Extraction method and application of high-stability immune globulin

Abstract

The invention discloses an extraction method and application of high-stability immune globulin, and belongs to the technical field of protein separation. The method comprises the following steps: adding a first protective agent into defatted bovine colostrum, performing enzymolysis curding, performing whey separation and concentration, and adding a second protective agent to obtain the high-stability immunoglobulin extracting solution, the first protective agent is composed of sodium caseinate and sodium citrate, and the second protective agent is fructo-oligosaccharide. After the immunoglobulin liquid prepared by the method is stored for 90 days at 4 DEG C, the activity retention rate reaches up to 85% or above, the protein advanced structure integrity is remarkably maintained, and the immunoglobulin liquid can be directly used as a functional ingredient to be stably added into a liquid dairy product. The method is suitable for preparing the dairy product containing the high-stability immunoglobulin.

Description

Technical Field

[0001] This invention belongs to the field of protein separation technology, specifically a method for extracting highly stable immunoglobulins and its application. Background Technology

[0002] Bovine colostrum is rich in immunoglobulins, among which IgG has attracted much attention due to its important immunomodulatory function. Currently, enzymatic hydrolysis is a common method for enriching IgG by hydrolyzing other proteins. However, immunoglobulins are proteins that are highly sensitive to physicochemical conditions, and are easily affected by various stresses during extraction, such as heating, pH changes, enzymatic treatment, and mechanical shearing. More importantly, even after successful extraction, they are still prone to denaturation, aggregation, and loss of activity in liquid storage, leading to a significant reduction in their biological stability.

[0003] In existing technologies, some extraction processes, such as Chinese patent CN115417929B, use ammonium sulfate salting-out for the precipitation and purification of immunoglobulins. However, this method still has significant limitations: firstly, ammonium sulfate is a chemical reagent that cannot be used in food, and its use in the extraction process will result in the product being unsuitable for direct application in the food industry; secondly, the process route is still relatively complex, involving multiple salting-out and centrifugation operations, which not only easily damages the native conformation and biological activity of immunoglobulins during repeated acid-base adjustments and centrifugal shearing, but also makes it difficult to meet the requirements of production efficiency and safety for industrial production. Furthermore, for ease of preservation, many immunoglobulin products are prepared as lyophilized powders, which brings inconvenience to fields that require the direct application of immunoglobulins in liquid form, such as adding them to liquid milk.

[0004] Therefore, developing an extraction method that maintains the structural stability of immunoglobulins, is suitable for liquid food systems, and is industrially feasible is of great technical significance and application demand. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for extracting highly stable immunoglobulins. By adding specific protective agents at specific stages, the long-term storage activity of immunoglobulin solutions can be improved, thereby achieving the goal of large-scale preparation of highly stable food-grade immunoglobulin raw materials.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for extracting highly stable immunoglobulins involves first adding a first protective agent to skimmed bovine colostrum, then enzymatically hydrolyzing the coagulated milk, separating and concentrating the whey, and adding a second protective agent to obtain a highly stable immunoglobulin extract. The first protective agent is composed of sodium caseinate and sodium citrate in a weight ratio of 0.6~4.5:0.4~1.5; the second protective agent is fructooligosaccharide.

[0007] Preferably, the weight ratio of sodium caseinate to sodium citrate is 1:0.4.

[0008] Sodium caseinate in the first protective agent preferentially adsorbs at the gas-liquid interface and on the surface of protein molecules. Through steric hindrance, it prevents IgG in skimmed bovine colostrum from undergoing interfacial denaturation and intermolecular aggregation during subsequent enzymatic hydrolysis and stirring. At the same time, it can competitively bind to proteases, reducing non-target hydrolysis of IgG. Sodium citrate, as a highly efficient pH buffer, establishes and maintains a neutral and stable environment for the enzymatic hydrolysis reaction, effectively preventing changes in IgG charge distribution and conformational instability caused by pH fluctuations.

[0009] The second protective agent, fructooligosaccharides, forms a stable, highly hydrated layer around IgG molecules, increasing the hydration level of the protein surface and making its structure more compact. This effectively combats conformational unfolding and aggregation caused by molecular thermal motion during liquid storage.

[0010] The first and second protective agents have complementary mechanisms of action, providing protection at the molecular interface, environmental pH, and solution thermodynamics levels, respectively, resulting in a synergistic effect, improving the stability of IgG molecules, effectively maintaining the secondary and tertiary structures of IgG, and preventing its denaturation and inactivation.

[0011] As a limitation of the present invention, the following steps are performed sequentially: S1. Adding the first protective agent: Adding a first protective agent at a weight percentage of 1% to 6% of the skimmed bovine colostrum to obtain a mixture; S2. Enzymatic hydrolysis of coagulant: Add 0.01% to 0.03% of rennet by weight of skimmed bovine colostrum to the mixture and hydrolyze at the hydrolysis temperature for 0.8 to 1.5 hours to obtain enzymatic hydrolyzed coagulant; The added rennet brought its activity concentration in the final reaction system to 620 IMCU / mL. S3. Whey separation and concentration: Mechanically cut the enzymatically hydrolyzed coagulated milk until the whey liquid flows out, let it stand for 0.5~1h, remove the precipitate by sieving, separate the whey liquid, and concentrate it by ultrafiltration membrane to obtain concentrated whey; S4. Add a second protective agent: Add a second protective agent to the concentrated whey, mix well, and obtain a highly stable immunoglobulin extract; The amount of the second protective agent is 2% to 7% of the weight of the skimmed bovine colostrum.

[0012] Preferably, the weight ratio of sodium caseinate, sodium citrate, and fructooligosaccharides is 1:0.4:2.

[0013] As a second limitation of the present invention, the defatted bovine colostrum is bovine milk produced by a cow within one day of calving, which has been defatted to a fat content of ≤0.5%.

[0014] As a further limitation of the present invention, the mesh size of the sieve is 200 mesh.

[0015] As another limitation of the present invention, sterilization is performed after adding a second protective agent.

[0016] As a further limitation of the present invention, the sterilization process is performed by filtration through a filter membrane.

[0017] As another limitation of the present invention, the molecular weight cutoff of the ultrafiltration membrane is 10 kDa.

[0018] The present invention also provides an application of a highly stable immunoglobulin extract, wherein the highly stable immunoglobulin extract obtained by the above extraction method is used to be added to dairy products.

[0019] As a further limitation of the present invention, the amount of the highly stable immunoglobulin extract added is 0.5~1g / 250mL.

[0020] The present invention also provides a milk containing a highly stable immunoglobulin extract, wherein any of the above-mentioned highly stable immunoglobulin extracts is added; The milk, when stored at 4°C for 60 days, retained ≥90.4% of the activity of immunoglobulin IgG. The milk was stored at 4°C for 90 days, and the activity retention rate of immunoglobulin IgG was ≥84.7%, while the content of irregularly folded immunoglobulins in the immunoglobulin structure was ≤42.3%.

[0021] Furthermore, the amount of highly stable immunoglobulin extract added to the milk was 0.6g / 250 mL.

[0022] Furthermore, the above-mentioned milk contains 25.90 mg / 100g of immunoglobulins, 103.00 mg / 100g of α-lactalbumin, 194.00 mg / 100g of β-lactoglobulin, and 2.43 mg / 100g of lactoferrin.

[0023] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows: This invention employs a composite protective agent system composed of sodium caseinate, sodium citrate, and fructooligosaccharides, and utilizes a staged addition strategy to protect immunoglobulins throughout the extraction process, significantly improving their long-term storage stability. Specifically, a first protective agent is added during the skimmed bovine colostrum stage. Sodium caseinate preferentially adsorbs at the gas-liquid interface and on the surface of protein molecules, effectively preventing interfacial denaturation and intermolecular aggregation of immunoglobulins during subsequent enzymatic hydrolysis and stirring through steric hindrance. It also competitively binds to proteases, reducing non-target hydrolysis. Sodium citrate acts as a highly efficient pH buffer, establishing and maintaining a neutral and stable environment, preventing changes in immunoglobulin charge distribution and conformational instability caused by pH fluctuations. After whey separation and concentration, a second protective agent, fructooligosaccharides, is further added. These form a stable, highly hydrated layer around the immunoglobulin molecules, increasing protein surface hydration and making the structure more compact, thereby resisting conformational unfolding and aggregation caused by molecular thermal motion during liquid storage.

[0024] The staged protection strategy and specific protective agent combination employed in this invention can synergistically act on multiple levels, including molecular interfaces, environmental pH, and solution thermodynamics, producing a significant synergistic effect. Circular dichroism spectroscopy and fluorescence spectroscopy analysis verified the protective effect of this invention at the molecular structure level: the α-helix and β-sheet content of the experimental group was closest to that of the fresh sample, with only a slight increase in random coils, indicating a high degree of preservation of its regular secondary structure; regarding the tertiary structure, the maximum fluorescence emission wavelength (336 nm) of the experimental group was almost identical to that of the fresh sample (335 nm), with only a small decrease in fluorescence intensity, proving its compact three-dimensional structure and intact hydrophobic core. In contrast, the control group without protective agent and the group with a single addition of protective agent both exhibited severe structural damage and insufficient immunoglobulin stability.

[0025] The highly stable immunoglobulin extract prepared by the extraction method of this invention showed a significantly higher bioactivity retention rate than the unprotected group after being stored at 4°C for 90 days. All the protective agents used were food-grade raw materials with neutral flavor, which did not affect the sensory quality of pure milk products and had good application compatibility. In particular, it was unexpectedly found that the staged addition strategy was highly effective in maintaining the structural integrity of immunoglobulins.

[0026] This invention is applicable to the large-scale preparation of highly stable food-grade immunoglobulin raw materials. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the process flow of a method for extracting highly stable immunoglobulins according to Embodiment 1 of the present invention. Detailed Implementation

[0028] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1 This embodiment discloses a method for extracting highly stable immunoglobulins and their application in VTIS sterilized milk, such as... Figure 1 Its process flow includes the following steps performed sequentially: S1. Add the first protective agent Milk produced by cows within one day of calving is collected after acceptance and degreased using a separator at a rate of 2 t / h until the fat content is ≤0.5%, thus completing the degreasing process and producing degreased bovine colostrum. 10 kg of sodium caseinate and 4 kg of sodium citrate were mixed evenly to obtain the first protective agent; Take 500 kg of skimmed bovine colostrum and add the first protective agent mentioned above (the amount of the first protective agent added is 2.8 wt.%), mix well to obtain a mixture; S2. Enzymatic hydrolysis of curd Place the mixture in a clean cheese tank, heat the mixture to the suitable enzymatic hydrolysis temperature of 40°C, add 50 g of rennet (0.01 wt.%) to the mixture, so that its activity concentration in the final reaction system reaches 620 IMCU / mL, and enzymatically hydrolyze at 40°C for 1.5 h to obtain enzymatically hydrolyzed curd; S3. Whey separation and concentration Maintain the temperature in the cheese vat at 40°C, mechanically cut the enzymatically hydrolyzed coagulated milk until the whey flows out, let it stand for 1 hour, pass it through a 200-mesh sieve, remove casein precipitate through a milk purifier, and separate the whey. Concentrate the whey through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain concentrated whey. If the whey is insufficient after the milk purifier removes casein precipitate or if there is a need for temporary storage, it can be frozen and stored at -18°C for later use.

[0031] S4. Add a second protective agent 20 kg of fructooligosaccharides (4 wt.%) were added to the concentrated whey as a second protective agent, mixed evenly, and then sterilized by filtration through a 0.22 μm filter membrane. After mixing, a highly stable immunoglobulin extract was obtained. The highly stable immunoglobulin extract was tested and found to contain 7400.0 mg / 100g of immunoglobulin, 3130.0 U / L of lactoperoxidase, and 33.1 mg / 100g of lactoferrin.

[0032] In this embodiment, the weight ratio of sodium caseinate to sodium citrate is 1:0.4.

[0033] A highly stable immunoglobulin extract was added at a rate of 0.6 g / 250 mL to VTIS-sterilized milk sterilized at 149°C for 1 s in an aseptic production line to obtain milk containing the highly stable immunoglobulin extract. Testing revealed that this milk contained 25.90 mg / 100g of immunoglobulins, 103.00 mg / 100g of α-lactalbumin, 194.00 mg / 100g of β-lactoglobulin, and 2.43 mg / 100g of lactoferrin. After storage at 4°C for 60 days, the milk retained 90.4% of the immunoglobulin IgG activity; after storage at 4°C for 90 days, the milk retained 84.7% of the immunoglobulin IgG activity, and the content of randomly folded immunoglobulins in the immunoglobulin structure was 42.3%.

[0034] Comparative Example 1 This comparative example is a blank control immunoglobulin extract, and its preparation method is basically the same as that in Example 1, except that no protective agent is added.

[0035] Comparative Example 2 This comparative example is an immunoglobulin extract with added low concentration of protective agent. Its preparation method is basically the same as that of Example 1, except that the protective agent components and dosage are added. The protective agent is 0.5 wt.% sodium caseinate, 0.2 wt.% sodium citrate, and 1 wt.% fructooligosaccharide, based on the weight percentage of skimmed bovine colostrum.

[0036] Comparative Example 3 This comparative example is an immunoglobulin extract with added high concentration of protective agent. Its preparation method is basically the same as that of Example 1. The only difference is the composition and amount of the added protective agent, which is 5 wt.% sodium caseinate, 2 wt.% sodium citrate, and 8 wt.% fructooligosaccharides, based on the weight percentage of skimmed bovine colostrum.

[0037] Comparative Example 4 This comparative example is an immunoglobulin extract, and its preparation method is basically the same as that of Example 1. The only difference is that all the protective agents, namely sodium caseinate (2 wt.% of the weight of skimmed bovine colostrum), sodium citrate (0.8 wt.% of the weight of the first protective agent), are added to the skimmed bovine colostrum all at once when the first protective agent is added, instead of being added separately as the first and second protective agents.

[0038] Comparative Example 5 This comparative example is an immunoglobulin extract, and its preparation method is basically the same as that of Example 1. The only difference is that all the protective agents, namely sodium caseinate (2 wt.% of the weight of skimmed bovine colostrum), sodium citrate (0.8 wt.% of the weight of the colostrum), are added to the concentrated whey all at once after ultrafiltration membrane concentration, instead of being added separately as first and second protective agents.

[0039] Effect Experiment Example 1 Equal amounts of immunoglobulin extracts obtained in Example 1 and Comparative Example 1 were stored at 4°C for 90 days. Every 30 days, the IgG activity retention rate was tested according to the group standard "BJC-FDD-FB2102". The results are shown in Table 1.

[0040] Table 1 Results of periodic IgG activity retention rate assay Note: Different letter subscripts for the numbers in the table indicate significant differences at the 5% level.

[0041] The results in Table 1 show that the first and second protective agents of the present invention can significantly delay the activity decay of IgG in liquid storage, and the IgG activity retention rate after 90 days is more than twice that of Comparative Example 1.

[0042] Effect Experiment Example 2 Equal amounts of immunoglobulin extracts obtained from Example 1, Comparative Example 2, and Comparative Example 3 were stored at 4°C for 60 days. The IgG activity retention rate was detected on day 60, referring to the group standard "BJC-FDD-FB2102". At the same time, the three newly produced immunoglobulin extracts were added at 0.6 g / 250 mL to VTIS sterilized milk sterilized at 149°C for 1 s in an aseptic production line and stored at room temperature of 25°C. Sensory evaluators were invited to conduct sensory evaluations, and the results are shown in Table 2.

[0043] Table 2. IgG activity retention rate and sensory evaluation results The results of Comparative Example 2 (low protectant concentration) showed that insufficient protectant concentration prevented the formation of a complete protective layer, leading to significant structural denaturation of immunoglobulins during storage and a significant reduction in bioactivity retention. While Comparative Example 3 (high protectant concentration) provided sufficient protection, excessive protectant concentration resulted in significant negative effects: firstly, it altered the osmotic pressure balance of the system, causing slight shrinkage of protein molecules; secondly, due to the flavor characteristics of the protectant itself, higher concentrations negatively impacted the product flavor and significantly increased raw material costs.

[0044] In contrast, the optimal ratio of protectants determined by this invention can ensure its biological activity while avoiding the problems of flavor degradation and cost increase caused by excessive protectants.

[0045] Effect Experiment Example 3 This experimental example was used to analyze the effect of different methods of adding the protective agent on the IgG activity retention rate after 60 days of storage. Specifically, equal amounts of immunoglobulin extracts obtained from Example 1, Comparative Example 4, and Comparative Example 5 were stored at 4°C for 60 days. The detection method was based on the group standard "BJC-FDD-FB2102", and the IgG activity retention rate was measured on day 60. The results are shown in Table 3.

[0046] Table 3. Comparison of effects of different addition methods after 60 days of storage. The results showed that the phased addition method of the present invention has a significantly better protective effect on IgG than the one-time addition. This is because adding fructooligosaccharides too early may result in losses due to Maillard reactions during enzymatic hydrolysis and heat treatment; while adding sodium caseinate and sodium citrate too late cannot provide immediate protection for IgG in the critical stages of enzymatic hydrolysis and pH adjustment.

[0047] Effect Experiment Example 4 This experimental example uses molecular-level analysis to examine the protective effect of the preparation method of the present invention on the immunoglobulin structure. Multiple spectroscopic methods were used to characterize the protein structural integrity of the immunoglobulin extracts from Example 1 (stored for 0 days), Example 1 (stored for 90 days), Comparative Example 1 (stored for 90 days), and Comparative Example 4 (stored for 90 days).

[0048] The analysis included: Primary structure analysis: High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used to analyze the peptide sequence coverage after enzymatic digestion and assess whether peptide chain breakage occurred. Secondary structure analysis: Circular dichroism (CD) was used to analyze the spectral changes in the far-ultraviolet region (190-250 nm) and calculate the changes in the content of regular structures such as α-helices and β-sheets. Tertiary structure analysis: Endogenous fluorescence spectroscopy was used to analyze the maximum emission wavelength shift of tryptophan (Trp) residues to determine whether the hydrophobic core of the protein's three-dimensional structure had unfolded.

[0049] Table 4. Characterization results of immunoglobulin structural integrity The results showed that the peptide coverage of all groups in the primary structure was high and the differences were small, indicating that the peptide backbone was less broken under the storage conditions of 4°C. Example 1 had the highest coverage, indicating that the composite protectant could slightly inhibit possible hydrolysis or chemical degradation.

[0050] The secondary structure data clearly showed the differences in protective effects. The α-helix and β-sheet content of Example 1 was closest to that of the fresh sample, with only a slight increase in random coils, indicating that its regular secondary structure was highly preserved. The α-helix and β-sheet content of Comparative Example 1 (without protectant) decreased significantly, while the random coils increased dramatically, indicating that the immunoglobulin underwent severe structural unfolding and denaturation, changing from an ordered structure to a disordered structure. The structural preservation of Comparative Example 4 (single addition) was between the two, but significantly worse than that of Example 1, demonstrating the superiority of the staged addition strategy at the molecular level.

[0051] In the tertiary structure, tryptophan (Trp) within immunoglobulins is typically encapsulated in a hydrophobic core, with its maximum fluorescence emission wavelength at 335 nm. When proteins unfold, Trp are exposed to a hydrophilic environment, leading to a redshift in λmax and quenching of fluorescence intensity. In Example 1, λmax (336 nm) was almost identical to that of the fresh sample (335 nm), with only a small decrease in fluorescence intensity, indicating a compact three-dimensional structure and intact hydrophobic core. In Comparative Example 1 (without protective agent), λmax redshifted to 348 nm, and fluorescence intensity was drastically quenched, a typical characteristic of severe protein unfolding and exposure of the hydrophobic core. Comparative Example 4 (single addition) also showed a significant redshift and quenching, indicating that its tertiary structure had been disrupted, but to a lesser extent than the group without protective agent.

[0052] In summary, by comparing the changes in the primary, secondary, and tertiary structures of immunoglobulins after storage, it is shown that the method of the present invention has significant advantages in maintaining the integrity of the higher-order structure of proteins. In particular, the strategy of adding protective agents in stages can effectively inhibit protein unfolding and denaturation, thereby ensuring the long-term stability of its biological activity.

[0053] Example 2 This embodiment discloses a method for extracting highly stable immunoglobulins and their application in pasteurized milk, which includes the following steps performed sequentially: S1. Add the first protective agent Milk produced by cows within one day of calving is collected after acceptance and degreased using a separator at a rate of 2 t / h until the fat content is ≤0.5%, thus completing the degreasing process and producing degreased bovine colostrum. 1.43 kg of sodium caseinate and 3.57 kg of sodium citrate were mixed evenly to prepare the first protective agent; Take 500 kg of skimmed bovine colostrum and add the first protective agent mentioned above (the amount of the first protective agent added is 1 wt.%), mix well to obtain a mixture; S2. Enzymatic hydrolysis of curd Place the mixture in a clean cheese tank and heat it to the appropriate hydrolysis temperature of rennet, 40°C. Add 100 g of rennet (0.02 wt.%) to the mixture to achieve an activity concentration of 620 IMCU / mL in the final reaction system. Hydrolyze the mixture at 40°C for 1 hour to obtain enzymatically hydrolyzed curd. S3. Whey separation and concentration Maintain the temperature in the cheese vat at 40°C, mechanically cut the enzymatically hydrolyzed coagulated milk until the whey flows out, let it stand for 0.7 hours, pass it through a 200-mesh sieve, remove casein precipitate through a milk purifier, and separate the whey. Concentrate the whey through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain concentrated whey. S4. Add a second protective agent 10 kg of fructooligosaccharides (2 wt.%) were added to the concentrated whey as a second protective agent, mixed evenly, and then filtered through a 0.22 μm filter membrane to sterilize. The mixture was then stirred to obtain a highly stable immunoglobulin extract. In this embodiment, the weight ratio of sodium caseinate to sodium citrate is 2:5.

[0054] A milk containing a highly stable immunoglobulin extract is obtained by adding 1 g / 250 mL of the extract to pasteurized milk in an aseptic production line.

[0055] Example 3 This embodiment discloses a method for extracting highly stable immunoglobulins and their application in pasteurized milk, which includes the following steps performed sequentially: S1. Add the first protective agent Milk produced by cows within one day of calving is collected after acceptance and degreased using a separator at a rate of 2 t / h until the fat content is ≤0.5%, thus completing the degreasing process and producing degreased bovine colostrum. 27.55 kg of sodium caseinate and 2.45 kg of sodium citrate were mixed evenly to obtain the first protective agent; Take 500 kg of skimmed bovine colostrum and add the first protective agent mentioned above (the amount of the first protective agent added is 6 wt.%), mix well to obtain a mixture; S2. Enzymatic hydrolysis of curd Place the mixture in a clean cheese tank and heat it to the appropriate hydrolysis temperature of rennet, 40°C. Add 150 g of rennet (0.03 wt.%) to the mixture to achieve an activity concentration of 620 IMCU / mL in the final reaction system. Hydrolyze the mixture at 40°C for 0.8 h to obtain enzymatically hydrolyzed curd. S3. Whey separation and concentration Maintain the temperature in the cheese vat at 40°C, mechanically cut the enzymatically hydrolyzed coagulated milk until the whey flows out, let it stand for 0.5 hours, pass it through a 200-mesh sieve, remove casein precipitate through a milk purifier, and separate the whey. Concentrate the whey through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain concentrated whey. S4. Add a second protective agent Add 35 kg of fructooligosaccharides (7 wt.%) as a second protective agent to concentrated whey, mix well, filter through a 0.22 μm filter membrane to sterilize, mix well, and obtain a highly stable immunoglobulin extract. In this embodiment, the weight ratio of sodium caseinate to sodium citrate is 4.5:0.4.

[0056] Adding 0.5 g / 250 mL of highly stable immunoglobulin extract to pasteurized milk in an aseptic production line yields a milk containing highly stable immunoglobulin extract.

[0057] The milk prepared in Examples 2 and 3 was subjected to IgG activity retention rate detection and immunoglobulin structural integrity characterization, as shown in Effect Experiment Example 5 and Effect Experiment Example 6.

[0058] Table 5. IgG activity retention rate under 4℃ storage conditions The results showed that the IgG activity retention rate of the milk prepared in Examples 2 and 3 after storage at 4°C for different times decreased in a basically consistent manner with that in Example 1, indicating that the protection system of the present invention is effective. After 90 days of storage, the IgG activity retention rates of Examples 2 and 3 were 83.0% and 81.5%, respectively, which were slightly lower than those of Example 1, but still maintained at a high level of over 80%, indicating that the method of the present invention can effectively maintain IgG activity under different amounts of protective agent.

[0059] Effect Experiment Example 6 The immunoglobulin extracts prepared in Examples 2 and 3 were stored for 90 days using spectroscopic methods to characterize the protein structural integrity. The results were then compared with those of the immunoglobulin structural integrity analysis of Example 1 after storage for 0 days and 90 days, as shown in Table 6.

[0060] Table 6. Characterization results of immunoglobulin structural integrity Table 6 shows that the secondary and tertiary structure data of the immunoglobulin extracts in Examples 2 and 3 are close to those in Example 1 (90 days) and are significantly better than those in the unprotected sample of Comparative Example 1, indicating that the protectant of the present invention can effectively inhibit the development and denaturation of immunoglobulins under different ratios.

[0061] In other embodiments, the amount of the first protective agent added is 1% or 3.5% or 4.6% or 5.5% or 6% of the weight of the skimmed bovine colostrum. The first protective agent is composed of sodium caseinate and sodium citrate in a weight ratio of 3:2 or 1:1.2 or 1:1 or 4:1 or 3:2 or 3:1. The amount of the second protective agent is 2% or 3.5% or 4.6% or 6.5% or 7% of the weight of the skimmed bovine colostrum. All of these methods successfully produce milk containing a highly stable immunoglobulin extract.

Claims

1. A method for extracting highly stable immunoglobulins, characterized in that, First, a first protective agent is added to the skimmed bovine colostrum, followed by enzymatic hydrolysis of the coagulated milk, whey separation and concentration, and the addition of a second protective agent to obtain a highly stable immunoglobulin extract. The first protective agent is composed of sodium caseinate and sodium citrate in a weight ratio of 0.6~4.5:0.4~1.5; the second protective agent is fructooligosaccharide.

2. The method for extracting highly stable immunoglobulins according to claim 1, characterized in that, This includes the following steps performed sequentially: S1. Adding the first protective agent: Adding a first protective agent at a weight percentage of 1% to 6% of the skimmed bovine colostrum to obtain a mixture; S2. Enzymatic hydrolysis of coagulant: Add 0.01% to 0.03% of rennet by weight of skimmed bovine colostrum to the mixture and hydrolyze at the hydrolysis temperature for 0.8 to 1.5 hours to obtain enzymatic hydrolyzed coagulant; S3. Whey separation and concentration: Mechanically cut the enzymatically hydrolyzed coagulated milk until the whey liquid flows out, let it stand for 0.5~1h, remove the precipitate by sieving, separate the whey liquid, and concentrate it by ultrafiltration membrane to obtain concentrated whey; S4. Add a second protective agent: Add a second protective agent to the concentrated whey, mix well, and obtain a highly stable immunoglobulin extract; The amount of the second protective agent is 2% to 7% of the weight of the skimmed bovine colostrum.

3. The method for extracting highly stable immunoglobulins according to claim 2, characterized in that, The defatted bovine colostrum is the milk produced by a cow within one day of calving, which has been defatted to a fat content of ≤0.5%.

4. The method for extracting highly stable immunoglobulins according to claim 3, characterized in that, The sieve mesh size is 200 mesh.

5. The method for extracting highly stable immunoglobulins according to claim 4, characterized in that, After adding the second protective agent, sterilization is performed.

6. The method for extracting highly stable immunoglobulins according to claim 5, characterized in that, Sterilization is achieved through membrane filtration.

7. A method for extracting highly stable immunoglobulins according to any one of claims 2 to 5, characterized in that, The ultrafiltration membrane has a molecular weight cutoff of 10 kDa.

8. An application of an extract containing highly stable immunoglobulins, characterized in that, The highly stable immunoglobulin extract according to any one of claims 1 to 7 is used for addition to dairy products.

9. The application of the highly stable immunoglobulin extract according to claim 8, characterized in that, The amount of the highly stable immunoglobulin extract added to dairy products is 0.5~1g / 250 mL.

10. A type of milk containing a highly stable immunoglobulin extract, characterized in that, Add the highly stable immunoglobulin extract according to any one of claims 1 to 7; The milk, when stored at 4°C for 60 days, retained ≥90.4% of the activity of immunoglobulin IgG. The milk was stored at 4°C for 90 days, and the activity retention rate of immunoglobulin IgG was ≥84.7%, while the content of irregularly folded immunoglobulins in the immunoglobulin structure was ≤42.3%.

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