A method for efficient separation of sheep blood immunoglobulins based on monosodium glutamate precipitation.

Abstract

This invention belongs to the field of immunoglobulin extraction, and specifically relates to a method for efficiently separating sheep blood immunoglobulins based on monosodium glutamate (MSG) salting-out. The separation method includes the step of adding a saturated MSG solution to sheep plasma for salting-out. The amount of sheep plasma added to the salting-out system is 35-50% of the total volume of the mixture. The saturation of the MSG solution in the salting-out system is 46-50%. This invention is the first to propose using MSG as a salting-out agent for sheep blood immunoglobulins, requiring only one salting-out step to obtain sheep blood immunoglobulins with a purity and recovery rate exceeding 70%. This method is simple to operate, has a large plasma processing capacity, fast processing speed, low requirements for equipment and environment, low cost, and is easy to scale up for production.

Description

Technical Field

[0001] This invention belongs to the field of immunoglobulin extraction technology, and specifically relates to a method for efficiently separating sheep blood immunoglobulins based on sodium glutamate precipitation. Background Technology

[0002] my country is one of the world's largest producers of mutton. Sheep blood, a key byproduct of sheep slaughtering, is produced in huge quantities, but most of it is directly discarded, resulting in low utilization and causing resource waste and environmental pollution. The small amount of sheep blood collected and utilized is mainly processed into low-value-added products such as animal feed and blood tofu, and its potential for high-value utilization has not been fully explored.

[0003] Sheep's blood is mainly composed of plasma and red blood cells. The protein content in plasma is 7-9%, of which immunoglobulins account for approximately 16-18%, making them one of the most valuable proteins in sheep's blood. Immunoglobulins possess significant immunomodulatory, antibacterial, and antiviral physiological activities, and can be used as functional ingredients in the food industry, particularly in nutritional and health food products.

[0004] Currently, common methods for separating immunoglobulins include salting out, organic solvent precipitation, and chromatography. However, these methods all have certain limitations. Regarding salting out, Chinese patent CN106749626B discloses an ammonium sulfate salting-out method for extracting bovine serum immunoglobulins. This process is cumbersome and lengthy, involving multiple ammonium sulfate salting-out, centrifugation, and dissolution operations. Furthermore, it requires a large cumulative amount of ammonium sulfate and generates a large amount of high-ammonia-nitrogen and high-salt waste liquid during the process, resulting in high subsequent treatment costs and a significant environmental burden. Chinese patent CN102321172B discloses a method for preparing animal serum immunoglobulin powder using iron salts. While the overall operation is simple, its immunoglobulin content is only 65.1%, and the recovery rate of immunoglobulins is not mentioned, making it impossible to assess its process yield and economic viability. The most commonly used organic solvent precipitation method is the low-temperature ethanol method. Chinese patent CN116731162B discloses a method for separating human immunoglobulins using this method. However, ethanol precipitation easily causes protein denaturation, so the entire process must be carried out under stringent low-temperature conditions, requiring sophisticated equipment and consuming a large amount of energy. Furthermore, the use of large quantities of flammable and explosive ethanol as a precipitant necessitates strict storage requirements and poses certain safety risks. Chromatographic methods include ion exchange chromatography and affinity chromatography. Chinese patent CN112111004B uses chromatography to extract rabbit immunoglobulins, achieving good separation results. However, the high cost of chromatographic equipment and media, coupled with limited throughput and processing speed, restricts large-scale application.

[0005] Therefore, developing a simple, environmentally friendly, safe, practical, and low-cost method for separating sheep blood immunoglobulins that is suitable for large-scale production is of great significance for realizing the high-value utilization of sheep blood resources. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention aims to propose a simple, green, environmentally friendly, safe, practical and low-cost method for separating sheep blood immunoglobulins, so as to solve the problems of complicated process, heavy environmental burden, high energy consumption and high cost in the separation of sheep blood immunoglobulins in the existing technology.

[0007] On one hand, the present invention provides a method for separating sheep blood immunoglobulins, the separation method comprising the step of adding a saturated sodium glutamate solution to sheep plasma for salting out.

[0008] Furthermore, the amount of sheep plasma added to the salting-out system is 35-50% of the total volume of the mixture (e.g., 35%, 38%, 40%, 42%, 45%, 48%, 50%), preferably 50%.

[0009] Furthermore, the saturation of the sodium glutamate solution in the salting-out system is 46-50%.

[0010] Furthermore, the salting-out step includes adding PBS solution and saturated sodium glutamate solution to sheep plasma, adjusting the pH value, stirring, allowing to stand, centrifuging, and collecting the precipitate.

[0011] Furthermore, the pH value is 7.4~9.0, and / or The stirring temperature is 25~55℃, and / or The settling time is more than 2 hours.

[0012] Furthermore, the method for preparing sheep plasma includes the following steps: adding an anticoagulant to sheep blood, mixing, centrifuging, and separating the supernatant to obtain sheep plasma.

[0013] Furthermore, the anticoagulant is heparin, sodium citrate, or ethylenediaminetetraacetic acid; Preferably, the volume ratio of the anticoagulant to sheep blood is 1:5 to 20 (e.g., 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20); more preferably, it is 1:9.

[0014] Furthermore, the separation method also includes the steps of dissolving, dialysis to remove salt, and drying the salt-precipitated product after salting out.

[0015] Furthermore, the separation method specifically includes the following steps: (1) Preparation of sheep plasma: Add 3.8% sodium citrate solution to fresh sheep blood as an anticoagulant, mix well, centrifuge, separate the supernatant to obtain sheep plasma; (2) Salting out precipitation: Add PBS solution and saturated monosodium glutamate solution to the sheep plasma obtained in step (1) until the saturation of monosodium glutamate solution is 46-50%, adjust the pH of the system to 7.4-9.0 (e.g. 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0), stir at 25-55 ℃ (e.g. 25 ℃, 35 ℃, 45 ℃, 55 ℃), let stand for more than 2 hours (e.g. 2h, 4h, 8h, 12h, 18h, 24h), centrifuge, and collect the precipitate; (3) Desalting and drying: The precipitate obtained in step (2) is dissolved, dialyzed to remove salt, and then dried to obtain sheep blood immunoglobulin.

[0016] Furthermore, the centrifugation conditions in steps (1) and (2) are as follows: centrifugation temperature is 1~10 ℃ (e.g., 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃), centrifugation speed is 3000~5000 rpm (e.g., 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm or 5000 rpm), and centrifugation time is 10~30 min (e.g., 10 min, 15 min, 20 min, 25 min or 30 min); Preferably, the centrifugation temperature is 4 ℃, the centrifugation speed is 4000 rpm, and the centrifugation time is 20 min.

[0017] Furthermore, the sheep plasma prepared in step (1) can be frozen and stored at -20 ℃ or -80 ℃. When it is ready for use, the frozen sheep plasma can be thawed in a water bath at 37 ℃.

[0018] Furthermore, in step (1), sheep blood plasma accounts for 35-50% (v / v) of the total volume of the mixture (e.g., 35%, 38%, 40%, 42%, 45%, 48%, 50%), preferably 50%.

[0019] Furthermore, the sodium glutamate saturation in the salting-out system described in step (2) is 46-50% (v / v).

[0020] Furthermore, the pH of the system is adjusted to 8 as described in step (2).

[0021] Furthermore, in step (2), the stirring temperature is 25~55 ℃, the stirring time is 30 min, and the settling temperature is 2~8℃; Preferably, the settling time is 2 to 8 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours).

[0022] Furthermore, the molecular weight of the sheep blood immunoglobulin is 145~155 kDa.

[0023] Furthermore, the reagent used for dissolution in step (3) is PBS solution; the dialysis in step (3) uses a dialysis bag with a molecular weight cutoff of 14 kDa.

[0024] Furthermore, in step (3), the freeze-drying temperature is -90 ~ -70 ℃ (e.g. -90 ℃, -85 ℃, -80 ℃, -75 ℃ or -70 ℃), and the freeze-drying time is 48~60 h (e.g. 48 h, 50 h, 52 h, 54 h, 56 h, 58 h or 60 h).

[0025] Furthermore, the pH of the PBS solution is 7.2-7.4, and the concentration is 0.01 M.

[0026] Secondly, the present invention provides the application of the above separation method in improving the separation effect of sheep blood immunoglobulins; Preferably, the separation effect includes the purity and / or recovery rate of sheep blood immunoglobulins.

[0027] Compared with the prior art, the present invention has the following advantages: (1) This invention is the first to propose using monosodium glutamate as a salting-out agent for sheep blood immunoglobulins, which can obtain sheep blood immunoglobulins with a purity and recovery rate of over 70% in just one salting-out step. This method is simple to operate, has a large plasma processing capacity and fast processing speed, low requirements for equipment and environment, low cost, and is easy to scale up for production.

[0028] (2) The monosodium glutamate used in this invention is a food-grade ingredient, which is highly safe and biodegradable with low environmental residue risk. The salting-out waste liquid does not contain ammonia nitrogen, has good biodegradability, and does not require special treatment such as deammoniation, which greatly reduces the difficulty and cost of waste liquid treatment. Compared with the traditional ammonium sulfate salting-out method, this invention is more green and environmentally friendly.

[0029] (3) The process of this invention is mild and the operating conditions are controllable, which can effectively protect the natural structure of sheep blood immunoglobulins.

[0030] (4) This invention uses sheep blood as raw material to transform slaughter waste into high value-added products, effectively reducing the waste of sheep blood resources and mitigating environmental pollution.

[0031] (5) The sheep blood immunoglobulin obtained by the present invention can be directly applied to functional foods or pet nutritional foods, providing a feasible path for the high-value utilization of sheep blood, with significant economic benefits and broad market prospects. Attached Figure Description

[0032] Figure 1 This is an electrophoresis diagram of immunoglobulins extracted with different salting-out agents in Example 1.

[0033] Figure 2 This is a comparison of the relative purity of immunoglobulins extracted with different salts based on grayscale analysis in Example 1.

[0034] Figure 3 This is an electrophoresis diagram of sheep blood immunoglobulins separated by sodium glutamate precipitation in Example 3.

[0035] Figure 4 The infrared spectrum of sheep blood immunoglobulins separated by sodium glutamate precipitation in Example 3.

[0036] Figure 5 The image shows the ultraviolet spectrum of sheep blood immunoglobulins separated by sodium glutamate precipitation in Example 3.

[0037] Figure 6 The fluorescence spectrum of sheep blood immunoglobulins separated by sodium glutamate precipitation in Example 3 is shown.

[0038] Figure 7 This is a comparison of the purity of sheep blood immunoglobulins obtained by different extraction methods in Example 4.

[0039] Figure 8 This is a comparison of the recovery rates of sheep blood immunoglobulins obtained by different extraction methods in Example 4. Detailed Implementation

[0040] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0041] In this invention, the saturation of the sodium glutamate solution in the salting-out system (hereinafter referred to as "sodium glutamate saturation") refers to the relative saturation calculated based on the volume ratio of the saturated sodium glutamate solution in the mixture.

[0042] In this invention, the amount of sheep plasma added (hereinafter referred to as "sheep plasma added amount") refers to the percentage of sheep plasma volume in the total volume of the salting-out system mixture.

[0043] The fresh sheep blood used in this embodiment of the invention was purchased from a local slaughterhouse.

[0044] Example 1: Screening of Salting-out Agents 1. Extraction method of sheep blood immunoglobulins: (1) After the fresh sheep blood passes the inspection and quarantine, the anticoagulant (3.8% sodium citrate solution) and sheep blood are mixed at a volume ratio of 1:9, centrifuged at 4 ℃ and 4000 rpm for 20 min, the supernatant is separated and stored at -20 ℃. (2) Before use, take out the frozen sheep blood plasma and thaw it in a water bath at 37 ℃; (3) At room temperature, take an appropriate amount of distilled water and add salts (sodium glutamate, sodium metabisulfite, ferrous sulfate and 27 other kinds of salts) while stirring until there are no more solids that dissolve. After standing, take the supernatant to obtain a saturated salt solution. (4) Slowly add saturated salt solution to sheep blood plasma while stirring, adjust the pH of the system to 8-9, stir for 30 min, and then let it stand at 4 ℃; (5) After standing, centrifuge at 4 ℃ and 4000 rpm for 20 min and collect the precipitate; (6) After the precipitate is dissolved in PBS solution, it is dialyzed and lyophilized to obtain sheep blood immunoglobulin.

[0045] 2. Methods for determining the purity and recovery rate of sheep blood immunoglobulins: The total protein content in the extract was determined by the BCA method, and the immunoglobulin content in the extract was determined by HPLC.

[0046] The HPLC conditions were as follows: Protein G column, 1 mL; column temperature, 30 ℃; mobile phase A was pH 6.5, 0.05 mol / L phosphate buffer, and mobile phase B was pH 2.5, 0.05 mol / L glycine hydrochloride buffer; gradient elution; flow rate, 0.4 mL / min; injection volume, 20 μL; and measurement wavelength, 280 nm.

[0047] The purity and recovery rate of sheep blood immunoglobulins were calculated using the following method:

[0048] 3. Results: 1) Screening of salting-out agents This embodiment used 27 different types of salts, each at five saturation levels (20%, 30%, 40%, 50%, and 60%) to precipitate immunoglobulins from sheep plasma. Some salts failed to form significant precipitation at specific saturation levels. For samples that produced precipitation, the precipitate was dissolved in PBS and analyzed by electrophoresis. The extraction efficiency of different salt types for sheep blood immunoglobulins was compared based on the electrophoresis results. The results are as follows: Figure 1 As shown, by comparing the electrophoresis patterns of each lane, it can be seen that the electrophoretic patterns of salts such as sodium metabisulfite, copper sulfate, sodium bisulfite, potassium carbonate, and ferrous sulfate are dominated by bands of miscellaneous proteins, resulting in poor extraction efficiency of sheep blood immunoglobulins. Salts such as monosodium glutamate, sodium acetate, and disodium succinate show clear immunoglobulin bands and fewer miscellaneous protein bands at partial saturation, indicating relatively better extraction efficiency of sheep blood immunoglobulins. Therefore, samples with fewer miscellaneous protein bands and a higher proportion of immunoglobulin bands are selected, and the relative purity of immunoglobulins is further calculated based on the grayscale analysis of the gel images, such as... Figure 2 As shown, the highest relative purity of immunoglobulins was obtained by electrophoresis when the monosodium glutamate (MSG) saturation was 50%. Therefore, MSG was selected as the salting-out agent for extracting immunoglobulins from sheep plasma, and its process conditions were further optimized.

[0049] 2) Screening of plasma addition amount The screening results (1) show that the separation effect of immunoglobulins is significantly worse when the monosodium glutamate (MSG) saturation is below 50%. Therefore, the upper limit of plasma addition was set at 50%. Under fixed experimental conditions, the effect of varying plasma addition within the range of 15% to 50% was investigated. As shown in Table 1, with the increase of plasma addition, the purity of immunoglobulins showed a trend of first increasing and then decreasing, while the recovery rate continued to rise. Considering both purity and recovery rate, a plasma addition of 50% can achieve better purity while ensuring a higher recovery rate, and this condition offers greater advantages in processing efficiency and economy.

[0050] Table 1. Effects of plasma addition amount on the purity and recovery rate of sheep blood immunoglobulins

[0051] 3) Screening for monosodium glutamate saturation After determining the plasma addition amount to be 50%, the monosodium glutamate (MSG) saturation could only be ≤50%. With other experimental conditions fixed, the effect of MSG saturation variations within the range of 34% to 50% was investigated. As shown in Table 2, with increasing MSG saturation, both the purity and recovery rate of immunoglobulins showed a trend of first increasing and then stabilizing. At saturations of 46% and 50%, both purity and recovery rates reached relatively high levels. Based on these results, and considering both reagent costs and process economics, a MSG saturation of 46% was selected.

[0052] Table 2. Effects of monosodium glutamate saturation on the purity and recovery rate of sheep blood immunoglobulins.

[0053] 4) pH value screening Under constant experimental conditions, the effect of pH (6–10) on immunoglobulin isolation was systematically evaluated. As shown in Table 3, the purity and recovery rate of immunoglobulins both showed a trend of first increasing and then decreasing with increasing pH, reaching the optimal level at pH=8.

[0054] Table 3. Effects of pH on the purity and recovery rate of sheep blood immunoglobulins

[0055] 5) Screening of extraction temperature Under constant experimental conditions, the effect of extraction temperature variations within the range of 4 ℃ to 55 ℃ was investigated. As shown in Table 4, with increasing extraction temperature, both the purity and recovery rate of immunoglobulins exhibited a trend of first increasing and then stabilizing. When the extraction temperature reached 25 ℃ or higher, both purity and recovery rate remained at high levels. Based on these results, and considering the energy efficiency and economy of practical operation, 25 ℃, close to room temperature, was selected.

[0056] Table 4. Effect of extraction temperature on the purity and recovery rate of sheep blood immunoglobulins

[0057] 6) Screening of settling time Under fixed experimental conditions, the effect of varying settling time within the range of 0–24 h was investigated. As shown in Table 5, with increasing settling time, both the purity and recovery rate of immunoglobulins showed a trend of first increasing and then decreasing. When the settling time reached 4 h, both purity and recovery rate were close to their optimal levels. Although extending the settling time could slightly improve the recovery rate, considering the balance between time efficiency and separation effect, 4 h was selected as the optimal settling time, which can significantly shorten the process cycle while ensuring high purity and recovery rate.

[0058] Table 5. Effects of settling time on the purity and recovery rate of sheep blood immunoglobulins.

[0059] Example 2: Separation of sheep blood immunoglobulins by monosodium glutamate precipitation method After further optimization of the reaction conditions based on response surface methodology, sheep blood immunoglobulins were separated using sodium glutamate precipitation, specifically including the following steps: (1) After the fresh sheep blood passes the inspection and quarantine, the anticoagulant (3.8% sodium citrate solution) and sheep blood are mixed at a volume ratio of 1:9, centrifuged at 4 ℃ and 4000 rpm for 20 min, the supernatant is separated and stored at -20 ℃. (2) Before use, take out the frozen sheep blood plasma and thaw it in a water bath at 37 ℃; (3) At room temperature, take an appropriate amount of distilled water, add sodium glutamate while stirring until there is no more solid that dissolves, let stand and take the supernatant to obtain a saturated sodium glutamate solution. (4) Add 0.2 mL of PBS solution to 10 mL of sheep blood plasma, slowly add 9.8 mL of saturated sodium glutamate solution while stirring. The saturation of sodium glutamate solution in the salting-out system is 49%. Adjust the pH of the system to 8, stir at 33 °C for 30 min, and then let stand at 4 °C for 4 h. (5) After standing, centrifuge at 4 ℃ and 4000 rpm for 20 min and collect the precipitate; (6) After the precipitate is dissolved in PBS solution, it is dialyzed and lyophilized to obtain sheep blood immunoglobulin.

[0060] The purity of the prepared immunoglobulin was 70.19±2.93% and the recovery rate was 82.61±2.13% as determined by the BCA method and HPLC method.

[0061] The PBS solution used in this embodiment has a pH of 7.2-7.4 and a concentration of 0.01 M.

[0062] Example 3: Structural Identification and Quality Analysis of Sheep Blood Immunoglobulins SDS-PAGE gel electrophoresis was used to compare the molecular weights of sheep plasma (plasma group), sheep immunoglobulin standard (standard group, brand "Beyotime", model A7056), and sheep blood immunoglobulin sample prepared in Example 2 (sample group).

[0063] The results are as follows Figure 3 As shown, the electrophoresis results indicate that, under reducing conditions, both the sample and the sheep blood immunoglobulin standard exhibit two characteristic bands, corresponding to the heavy chain (~50 kDa) and light chain (~25 kDa) of immunoglobulin, respectively. The other bands in the sample and standard are impurities.

[0064] Fourier transform infrared (FTIR) analysis was performed on the immunoglobulins in the sample and sheep blood immunoglobulin standards using a Fourier transform infrared spectroscopy (FTIR) instrument. 2 mg of the sample was ground and mixed with 200 mg of potassium bromide in an agate mortar, and then pressed into transparent thin slices. FTIR spectra were analyzed in the range of 4000–400 cm⁻¹. -1Measurements were taken within the wavenumber range with a resolution of 4 cm. -1 Collect at least 32 scans, and the results are as follows: Figure 4 As shown.

[0065] Immunoglobulins from the samples and sheep blood immunoglobulin standards were analyzed using ultraviolet (UV) spectroscopy. The samples and standards were dissolved in PBS solution to prepare a 1 mg / mL solution. Using PBS solution as a blank, the UV-Vis spectrophotometer was used to scan in the wavelength range of 250–400 nm. The results are as follows: Figure 5 As shown.

[0066] Fluorescence spectra of immunoglobulins from the sample and sheep blood immunoglobulin standards were plotted using a fluorescence spectrometer. A protein solution of 0.2 mg / mL was used, with an excitation wavelength of 280 nm, an emission wavelength of 300–500 nm, and a slit width of 10 nm. The results are as follows: Figure 6 As shown.

[0067] based on Figures 4-6 It can be seen that the immunoglobulin structure of the sample is consistent with that of the standard, indicating that the extraction method of Example 2 causes less damage to the protein structure, can effectively protect the natural structure of immunoglobulins, and is conducive to preserving their functional properties and biological activity.

[0068] Example 4: Purity and recovery rate of immunoglobulins obtained by different extraction methods

[0069] Group 1 Monosodium glutamate precipitation method Sheep blood immunoglobulins were separated using the sodium glutamate precipitation method described in Example 2.

[0070] Group 2 Octyl acid precipitation method Using the same sheep plasma as in Example 2, and referring to the basic principle of the caprylic acid precipitation method in the existing technology (Liu Shengjie, Zhu Maoying, Gu Shibin, et al. Comparison of three extraction methods of immunoglobulin G (IgG) [J]. Chinese Agricultural Science Bulletin, 2007, (11): 38-43.), the specific steps were as follows: Take 4 mL of plasma, add 16 mL of acetate-sodium acetate buffer to dilute, and adjust the pH to 4.5 with 0.1 mol / L NaOH. Slowly add 0.5 mL of caprylic acid while stirring at room temperature, and continue stirring for 30 min. Centrifuge at 10000 rpm for 30 min, discard the precipitate, and collect the supernatant. Filter the supernatant through multi-layer gauze and collect the filtrate. Put the filtrate into a dialysis bag and dialyze for 48 h, changing the dialysate every 8-10 h. Freeze-dry to obtain sheep blood immunoglobulin.

[0071] Group 3 Ammonium sulfate precipitation method Using the same sheep plasma as in Example 2, the ammonium sulfate precipitation method was performed based on the principles of existing techniques (Liu Shengjie, Zhu Maoying, Gu Shibin, et al. Comparison of three extraction methods for immunoglobulin G (IgG) [J]. Chinese Agricultural Science Bulletin, 2007, (11): 38-43.). The specific steps were as follows: Take 20 mL of plasma, add 20 mL of PBS, and slowly add 40 mL of saturated ammonium sulfate solution dropwise under magnetic stirring until the final ammonium sulfate concentration is 50%, adjusting the pH to 7.0. After stirring overnight at 4 ℃, let stand for 1 h, centrifuge at 4000 rpm for 30 min, and discard the supernatant. Dissolve the precipitate in 40 mL of PBS, and slowly add 20 mL of saturated ammonium sulfate solution dropwise under magnetic stirring until the final concentration is 33%, adjusting the pH to 7.0, stirring at 4 ℃ for 6 h, letting stand for 1 h, centrifuging at 4000 rpm for 30 min, and discarding the supernatant. Repeat this 33% ammonium sulfate precipitation step once. The final precipitate was dissolved in 20 mL PBS, dialyzed at 4 °C for 48 h, and then lyophilized to obtain sheep blood immunoglobulin.

[0072] As shown in Table 6, monosodium glutamate used in Group 1 has no GHS hazard classification, while octanoic acid used in Group 2 and ammonium sulfate used in Group 3 both pose risks such as skin corrosion / irritation and aquatic environmental hazards.

[0073] Table 6 Comparison of the hazardous properties of reagents used in different extraction methods

[0074] Note: The information in the table is sourced from the PubChem database and the National Hazardous Chemicals Safety Public Service Internet Platform.

[0075] like Figure 7 , Figure 8 As shown, the purity (70.19%) and recovery rate (82.61%) of immunoglobulins obtained by the sodium glutamate precipitation method in group 1 were significantly better than those obtained by the octanoic acid precipitation method in group 2 and the ammonium sulfate precipitation method in group 3.

[0076] In summary, this invention is the first to use monosodium glutamate (MSG) as a salting-out agent, achieving sheep blood immunoglobulin with a purity and recovery rate exceeding 70% in a single salting-out process. This method is simple to operate, has a large processing capacity, is fast, requires minimal equipment and environmental conditions, is low-cost, and is easily scalable for large-scale production. The MSG used is food-grade, safe, and biodegradable. The salting-out wastewater is ammonia-free, has good biodegradability, and requires no deammoniation treatment. Compared to the traditional ammonium sulfate salting-out method, this significantly reduces wastewater treatment costs and is more environmentally friendly. The process is mild and controllable, effectively protecting the natural structure of immunoglobulins. Using sheep blood as raw material, slaughter waste is transformed into high-value-added products, reducing resource waste and environmental pollution. The resulting products can be directly applied to functional foods or pet nutrition foods, providing a feasible path for the high-value utilization of sheep blood, with significant economic benefits and broad market prospects.

[0077] The preferred embodiments of the present invention have been described in detail above. It should be understood that the detailed description of the technical solutions of the present invention by means of preferred embodiments is illustrative and not restrictive. Those skilled in the art can modify the technical solutions described in the embodiments or make equivalent substitutions for some of the technical features based on reading the present invention specification; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating sheep blood immunoglobulins, characterized in that, The separation method includes the step of adding a saturated sodium glutamate solution to sheep plasma for salting out.

2. The separation method according to claim 1, characterized in that, The amount of sheep plasma added to the salting-out system is 35-50% of the total volume of the mixture.

3. The separation method according to claim 1, characterized in that, The sodium glutamate solution in the salting-out system has a saturation of 46-50%.

4. The separation method according to claim 1, characterized in that, The salting-out process includes adding PBS solution and saturated sodium glutamate solution to sheep plasma, adjusting the pH, stirring, allowing to stand, centrifuging, and collecting the precipitate.

5. The separation method according to claim 4, characterized in that, The pH value is 7.4~9.0, and / or The stirring temperature is 25~55℃, and / or The settling time is more than 2 hours.

6. The separation method according to claim 1, characterized in that, The method for preparing sheep plasma includes the following steps: adding an anticoagulant to sheep blood, mixing, centrifuging, and separating the supernatant to obtain sheep plasma.

7. The separation method according to claim 6, characterized in that, The anticoagulant is heparin, sodium citrate, or ethylenediaminetetraacetic acid; the volume ratio of the anticoagulant to sheep blood is 1:5~20.

8. The separation method according to claim 1, characterized in that, The separation method further includes the steps of dissolving, dialysis to remove salt, and drying the salt-precipitated product after salting out.

9. The separation method according to claim 1, characterized in that, The separation method includes the following steps: (1) Preparation of sheep plasma: Add 3.8% sodium citrate solution to fresh sheep blood as an anticoagulant, mix well, centrifuge, separate the supernatant to obtain sheep plasma; (2) Salting out precipitation: Add PBS solution and saturated monosodium glutamate solution to the sheep plasma obtained in step (1) until the monosodium glutamate solution saturation is 46~50%, adjust the pH of the system to 7.4~9.0, stir at 25~55 ℃, let stand for more than 2 hours, centrifuge, and collect the precipitate; (3) Desalting and drying: The precipitate obtained in step (2) is dissolved, dialyzed to remove salt, and then dried to obtain sheep blood immunoglobulin.

10. The application of the separation method according to any one of claims 1 to 9 in improving the separation effect of sheep blood immunoglobulins; the separation effect includes the purity and / or recovery rate of sheep blood immunoglobulins.

Images