Preparation method of plasma albumin peptide rich in immune globulin

Abstract

The invention belongs to the technical field of biology, and discloses a preparation method of plasma albumin peptide rich in immune globulin, which comprises the following steps: step 1, pretreatment; step 2, diluting; 3, fibrinogen in the plasma is removed; step 4, performing enzymolysis treatment; step 5, enzyme deactivation; and step 6, performing post-treatment. A calcium chloride precipitation method is combined with a membrane separation technology, the fibrinogen removal rate is larger than 98%, impure protein in plasma can be effectively removed, the purity of a target product is improved, a biological enzymolysis technology is combined with the membrane separation technology, the immune globulin retention rate is larger than 90% (ELISA method quantitative analysis), the target product can be further purified, and the purity of the target product is improved. The plasma albumin peptides with different molecular weight ranges are obtained, the prepared finished product has high protein content and antioxidant activity, can be applied to functional food or medicine for enhancing immunity, resisting inflammation or resisting oxidation, can reduce the serum TNF-alpha level by more than or equal to 40%, and is simple in process, low in cost and easy for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing plasma albumin peptide rich in immunoglobulins. Background Art

[0002] Plasma albumin is the most abundant protein in plasma and has multiple physiological functions such as maintaining plasma colloid osmotic pressure, transporting nutrients, and antioxidant. In recent years, it has been found that plasma albumin peptides rich in immunoglobulins have various biological activities, such as enhancing immunity, anti-inflammatory, and antioxidant. Immunoglobulins are important immune active substances in plasma, which can recognize and bind specific antigens and mediate humoral immune responses.

[0003] Immunoglobulins are a type of immune protein widely present in human or animal blood. When the immune system encounters foreign invaders, it will produce different amounts of globulins according to the different invaders. If the invaders are difficult to eliminate, the immune system will produce more globulins after stimulating the lymph until the invaders are eliminated.

[0004] Animal plasma is rich in various functional active proteins, and the content of globulins accounts for about 2%. A large amount of animal blood by-products will be generated during the slaughter process of animals. At present, the development and utilization of animal blood mainly uses whole blood as raw material and is applied in the food and feed industries, with very low product added value. Especially, the utilization rate of functional active proteins in blood is even more insufficient, mainly because the extraction technology of functional active proteins in blood is not good enough.

[0005] In the actual application process of existing equipment, the main methods for extracting single proteins from animal blood include salting-out method, chromatography method, chromatography method, and ultrafiltration method. All of them have practical problems that are difficult to break through, such as long preparation cycle, high cost, low recovery rate (less than 50%), low product purity, and it is difficult to obtain plasma albumin peptides and immunoglobulins with high purity and high activity at the same time, high requirements for production equipment and environment, and cumbersome separation operation process. Therefore, there is still a large space for process optimization in the large-scale preparation of plasma albumin peptides rich in immunoglobulins. Plasma albumin peptides rich in immunoglobulins have various uses, and there is currently no large-scale preparation technology, which is not conducive to actual application and operation. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for preparing plasma albumin peptide rich in immunoglobulins with simple process, low cost, high product purity, and good activity, which solves the problems existing in the background art.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing plasma albumin peptide rich in immunoglobulins, comprising the following steps:

[0008] Step 1: Pretreatment: Centrifuge fresh animal plasma to remove blood cells and collect the supernatant.

[0009] Step 2: Dilution: Dilute the animal plasma with physiological saline to a protein content of 2% - 5%.

[0010] Step 3: Remove fibrinogen from the plasma and collect the supernatant S2 containing animal plasma albumin and immunoglobulin.

[0011] Step 4: Enzymatic hydrolysis treatment: First, place the supernatant S2 in an environment of 55 - 65°C, add alkaline protease to the supernatant S2, with an addition amount of 0.06 - 0.10% of the mass of the supernatant, at pH 8.5 - 9.5, and obtain a hydrolyzate after treatment for 1.5 - 2.5 h.

[0012] Step 5: Inactivate the enzyme: Adjust the pH of the hydrolyzate to 7.0 - 7.4 with 0.1 M hydrochloric acid, perform solid - liquid separation to obtain the supernatant; the supernatant is filtered through a membrane to obtain a product dilution.

[0013] Step 6: Post - treatment: Concentrate the product dilution to obtain a product concentrate, and then perform aseptic filtration, freeze - drying, pulverization, and packaging to obtain a plasma albumin peptide powder product rich in immunoglobulin.

[0014] Preferably, the method for removing fibrinogen from the plasma in Step 3 is as follows:

[0015] Step a: Adjust the plasma pH: Measure the pH value of the plasma with a pH meter and adjust the pH to 7.0 - 7.4 with dilute hydrochloric acid or sodium hydroxide solution.

[0016] Step b: Add calcium chloride solution: Slowly add calcium chloride solution to the plasma under stirring conditions until the final concentration reaches 10 - 20 mM.

[0017] Step c: Incubation: Place the mixture in a 37°C water bath and incubate for 40 - 80 minutes to fully convert fibrinogen into fibrin.

[0018] Step d: Centrifugation: Centrifuge the incubated mixture at 4000 - 6000 rpm for 15 - 25 minutes and collect the supernatant S1 (containing plasma albumin and immunoglobulin).

[0019] Step e: Filtration: To further remove residual fibrin, filter the supernatant S1 through a 0.5 - μm filter membrane to obtain the supernatant S2.

[0020] Preferably, the concentration of the calcium chloride solution in Step 2 is 1 M; the incubation time in Step 3 is 40 - 80 minutes; the centrifugation speed in Step 4 is 5000 rpm and the centrifugation time is 20 minutes.

[0021] Preferably, the temperature of the enzymatic hydrolysis treatment system is maintained at 55-65°C; during the enzyme inactivation process, ultrafiltration membranes with a molecular weight cut-off greater than 5000 D are used for filtration; nanofiltration membrane equipment with a molecular weight cut-off of 100-300 D is used for concentration; the animal plasma is animal plasma, bovine plasma or ovine plasma.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) By adopting the calcium chloride precipitation method in combination with the membrane separation technology, the fibrinogen removal rate of the present invention is >98%, which can effectively remove the miscellaneous proteins in the plasma and improve the purity of the target product.

[0024] (2) By adopting the biological enzymatic hydrolysis technology in combination with the membrane separation technology, the immunoglobulin retention rate of the present invention is >90% (quantitative analysis by ELISA method), which can further purify the target product and obtain plasma albumin peptides with different molecular weight ranges.

[0025] (3) The finished product prepared by the present invention has both high protein content and antioxidant activity, can be applied to functional foods or drugs for enhancing immunity, anti-inflammatory or antioxidant purposes, and can reduce the serum TNF-α level by ≥40%. At the same time, the process is simple, the cost is low, and it is easy to industrialize production. Description of the Drawings

[0026] Figure 1 It is the experimental result of fibrinogen removal of the present invention.

[0027] Figure 2 It is the test result of the basic indexes of the product of the present invention.

[0028] Figure 3 It is the experimental result of immunity enhancement of the present invention.

[0029] Figure 4 It is the experimental result of anti-inflammatory activity of the present invention.

[0030] Figure 5 It is the experimental result of in vivo antioxidant activity of the present invention.

[0031] Figure 6 It is the experimental result of acute toxicity of the present invention. Detailed Embodiments

[0032] Next, in combination with the detailed embodiments, the present invention will be further described. It should be noted that, on the premise of no conflict, any combination of the following-described embodiments or technical features can form a new embodiment.

[0033] In the description of the present invention, it should be noted that for the orientation terms, such as the terms "center", "lateral", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., which indicate the orientation and positional relationship are based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and should not be construed as limiting the specific protection scope of the present invention.

[0034] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention are used to distinguish similar objects and do not necessarily need to describe a specific order or sequence.

[0035] One of the preferred embodiments of the present invention is, as Figures 1 to 6 shown, a preparation method of plasma albumin peptide rich in immunoglobulin;

[0036] Example 1 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Animal Plasma

[0037] 1. Pretreatment: Take 1000 mL of fresh animal blood and centrifuge to remove blood cells;

[0038] 2. Dilution: Dilute the supernatant with physiological saline to a protein content of 3%;

[0039] 3. Removal of fibrinogen:

[0040] Add 1M calcium chloride solution to a final concentration of 15 mM and incubate at 37 °C for 60 minutes;

[0041] Centrifuge at 5000 rpm for 20 minutes and filter through a 0.5 μm filter membrane to obtain the clear liquid S2 (sample for detecting the content of fibrinogen, total protein and albumin, and the results are shown in Figure 1 );

[0042] 4. Enzymatic hydrolysis: Heat the S2 solution to 60 °C, adjust the pH to 9.0, add 0.08% alkaline protease (Novozymes (Denmark) Alcalase 2.4L), and carry out enzymatic hydrolysis for 2 hours;

[0043] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate through a 100D nanofiltration membrane;

[0044] 6. Post-treatment: Freeze-dry the concentrated solution to obtain a white powder.

[0045] Example 2 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Animal Plasma

[0046] 1. Pretreatment: Take 800 mL of fresh animal blood and centrifuge to remove blood cells;

[0047] 2. Dilution: Dilute with physiological saline to a protein content of 5%;

[0048] 3. Removal of fibrinogen:

[0049] Add 1 M calcium chloride to a final concentration of 20 mM and incubate at 37 °C for 80 minutes;

[0050] Centrifuge at 6000 rpm for 25 minutes and filter through a 0.5 μm filter membrane (sample and detect the contents of fibrinogen, total protein, and albumin. The results are shown in Figure 1 );

[0051] 4. Enzymatic hydrolysis: Add 0.10% alkaline protease (Novozymes (Denmark) Alcalase 2.4L) under the conditions of 65 °C and pH 9.5 and hydrolyze for 2.5 hours;

[0052] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate through a 300D nanofiltration membrane;

[0053] 6. Post-treatment: Membrane filtration (5000D), concentration (300D), and freeze-drying.

[0054] Example 3 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Bovine Plasma

[0055] 1. Pretreatment: Take 1200 mL of fresh bovine blood and centrifuge to remove blood cells;

[0056] 2. Dilution: Dilute with physiological saline to a protein content of 2%;

[0057] 3. Removal of fibrinogen:

[0058] Add 1 M calcium chloride to a final concentration of 10 mM and incubate at 37 °C for 40 minutes;

[0059] Centrifuge at 4000 rpm for 15 minutes and filter through a 0.5 μm filter membrane (sample and detect the contents of fibrinogen, total protein, and albumin. The results are shown in Figure 1 );

[0060] 4. Enzymatic hydrolysis: Add 0.06% alkaline protease (Novozymes (Denmark) Alcalase 2.4L) under the conditions of 55 °C and pH 8.5 and hydrolyze for 1.5 hours;

[0061] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate through a 100D nanofiltration membrane;

[0062] 6. Post-treatment: Freeze-dry the concentrated solution to obtain a white powder.

[0063] Example 4 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Animal Plasma

[0064] 1. Pretreatment: Take 1500 mL of fresh animal blood, centrifuge to remove blood cells;

[0065] 2. Dilution: Dilute the supernatant with physiological saline to a protein content of 4%;

[0066] 3. Removal of fibrinogen:

[0067] Add 1M calcium chloride solution to a final concentration of 18 mM, incubate at 37 °C for 70 minutes;

[0068] Centrifuge at 5000 rpm for 20 minutes, filter through a 0.5 μm filter membrane to obtain the clear liquid S2;

[0069] 4. Enzymatic hydrolysis: Heat the S2 solution to 58 °C, adjust the pH to 8.8, add 0.07% alkaline protease (Novozymes (Denmark) Alcalase 2.4L), and carry out enzymatic hydrolysis for 2.2 hours;

[0070] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate with a 200D nanofiltration membrane;

[0071] 6. Post-treatment: Freeze-dry the concentrated solution to obtain a white powder.

[0072] Example 5 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Bovine Plasma

[0073] 1. Pretreatment: Take 1000 mL of fresh bovine blood, centrifuge to remove blood cells;

[0074] 2. Dilution: Dilute the supernatant with physiological saline to a protein content of 3.5%;

[0075] 3. Removal of fibrinogen:

[0076] Add 1M calcium chloride solution to a final concentration of 12 mM, incubate at 37 °C for 50 minutes;

[0077] Centrifuge at 4500 rpm for 18 minutes, filter through a 0.5 μm filter membrane to obtain the clear liquid S2;

[0078] 4. Enzymatic hydrolysis: Heat the S2 solution to 62 °C, adjust the pH to 9.2, add 0.09% alkaline protease (Novozymes (Denmark) Alcalase 2.4L), and carry out enzymatic hydrolysis for 2.3 hours;

[0079] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate with a 150D nanofiltration membrane;

[0080] 6. Post-treatment: The concentrated solution was freeze-dried to obtain a white powder.

[0081] Example 6 Preparation of Plasma Albumin Peptide Rich in Immunoglobulin from Sheep Plasma

[0082] 1. Pretreatment: 1200 mL of fresh sheep blood was taken and centrifuged to remove blood cells.

[0083] 2. Dilution: The supernatant was diluted with physiological saline to a protein content of 4.5%.

[0084] 3. Removal of fibrinogen:

[0085] 1 M calcium chloride solution was added to a final concentration of 16 mM and incubated at 37 °C for 65 minutes.

[0086] Centrifuge at 5500 rpm for 22 minutes and filter through a 0.5 μm filter membrane to obtain the clear liquid S2.

[0087] 4. Enzymolysis: The temperature of the S2 solution was raised to 63 °C, the pH was adjusted to 9.1, and 0.08% alkaline protease (Alcalase 2.4L from Novozymes, Denmark) was added for enzymolysis for 2.4 hours.

[0088] 5. Enzyme inactivation and purification: Adjust the pH to 7.2, filter through a 5000D ultrafiltration membrane, and concentrate with a 250D nanofiltration membrane.

[0089] 6. Post-treatment: The concentrated solution was freeze-dried to obtain a white powder. Working principle:

[0090] Comparative Example 1

[0091] Compared with Example 1, in Comparative Example 1, the addition of calcium chloride solution in Step 3.1 was not carried out, and the other steps were the same.

[0092] Comparative Example 2

[0093] Compared with Example 2, in Comparative Example 2, the 0.5 μm filter membrane filtration in Step 3.2 was not carried out, and the other steps were the same.

[0094] Comparative Example 3

[0095] Compared with Example 3, in Comparative Example 3, the addition of calcium chloride solution in Step 3.1 was not carried out, and the other steps were the same.

[0096] Comparative Example 4

[0097] Compared with Example 4, in Comparative Example 4, the membrane filtration in Step 3 (i.e., omission of 0.5 μm filter membrane filtration) was not carried out, and the other steps were the same.

[0098] Comparative Example 5

[0099] Compared with Example 5, in Comparative Example 5, the calcium chloride precipitation in Step 3 was not carried out (i.e., the addition of calcium chloride solution was omitted), and the remaining steps were the same.

[0100] Comparative Example 6

[0101] Compared with Example 6, in Comparative Example 6, the membrane filtration in Step 3 was not carried out (i.e., the 0.5 μm membrane filtration was omitted), and the remaining steps were the same.

[0102] When in use, 1. Determination of fibrinogen content: Measured by ELISA method (kit: Human Fibrinogen Assay Kit, Abcam, product number: ab108842).

[0103] 2. Determination of total protein content: Measured by BCA method (kit: Pierce TM BCA Protein Assay Kit, Thermo Scientific, product number: 23225);

[0104] 3. Determination of albumin content: Measured by immunoturbidimetry method (kit: Human Albumin ELISA Kit, Abcam, product number: ab108788);

[0105] 4. Calculation of fibrinogen removal rate:

[0106]

[0107] The initial concentration was uniformly 3.50 mg / mL (average value of Examples 1-3 and Comparative Examples 1-3).

[0108] 5. Statistical analysis:

[0109] Homogeneity of variance test (Levene test):

[0110] Fibrinogen content: F = 1.24, P = 0.35 (homogeneity of variance holds, P > 0.05).

[0111] One-way analysis of variance (ANOVA):

[0112] Fibrinogen removal rate: F = 2850.6, P < 0.001 (extremely significant difference between groups).

[0113] Post hoc test (Tukey HSD):

[0114] Examples 1-3 vs Comparative Examples 1-3: P < 0.001 (extremely significant difference).

[0115] Conclusion:

[0116] 1. The fibrinogen removal rates of Examples 1-6 were all >98%, significantly higher than those of the control group (P<0.01). The synergistic effect of the calcium chloride precipitation method and the membrane filtration technology can efficiently remove fibrinogen.

[0117] 2. The removal rates of Control Example 1 (without adding calcium chloride), Control Example 3 (without adding calcium chloride), and Control Example 5 (without adding calcium chloride) were 21.1%, 22.1%, and 20.0% respectively, indicating the key role of calcium chloride in fibrinogen removal.

[0118] 3. The removal rate of Control Example 2 (without membrane filtration) was 64.3%, the removal rate of Control Example 4 (without membrane filtration) was 58.63%, and the removal rate of Control Example 6 (without membrane filtration) was 58.63%, indicating the importance of membrane filtration in removing residual fibrinogen fragments.

[0119] The immunoglobulin-rich plasma albumin peptides prepared in Examples 1-6 and Control Examples 1-6 were subjected to the following tests:

[0120] 1. Protein content determination: The BCA method was used to determine the protein content;

[0121] 2. Immunoglobulin content determination: The ELISA method was used to determine the immunoglobulin content;

[0122] 3. Antioxidant activity determination: The DPPH free radical scavenging method was used to determine the antioxidant activity.

[0123] Figure 2 Results of the statistical analysis of the data:

[0124] 1. Homogeneity of variance test (Levene test)

[0125] Immunoglobulin content: F = 1.24, P = 0.35 (homogeneity of variance holds, P>0.05)

[0126] DPPH free radical scavenging rate: F = 0.89, P = 0.52 (homogeneity of variance holds, P>0.05)

[0127] 2. One-way analysis of variance (ANOVA)

[0128] Immunoglobulin content: F(5,24) = 256.7, P<0.001 (extremely significant difference between groups)

[0129] DPPH free radical scavenging rate: F(5,24) = 198.4, P<0.001 (extremely significant difference between groups)

[0130] 3. Post hoc test (Tukey HSD)

[0131] Example group (1 - 6) vs Control group (1 - 6)

[0132] Immunoglobulin content:

[0133] Example 1 vs Control 1: P < 0.001 (mean difference Δ = 44.7 mg / g)

[0134] Example 2 vs Control 2: P < 0.001 (mean difference Δ = 38.4 mg / g)

[0135] Example 3 vs Control 3: P < 0.001 (mean difference Δ = 40.7 mg / g)

[0136] Example 4 vs Control 4: P < 0.001 (mean difference Δ = 37.7 mg / g)

[0137] Example 5 vs Control 5: P < 0.001 (mean difference Δ = 43.2 mg / g)

[0138] Example 6 vs Control 6: P < 0.001 (mean difference Δ = 40.2 mg / g)

[0139] DPPH free radical scavenging rate:

[0140] Example 1 vs Control 1: P < 0.001 (mean difference Δ = 13.0%)

[0141] Example 2 vs Control 2: P < 0.001 (mean difference Δ = 9.1%)

[0142] Example 3 vs Control 3: P < 0.001 (mean difference Δ = 10.6%)

[0143] Example 4 vs Control 4: P < 0.001 (mean difference Δ = 8.7%)

[0144] Example 5 vs Control 5: P < 0.001 (mean difference Δ = 11.3%)

[0145] Example 6 vs Control 6: P < 0.001 (mean difference Δ = 10.3%)

[0146] 4. Significance conclusion

[0147] Significance conclusion

[0148] The immunoglobulin content of Examples 1 - 6 (mean 119.6 mg / g) was significantly higher than that of Controls 1 - 6 (mean 78.2 mg / g), and the difference was statistically significant (P < 0.01);

[0149] The DPPH radical scavenging rates of Examples 1-6 (mean 84.9%) were significantly higher than those of Comparative Examples 1-6 (mean 74.1%), and the difference was statistically significant (P<0.01).

[0150] Summary:

[0151] Through Tukey HSD post hoc test analysis, the significant differences in immunoglobulin content and DPPH radical scavenging rate between the Example group (1-6) and the Comparative Example group (1-6) were further verified. The immunoglobulin content and antioxidant activity of the Example group were significantly better than those of the Comparative Example group, fully demonstrating the technical advantages of the method of the present invention in efficiently preparing plasma albumin peptides rich in immunoglobulins.

[0152] The method of the present invention can efficiently remove fibrinogen in plasma, retain the activity of immunoglobulins, and improve the purity and antioxidant activity of the product by combining the calcium chloride precipitation method with membrane filtration technology; the process advantages of the calcium chloride precipitation combined with membrane filtration technology were fully verified. This result further supports the innovation of the method of the present invention in efficiently retaining the activity of immunoglobulins and improving the purity of the product.

[0153] Application data:

[0154] Animal experiment and clinical trial design (all plasma albumin peptides rich in immunoglobulins used below were prepared in Example 1)

[0155] 1. Immunity enhancement experiment (mouse model)

[0156] 1.1 Experimental design

[0157] 1.1.1 Animal grouping and sample size:

[0158] Blank control group: 10 ICR mice, intragastrically administered normal saline (0.2 mL / 10 g body weight) daily.

[0159] Low-dose group: 10 ICR mice, intragastrically administered 50 mg / kg·d plasma albumin peptide solution rich in immunoglobulins daily.

[0160] High-dose group: 10 ICR mice, intragastrically administered 200 mg / kg·d plasma albumin peptide solution rich in immunoglobulins daily.

[0161] Positive control group: 10 ICR mice, intragastrically administered 100 mg / kg·d Ganoderma lucidum polysaccharide (referring to the Chinese Pharmacopoeia standard) daily.

[0162] 1.1.2 Model establishment:

[0163] All mice (except the blank control group) were intraperitoneally injected with cyclophosphamide (80 mg / kg·d) for 3 consecutive days to induce an immunosuppression model.

[0164] Medication started on the 4th day after injection and continued for 14 consecutive days.

[0165] 1.1.3 Detection indicators and methods:

[0166] Immune organ index:

[0167] Determination of serum immunoglobulins:

[0168] Collect serum and detect the levels of IgG, IgA, and IgM by ELISA method (kit brand: BIOSAMITE, catalog number: BSA20376).

[0169] Lymphocyte proliferation ability: ConA-induced splenic lymphocyte proliferation assay (MTT method)

[0170] Take the spleen to prepare a single cell suspension, stimulate and culture with ConA (5 μg / mL) for 48 hours, measure the absorbance (OD570nm) by MTT method, and calculate the proliferation rate (OD of experimental group / OD of blank group × 100%).

[0171] 1.2. Data significance analysis

[0172] Statistical method:

[0173] Data are presented as mean ± standard deviation (Mean ± SD) and analyzed by one-way ANOVA and Tukey's post hoc test. The significance level is set as *P < 0.05, **P < 0.01.

[0174] Compared with the blank control group, *P < 0.05, **P < 0.01;

[0175] There was no significant difference between the high-dose group and the positive control group (P > 0.05)

[0176] Conclusion: Plasma albumin peptide rich in immunoglobulins significantly increases the spleen index, serum IgG level, and lymphocyte proliferation ability of immunosuppressed mice, indicating its potential to enhance immune function.

[0177] 2. Anti-inflammatory activity experiment (rat paw swelling model)

[0178] 2.1 Experimental design:

[0179] 2.1.1 Animal grouping and sample size:

[0180] Blank control group: 10 male SD rats (weight 200 - 220 g), intragastrically administered normal saline (1 mL / 100 g body weight) daily.

[0181] Low-dose group: 10 SD rats were intragastrically administered with 50 mg / kg·d of plasma albumin peptide solution rich in immunoglobulins (dissolved in normal saline) daily.

[0182] High-dose group: 10 SD rats were intragastrically administered with 200 mg / kg·d of plasma albumin peptide solution rich in immunoglobulins daily.

[0183] Positive control group: 10 SD rats were intragastrically administered with 10 mg / kg·d of dexamethasone daily (referring to the standards of the Chinese Pharmacopoeia).

[0184] 2.1.2 Model establishment:

[0185] All rats were subcutaneously injected with 1% carrageenan solution (0.1 mL / rat) into the right hind paw to induce acute inflammation.

[0186] Medication was started immediately after injection and observed continuously for 5 hours.

[0187] 2.1.3 Detection indexes and methods:

[0188] 2.1.3.1 Paw swelling rate:

[0189] The volume of the right hind paw was measured at 1, 3, and 5 hours after injection using a plethysmometer (model: Plethysmometer 7140, Ugo Basile).

[0190] Calculation formula: Paw swelling rate (%) = (paw volume after injection - paw volume before injection) / paw volume before injection × 100%.

[0191] 2.1.3.2 Detection of inflammatory factors:

[0192] After the experiment, serum was collected, and the levels of TNF-α and IL-6 were detected by ELISA method (kit brand: R&D Systems, cat. no.: DY506-05 DY510-05).

[0193] 2.2. Data significance analysis

[0194] Statistical method:

[0195] Data were presented as mean ± standard deviation (Mean ± SD) and analyzed by one-way analysis of variance (ANOVA) and Tukey's post hoc test. The significance level was set as *P < 0.05, **P < 0.01.

[0196] 3. In vivo antioxidant activity verification (aging mouse model)

[0197] 3.1 Experimental design:

[0198] 3.1.1 Animal grouping and sample size:

[0199] Blank control group: 10 naturally aged C57BL / 6 mice (18 months old, male), intragastrically administered with normal saline (0.2 mL / 10 g body weight) daily.

[0200] Low-dose group: 10 naturally aged C57BL / 6 mice (18 months old, male), intragastrically administered with 50 mg / kg·d immunoglobulin-rich plasma albumin peptide solution (dissolved in normal saline) daily.

[0201] High-dose group: 10 naturally aged C57BL / 6 mice (18 months old, male), intragastrically administered with 200 mg / kg·d immunoglobulin-rich plasma albumin peptide solution daily.

[0202] Positive control group: 10 naturally aged C57BL / 6 mice (18 months old, male), intragastrically administered with 100 mg / kg·d vitamin E (referring to the standards of the Chinese Pharmacopoeia) daily.

[0203] 3.1.2 Administration period:

[0204] Administer the drug continuously for 28 days, and intragastrically administer at a fixed time every day.

[0205] 3.1.3 Detection indexes and methods:

[0206] 3.1.3.1 Detection of antioxidant enzyme activity:

[0207] Superoxide dismutase (SOD)*: The WST-1 method (kit brand: Beyotime, product number: S0101) was used to measure the SOD activity in plasma (unit: U / mL).

[0208] Glutathione peroxidase (GSH-Px): The DTNB method (kit brand: Solarbio, product number: BC1175) was used to measure the GSH-Px activity in plasma (unit: U / mL).

[0209] 3.1.3.2 Detection of lipid peroxidation products**:

[0210] Malondialdehyde (MDA): The thiobarbituric acid (TBA) method (kit brand: Jiancheng Bioengineering, product number: A003-1) was used to measure the MDA content in plasma (unit: nmol / mL).

[0211] 3.2 Data significance analysis

[0212] Statistical method:

[0213] Data were presented as mean ± standard deviation (Mean ± SD), and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The significance level was set at *P < 0.05 and **P < 0.01.

[0214] 4. Safety evaluation (acute toxicity experiment)

[0215] 4.1 Experimental design

[0216] 4.1.1 Experimental animals and grouping:

[0217] Animal strain and number: Forty healthy ICR mice (20 males and 20 females, weighing 18 - 22 g) were selected and randomly divided into two groups:

[0218] Experimental group: Twenty mice were given a single intragastric administration of 5000 mg / kg of plasma albumin peptide solution rich in immunoglobulin (dissolved in normal saline, concentration 50 mg / mL).

[0219] Control group: Twenty mice were given a single intragastric administration of an equal volume of normal saline (0.2 mL / 10 g body weight).

[0220] 4.1.2 Animal feeding conditions:

[0221] Feeding environment: Temperature 22 ± 2°C, relative humidity 50 ± 10%, 12-hour light / dark cycle.

[0222] Feed and drinking water: Standard rodent feed (SPF grade) and sterilized drinking water, ad libitum.

[0223] Adaptive feeding: The animals were adaptively fed in the above environment for 7 days before the experiment.

[0224] 4.1.3 Administration method:

[0225] A disposable sterile intragastric needle (specification 1.2 mm) was used to complete the intragastric administration operation from 9:00 to 11:00 every morning.

[0226] After intragastric administration, the immediate reactions of the animals were observed (such as activity status, respiration, mucosal color, etc.).

[0227] 4.1.4 Observation indicators and time points:

[0228] Mortality rate: The animals were continuously observed for 14 days, and the daily survival status was recorded.

[0229] Body weight change: The body weights were measured before administration (day 0) and on days 1, 3, 7, and 14 after administration, and the body weight growth rate (%) was calculated.

[0230] Organ pathological examination:

[0231] Sampling time: After the experiment ended (on the 14th day), all mice were anesthetized and sacrificed.

[0232] Organ collection: The heart, liver, spleen, lungs, and kidneys were completely removed, rinsed with physiological saline, and then fixed in 4% paraformaldehyde for 24 hours.

[0233] Pathological section: Paraffin embedding, HE staining, and observation of histopathological changes (such as inflammation, necrosis, congestion, etc.) under an optical microscope. A semi - quantitative scoring system (0 - 3 points, 0 = no abnormality, 1 = mild, 2 = moderate, 3 = severe) was used.

[0234] 4.2 Data significance analysis

[0235] Statistical methods:

[0236] The weight growth rate and pathological scores were plotted as Mean ± SD, and an independent - samples t - test was used.

[0237] The survival rate and the rate of organ pathological abnormalities were count data, and a chi - square test was used for analysis.

[0238] Significance criteria:

[0239] The significance level was set as *P < 0.05, **P < 0.01.

[0240] General conclusion:

[0241] 1. The protein content of the plasma albumin peptide rich in immunoglobulin prepared in Examples 1 - 6 of the present invention all exceeded 940 mg / g, the immunoglobulin content was ≥118 mg / g, and the DPPH free - radical scavenging rate was ≥84%. It has high purity, high immunological activity, and significant antioxidant capacity;

[0242] 2. In the finished products of Comparative Example 1, Comparative Example 3, and Comparative Example 5 without adding calcium chloride, and Comparative Example 2, Comparative Example 4, and Comparative Example 6 without membrane filtration, the immunoglobulin content and DPPH free - radical scavenging rate were significantly lower than those of the Example group. Thus, it can be seen that the synergistic effect of the calcium chloride precipitation method and the membrane filtration technology is crucial for retaining the immunoglobulin activity and improving the product purity;

[0243] 3. As Figure 2 shown, the immunoglobulin content and antioxidant activity of Examples 1 - 6 were significantly better than those of Comparative Examples 1 - 6 (P < 0.01), fully verifying the superiority of the process of the present invention;

[0244] 4. Further confirmation through animal experiments showed that the plasma albumin peptide rich in immunoglobulins prepared in Example 1 could significantly increase the spleen index of immunosuppressed mice (5.5 mg / g vs 3.2 mg / g in the blank group), the serum IgG level (22.3 μg / mL vs 12.5 μg / mL in the blank group), and the lymphocyte proliferation rate (158% vs 100% in the blank group), and effectively inhibit the inflammatory factors TNF-α (68.4 pg / mL vs 120.5 pg / mL in the blank group) and IL-6 (45.2 pg / mL vs 85.6 pg / mL in the blank group). At the same time, it increased the SOD (128.7 U / mL vs 85.3 U / mL in the blank group) and GSH-Px activities (65.2 U / mL vs 35.6 U / mL in the blank group) of aged mice, and decreased the MDA content (2.9 nmol / mL vs 5.8 nmol / mL in the blank group). Overall, it has multiple biological activities of enhancing immunity, anti-inflammation, and anti-oxidation;

[0245] 5. The acute toxicity experiment showed that there were no significant differences in the survival rate, body weight growth rate, and organ pathological indexes of the experimental group mice under a single gavage dose of 5000 mg / kg compared with the control group (P>0.05), demonstrating the good safety of this product.

[0246] The above examples and data fully verified the technical advantages of the method of the present invention in the efficient preparation of plasma albumin peptides rich in immunoglobulins, as well as the application potential of the end product in the fields of functional foods and pharmaceuticals.

[0247] The basic principles, main features, and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing plasma albumin peptides rich in immunoglobulins, characterized in that: The following steps are involved: Step 1: Pretreatment: Centrifuge fresh animal plasma, remove blood cells, and collect the supernatant; Step 2: Dilution: dilute the animal plasma with normal saline to a protein content of 2% to 5%; Step 3: removing fibrinogen from the plasma and collecting the clear fluid S2 containing animal plasma albumin and immunoglobulin; Step 4: Enzymatic hydrolysis: firstly place the clear liquid S2 at 55-65°C, add alkaline protease to the clear liquid S2, the addition amount is 0.06-0.10% of the mass of the supernatant, pH 8.5-9.5, and after treating for 1.5-2.5h, obtain an enzymatic hydrolyzate; Step 5: Inactivating the enzyme: adjusting the pH of the enzymatic solution to 7.0-7.4 with 0.1M hydrochloric acid, and performing solid-liquid separation to obtain a supernatant; filtering the supernatant with a membrane to obtain a product dilution; Step 6: Post-treatment: The product dilution is concentrated to obtain a product concentrate, which is then sterile filtered, freeze-dried, crushed and packaged to obtain a plasma albumin peptide powder product rich in immunoglobulins.

2. The method for preparing an immunoglobulin-rich plasma albumin peptide according to claim 1, characterized in that: The method for removing fibrinogen in plasma in step 3 is as follows: Step a: Adjusting the plasma pH: Measure the pH of the plasma with a pH meter and adjust the pH to 7.0-7.4 with dilute hydrochloric acid or sodium hydroxide solution; Step b: Adding calcium chloride solution: slowly adding calcium chloride solution to the plasma under stirring conditions to a final concentration of 10-20 mM; Step c: Incubation: Place the mixture in a 37°C water bath and incubate for 40 to 80 minutes to allow the fibrinogen to be fully converted into fibrin; Step d: Centrifugation: Centrifuge the incubated mixture at 4000-6000 rpm for 15-25 minutes and collect the supernatant S1 (containing plasma albumin and immunoglobulin); Step e: Filtration: To further remove residual fibrin, the supernatant S1 is filtered through a 0.5 μm filter membrane to obtain the supernatant S2.

3. The method for preparing a plasma albumin peptide rich in immunoglobulins according to claim 1, characterized in that: The concentration of the calcium chloride solution in step 2 is 1 M; the incubation time in step 3 is 40 to 80 minutes; and the centrifugation speed in step 4 is 5000 rpm and the centrifugation time is 20 minutes.

4. The method for preparing an immunoglobulin-rich plasma albumin peptide according to claim 1, characterized in that: The enzymatic hydrolysis treatment maintains the system temperature at 55-65°C; the enzyme inactivation process uses an ultrafiltration membrane with a molecular weight cutoff greater than 5000D for filtration; the concentration uses a nanofiltration membrane device with a molecular weight cutoff of 100-300D; the animal plasma is animal plasma, bovine plasma or sheep plasma.

5. A method for preparing an immunoglobulin-rich plasma albumin peptide according to any one of claims 1 to 4, characterized in that: In the step b, the protein content is ≥900 mg / g, the immunoglobulin content is ≥100 mg / g, and the DPPH free radical scavenging rate is ≥84%.

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