrogen phosphate in the mixture is 18%.

[0106] Table 4 Results Analysis of the Recovery Rate of Sheep Serum Albumin with Different Proportions of Sodium Dihydrogen Phosphate

[0107]

[0108] 5) Addition of Multiple Inorganic Salts

[0109] The quantity and mass 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 at 4%, the mass percentage of ethanol in the mixture was set at 28%, the mass percentage of sodium dihydrogen phosphate in the mixture was set at 18%, and then different proportions of sodium chloride were added to the aqueous two-phase system. The mass percentage of sodium chloride in the mixture was 0%, 3%, 5%, 7% and 10%, and the other extraction steps were the same as the above extraction method.

[0110] The screening results of the proportion of sodium chloride are shown in Table 5. When in the aqueous two-phase system, the mass percentage of sheep plasma in the mixture 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 fraction of sodium chloride in the mixture is 3%, the aqueous two-phase system formed at this time has the best separation effect on sheep serum albumin, and the recovery rate can reach 92.05%.

[0111] Table 5 Results Analysis of the Recovery Rate of Sheep Serum Albumin with Sodium Chloride

[0112]

[0113] Example 3 Structure Identification and Quality Analysis of Sheep Serum Albumin

[0114] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to conduct a comparative detection of protein molecular weight standards on sheep plasma, sheep blood albumin standard (Solarbio, SP084), and sample A prepared according to the extraction conditions in Example 2 (the mass percentage of sheep plasma in the mixture was 4%, the mass percentage of ethanol in the mixture was 28%, the mass percentage of sodium dihydrogen phosphate in the mixture was 18%, and the mass fraction of sodium chloride in the mixture was 3%).

[0115] The detection results are as Figure 6 shown. The electrophoresis results indicate that the molecular weight of the albumin obtained from sample A is consistent with that of the standard albumin, approximately 66 kDa, and the electrophoresis purity is higher than 90%.

[0116] Fourier transform infrared absorption spectroscopy analysis was performed on the albumin of sample A and the sheep blood albumin standard using a Fourier transform infrared spectrometer. The spectra of the samples were scanned at a resolution of 4 cm -1 within the frequency scanning range of 400 - 4000 cm -1 .

[0117] The fluorescence spectra of the albumin of sample A and the sheep blood albumin standard were plotted using a fluorescence spectrometer. The excitation wavelength was 280 nm, the emission wavelength was 300 - 450 nm, and both the excitation slit and the emission slit were 3 nm.

[0118] The results are as Figure 7 shown. The albumin structure of sample A is consistent with that of the standard product, indicating that the extraction method in Example 2 causes less damage to the protein structure.

[0119] Example 4 Stability determination

[0120] The thermodynamic stability of the sheep blood albumin in sample A was determined by differential scanning calorimetry and compared with the standard albumin.

[0121] Approximately 8 mg of albumin powder and sample A were weighed separately and pressed into tablets in an aluminum crucible. They were placed in a DSC instrument with a nitrogen flow rate of 50 ml / min. The temperature program was set to 20 - 120 °C, and the heating rate was 10 °C / min.

[0122] The results are as Figure 8 shown. The albumin molecules in sample A have the same thermodynamic stability as the standard product. Their secondary structure is intact and they have good heat resistance, meeting the stability requirements of biological products. This result further verifies the protective effect of this extraction method on the native conformation of proteins.

[0123] Example 5 Protein emulsifying property and emulsion stability

[0124] Dissolve sample A and albumin pure products of other different species in deionized water to prepare a 1 mg / mL solution. Albumin pure products of other species include bovine, porcine, and chicken albumins. Emulsify the sample with a high-speed shearer at a rotation speed of 10,000 r / min, and slowly add corn oil to the solution while homogenizing. Finally, the ratio of the aqueous phase to the oil phase is 3:1. After homogenization, at the 0th minute and the 10th minute, respectively, aspirate 50 μL of the liquid from the bottom of the tube and add it to 5 mL of SDS solution (0.1%, w / v). Then measure the absorbance of the diluted solution at 500 nm.

[0125] The calculation formula for emulsion stability (ESI) is as follows:

[0126]

[0127] where A0 is the absorbance value at the 0th minute;

[0128] A 10 is the absorbance value at the 10th minute;

[0129] The results are as Figure 9 shown. Sheep blood albumin is significantly superior to bovine, porcine, and chicken albumins in terms of emulsion stability, indicating that sheep blood albumin has better emulsifying properties and can provide higher performance in food processing and related applications.

[0130] Example 6 Determination of Protein Gel Strength

[0131] 1. Preparation of albumin gel:

[0132] (1) Dissolve sample A in distilled water and stir magnetically for 30 min to reach a protein concentration of 10% (w / v), and place it at 4 °C overnight for sufficient hydration.

[0133] (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 a heat-induced gel.

[0134] 2. Method for determining gel strength:

[0135] Cut the gel sample into cylinders with a diameter of 12 mm and the same height, and measure it with a TA50 spherical probe at a 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).

[0136] 3. Results

[0137] The results are as Figure 10 shown. Sheep blood albumin has obvious significance in terms of gel strength compared to other species and has better gel strength.

[0138] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0139] In addition, it should be noted that in the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without conflict. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A method for rapid separation of sheep serum albumin based on a two-phase aqueous system, characterized in that: The method comprises the following steps: (1) adding an aqueous two-phase system to sheep plasma, stirring the resulting mixture, allowing it to stand, centrifuging it, and collecting the upper phase solution, wherein the aqueous two-phase system consists of ethanol, sodium dihydrogen phosphate, and water, or; The two-phase aqueous system consists of ethanol, sodium dihydrogen phosphate, water and sodium chloride; (2) Removal of ethanol from the upper phase solution; The mass percentage of the sheep plasma in the mixture is 1-5%; the mass percentage of ethanol in the mixture is 25-30%; the mass percentage of sodium dihydrogen phosphate in the mixture is 16-24%; and the mass percentage of the sodium chloride in the mixture is 1-5%.

2. 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%.

3. The method according to claim 1, 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%.

4. The method according to claim 3, 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%.

5. The method according to claim 1, wherein The standing time is 10 to 60 minutes.

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

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

8. The method according to claim 7, characterized in that The freeze-drying operation conditions are: freeze-drying temperature is -90~-70℃, and freeze-drying time is 36~60h.

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