Preparation method of pig spleen oligopeptide capable of improving immunity

Abstract

The invention is applicable to the field of food processing, and provides a preparation method of pig spleen oligopeptide capable of improving immunity, which comprises the following steps: step 1, selecting qualified pig spleen after inspection and quarantine, cleaning, adding a proper amount of clear water for homogenization treatment, then heating to 90-95 DEG C, and keeping the temperature for 15 minutes; step 2, cooling the tissue homogenate after enzyme deactivation to a proper temperature, and carrying out enzymolysis treatment; step 3, adjusting the pH value and temperature of the enzymolysis system to optimal reaction conditions, and continuing enzymolysis; 4, after enzymolysis is completed, standing and precipitating, and separating supernatant enzymatic hydrolysate from residual precipitates; and step 5, performing concentration, dialysis and drying treatment on the enzymatic hydrolysate obtained by separation to finally obtain the pig spleen oligopeptide. The method is simple and convenient to operate, low in cost and high in raw material utilization rate, and the obtained product has the multiple advantages of high purity, easiness in absorption, rich nutrition, strong immunoregulation function and the like.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a method for preparing porcine spleen oligopeptides with enhanced immunity. Background Art

[0002] Peptides are polymers formed by amino acids linked by peptide bonds. Depending on the number of amino acids they contain, they can be called oligopeptides of a certain number. They are generally divided into oligopeptides composed of 2 to 20 amino acids and polypeptides composed of more than 20 amino acids. The traditional theory of protein metabolism holds that proteins must be degraded into free amino acids in the gastrointestinal tract before they can be absorbed and further transported to various tissues of the body to participate in metabolism.

[0003] With the continuous in-depth research on protein and amino acid nutrition, researchers have gradually realized that peptides also have important nutritional value. In 1953, Agar found that peptides could also be directly absorbed by the intestine when studying the absorption efficiency of different amino acids. In 1959, Newey and Smyth further confirmed that the intestine could directly absorb and utilize several dipeptides. With the wide acceptance of this view, more and more research has begun to focus on the nutritional role and physiological functions of peptides in the body.

[0004] In 1982, Tranter et al. defined peptides that have a positive impact on life activities, are bioactive, and ultimately beneficial to health as bioactive peptides. With the development of research and technology, bioactive peptides have shown broad application prospects in multiple fields such as food, medicine, and industry.

[0005] China is the world's largest producer of live pigs, and pork production accounts for about 44.47% of the global total output. As a by-product of pig slaughter and processing, porcine spleen is rich in high-quality protein and has high nutritional value, but it is often disposed of at a low price or discarded and not fully developed and utilized. It is recorded in "Chinese Herbal Medicine" that porcine spleen has the effects of strengthening the spleen and stomach, eliminating indigestion and stagnation, etc.

[0006] Transfer factor is a mixture of functional peptides with a molecular weight less than 10 kDa, mainly composed of free amino acids, nucleic acids, and peptides, and has good antigenicity, stable chemical properties, and non-toxic and safe characteristics. In 1949, Lawrence first discovered that leukocyte extracts had the effect of enhancing the immunity of the recipient. Since then, transfer factors from various animal sources have been successively discovered and applied clinically, and common sources include pigs, cows, sheep, chickens, and dogs, etc.

[0007] Therefore, preparing porcine spleen oligopeptides from fresh and healthy porcine spleen not only helps to make full use of porcine spleen resources, but also can develop its functions of regulating immunity and enhancing physical fitness, which is a method with practical value.

[0008] At present, the protein enzymatic hydrolysis method is the main means for industrial production of bioactive peptides. According to the data in the "Food-derived Bioactive Peptides" database, most of the functional peptides derived from food are concentrated in oligopeptides. Therefore, in view of the above situation, there is an urgent need to develop a method for preparing oligopeptides from porcine spleen through protease hydrolysis technology, aiming to realize the high-value utilization of porcine spleen and promote the technological progress and sustainable development of related industries. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for preparing porcine spleen oligopeptides with enhanced immunity, aiming to solve the problems mentioned in the above background technology.

[0010] The present invention is realized as follows: A method for preparing porcine spleen oligopeptides with enhanced immunity includes the following steps: Step 1: Select porcine spleen that has passed inspection and quarantine, wash it, add an appropriate amount of water for homogenization treatment, and then raise the temperature to 90 - 95 °C and keep it warm for 15 minutes. Step 2: Cool the inactivated tissue homogenate to an appropriate temperature for enzymatic hydrolysis treatment. Step 3: Adjust the pH value and temperature of the enzymatic hydrolysis system to the optimal reaction conditions and continue enzymatic hydrolysis. Step 4: After enzymatic hydrolysis is completed, let it stand for precipitation, and separate the supernatant enzymatic hydrolysis solution and the residual precipitate. Step 5: Concentrate, dialyze, and dry the separated enzymatic hydrolysis solution to finally obtain porcine spleen oligopeptides.

[0011] A further technical solution is that in Step 1, the porcine spleen and water are mixed for homogenization at a weight ratio of 1:2.

[0012] A further technical solution is that the specific content of Step 2 includes: Step 21: Keep the tissue homogenate at 45 - 50 °C and adjust the pH to 2.5 - 3.5. Step 22: Add acid protease to the system, with an enzyme addition amount of 6200 U / g, and stir and react uniformly under constant temperature conditions for 5 - 6 hours. Step 23: After the reaction is completed, raise the temperature of the enzymatic hydrolysis system to 90 - 95 °C and maintain it for 15 minutes to inactivate the protease and terminate the reaction.

[0013] A further technical solution is that in Step 3, the pH value of the enzymatic hydrolysis system is adjusted to 2.

[0014] A further technical solution is that the specific content of Step 5 includes: Step 51: Vacuum concentrate the enzymatic hydrolysis solution at 65 - 75 °C to make the solid content 30% - 45%. Step 52: Dialyze the concentrated solution against a dialysis membrane with a molecular weight cut-off of 100 Da for 24 - 48 h to remove small molecule impurities; Step 53: Lyophilize the dialyzed concentrated solution to obtain the finished product of oligopeptide powder.

[0015] In a further technical solution, in Step 5, the content of oligopeptides in the obtained porcine spleen oligopeptides accounts for more than 70% of the total peptide components, and the proportion of peptide segments with a molecular weight less than 1100 Da exceeds 80%.

[0016] Another object of the present invention is the application of the porcine spleen oligopeptides prepared by the preparation method of porcine spleen oligopeptides with improved immunity in regulating the immune function of the body and as a basic raw material for the development of immune adjuvants.

[0017] A preparation method of porcine spleen oligopeptides with improved immunity provided by the present invention has the following beneficial effects: The present invention uses porcine spleen that has passed inspection and quarantine as the raw material, and through process steps such as thawing, grinding, sterilization, enzymatic hydrolysis, enzyme inactivation, separation, concentration, dialysis, and drying, converts porcine spleen into oligopeptide powder that is easily absorbed by the human body, solves the taste problem, and can exert its function with a small consumption amount.

[0018] The prepared porcine spleen oligopeptide powder is rich in essential amino acids, providing rich nutritional support for the body; the length of its peptide chains is mainly concentrated in tripeptides, tetrapeptides, and pentapeptides, which is conducive to penetrating the intestinal barrier and being directly absorbed; the N-terminus is mainly valine and leucine, and the C-terminus is rich in proline, leucine, and phenylalanine, and these characteristics help to exert its physiological functions; in addition, since it is mainly hydrophobic polypeptides, it can maintain a certain stability during gastrointestinal digestion and maintain its physiological functions.

[0019] This method not only has the advantages of simple operation, low production cost, high raw material utilization rate, and high product yield, but also endows the product with the advantages of high purity, safety and non-toxicity, and easy absorption by the body. Description of the Drawings

[0020] Figure 1 It is the process flow chart of the preparation method of porcine spleen oligopeptides with improved immunity provided by the embodiment of the present invention; Figure 2 It is the bar chart of the regulation of cytokines in immunosuppressed mice by porcine spleen oligopeptides; Figure 3 It is the schematic diagram of the regulation of intestinal macrophages in immunosuppressed mice by porcine spleen oligopeptides; Figure 4 It is the schematic diagram of the regulation of spleen B lymphocytes in immunosuppressed mice by porcine spleen oligopeptides. Detailed Embodiments

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] The following describes in detail the specific implementation of the present invention with reference to specific embodiments.

[0023] As Figure 1 shown, a preparation method of porcine spleen oligopeptide with enhanced immunity provided by an embodiment of the present invention uses porcine spleen as the main raw material, and includes the following steps: Step 1: Select porcine spleens that have passed inspection and quarantine, wash them, add an appropriate amount of water for homogenization, then heat up to 90-95 °C and keep warm for 15 minutes to sterilize and remove potential impurities; Step 2: Cool the tissue homogenate after inactivating enzymes to an appropriate temperature and perform enzymatic hydrolysis treatment; Step 3: Adjust the pH value and temperature of the enzymatic hydrolysis system to the optimal reaction conditions and continue enzymatic hydrolysis; Step 4: After enzymatic hydrolysis is completed, let it stand for precipitation, and separate the supernatant enzymatic hydrolysate and the residual precipitate; Step 5: Concentrate, dialyze and dry the separated enzymatic hydrolysate to finally obtain porcine spleen oligopeptide.

[0024] Among them, in Step 1, the porcine spleen and water are mixed for homogenization at a weight ratio of 1:2.

[0025] In Step 2, it was found in the study that different commercial enzymes have different enzymatic hydrolysis efficiencies for porcine spleen. Therefore, after detecting the enzymatic hydrolysis efficiencies of some commonly used commercial enzymes for porcine spleen through experiments, commercial acidic protease was selected. The specific steps of Step 2 include: Step 21: Keep the tissue homogenate at 45-50 °C and adjust the pH to 2.5-3.5; Step 22: Add acidic protease to the system, with the enzyme addition amount being 6200 U / g, and stir and react evenly under constant temperature conditions for 5-6 hours; Step 23: After the reaction is completed, raise the temperature of the enzymatic hydrolysis system to 90-95 °C and maintain it for 15 minutes to inactivate the protease and terminate the reaction.

[0026] In Step 3, adjust the pH value of the enzymatic hydrolysis system to 2.

[0027] The specific steps of Step 5 include: Step 51: Vacuum concentrate the enzymatic hydrolysate at 65-75 °C to make the solid content 30%-45%; Step 52: Dialyze the concentrated solution using a dialysis membrane with a molecular weight cut-off of 100 Da for 24-48 h to remove small molecule impurities; Step 53: Freeze-dry the concentrated solution after dialysis to obtain the finished product of oligopeptide powder.

[0028] In Step 5, the oligopeptide content in the obtained porcine spleen oligopeptide accounts for more than 70% of the total peptide components, and the proportion of peptide segments with a molecular weight less than 1100 Da exceeds 80%, showing good bioavailability. It has a certain stability during gastrointestinal digestion and can maintain its original physiological activity; at the same time, it is enriched in essential amino acids and hydrophobic amino acids, with their proportions exceeding 40% and 60% respectively. The peptide chain length is relatively short, mainly concentrated in the range of tripeptides, tetrapeptides and pentapeptides, which is beneficial to cross the intestinal barrier and achieve rapid absorption. The N-terminus is mainly composed of valine and leucine, and the C-terminus is mainly composed of proline, leucine and phenylalanine, which helps to exert specific physiological functions.

[0029] An embodiment of the present invention also provides the application of the porcine spleen oligopeptide prepared by the preparation method of the porcine spleen oligopeptide with enhanced immunity in regulating the immune function of the body and as a basic raw material for the development of immune adjuvants. That is, it can be used to regulate the immune function of the body, has a potential immune-enhancing effect, and can be used as one of the basic raw materials for the development of immune adjuvants. For example, it can be used as a functional raw material in industrial production, and can also be further processed into different pharmaceutically acceptable dosage forms, including but not limited to oral liquids, small volume injections, large volume injections, tablets, capsules or powders, etc., and is widely used in the fields of food, medicine and health products.

[0030] Example 1: Preparation of porcine spleen oligopeptide powder The porcine spleen oligopeptide powder is prepared from fresh porcine spleen that has passed quarantine by the above process method. Its components mainly include 83.97% protein, 1.52% fat, 5.68% total sugar, 7.19% moisture and 4.22% ash. Among them, the content of oligopeptide is greater than 75%, and the proportion of peptide segments with a molecular weight less than 1100 Da exceeds 80%.

[0031] Example 2: Amino acid characteristics of porcine spleen oligopeptide For the porcine spleen oligopeptide powder prepared in Example 1 above, the amino acid sequence of the oligopeptide was analyzed using a high-performance liquid chromatography-mass spectrometry (QExactiveHF-X) instrument. It was found that essential amino acids and hydrophobic amino acids were enriched in the oligopeptide. The proportion of essential amino acids was 43.99%, and the proportion of hydrophobic amino acids was 64.66%.

[0032] Example 3: Chain length characteristics of porcine spleen oligopeptide Further analysis of the polypeptide sequences detected in Example 2 revealed that the oligopeptides had short chain lengths, mainly concentrated in tripeptides, tetrapeptides, and pentapeptides. They were easily able to penetrate the intestinal barrier, be directly absorbed by the body, and exert physiological functions. A total of 1509 different sequences of peptides were detected in the oligopeptides from porcine spleen, including 320 tripeptides, accounting for 21.21%; 679 tetrapeptides, accounting for 45.00%; 124 pentapeptides, accounting for 8.22%. The total proportion of tripeptides - pentapeptides was 74.42%.

[0033] Example 4. Digestive characteristics of oligopeptides from porcine spleen After the in vitro digestion experiment of the porcine spleen oligopeptide powder, the supernatant was taken by centrifugation, and the amino acid sequences of the oligopeptides were analyzed by high performance liquid chromatography - mass spectrometry (QExactiveHF-X). A total of 1450 different sequences of polypeptides were detected in the supernatant after digestion of the porcine spleen oligopeptide powder, including 244 tripeptides, accounting for 16.83%; 548 tetrapeptides, accounting for 37.79%; 144 pentapeptides, accounting for 9.93%. The total proportion of tripeptides - pentapeptides was 64.55%. Compared with before digestion, 537 polypeptide sequences were the same, and 376 were hydrophobic polypeptides, accounting for 70%. In summary, the porcine spleen oligopeptides can maintain a certain stability during the gastrointestinal digestion process and maintain the physiological functions of the peptides.

[0034] Example 5. Structural characteristics of oligopeptides from porcine spleen Further analysis of the polypeptide sequences detected in Examples 2 and 4 found that the N-terminus of the porcine spleen oligopeptides was mainly leucine and valine, and the C-terminus was mainly proline, leucine, and phenylalanine, which exerted physiological functions. In the pre-digestion sequences, the total proportion of valine and leucine at the N-terminus was 42.48%, and the total proportion of proline, leucine, and phenylalanine at the C-terminus was 49.97%. In the post-digestion sequences, the total proportion of valine and leucine at the N-terminus was 35.38%, and the total proportion of proline, leucine, and phenylalanine at the C-terminus was 47.37%.

[0035] Example 6. Immunomodulatory function of active oligopeptides from porcine spleen As Figures 2 - 4 shown, an immunosuppressed mouse model was constructed using cyclophosphamide. The porcine spleen oligopeptide powder was continuously fed to the mice once a day for one month at doses of 10, 100, and 200 mg / kg, and the immunological indexes of the mice were detected. The grouping method was as follows: control group, gavaged with normal saline; model group: gavaged with normal saline; 10 mg group: gavaged with porcine spleen oligopeptides at a dose of 10 mg / kg / d; 100 group, gavaged with porcine spleen oligopeptides at a dose of 100 mg / kg / d; 200 group, gavaged with porcine spleen oligopeptides at a dose of 200 mg / kg / d; positive group, gavaged with levamisole hydrochloride at a dose of 40 mg / kg / d.

[0036] As can be seen from the ELISA test results in Table 2 (attached Figure 2 ). Compared with the control group, the concentrations of TNF-α, IL-4 and IFN-γ in the spleen, blood and intestine of the model group were significantly decreased (P<0.05), indicating a decrease in the immune resistance of mice. Compared with the model group, the concentrations of TNF-α, IL-4 and IFN-γ in the spleen, blood and intestine of the 200mg / kg group and the positive group were significantly increased (P<0.05), indicating an increase in the immune resistance of mice.

[0037] Table 1 Effects of porcine spleen bioactive oligopeptides on cytokines in immunosuppressed mice

[0038] Note: Compared with the control group, #<0.05; compared with the model group, *<0.05.

[0039] Secondly, as can be seen from the immunofluorescence results (attached Figure 3 and 4 ), compared with the control group, the number of intestinal macrophages in the model group was significantly decreased, and the number of splenic B lymphocytes was also decreased, indicating a decrease in the immune capacity of mice. In the 200mg group and the positive group, the number of intestinal macrophages in mice was significantly increased compared with the model group, and the number of splenic B lymphocytes was also increased, indicating that both porcine spleen oligopeptides and levamisole hydrochloride can improve the immunity of immunosuppressed mice.

[0040] The above two experimental results show that the porcine spleen oligopeptides obtained by enzymatic hydrolysis of the present invention can improve immunity.

[0041] The present invention provides a preparation method of porcine spleen oligopeptides with improved immunity in the above embodiments, and the main advantages are as follows: The present invention uses porcine spleen that has passed inspection and quarantine as raw materials, and through processes such as thawing, grinding, sterilization, enzymatic hydrolysis, enzyme inactivation, separation, concentration, dialysis and drying, converts porcine spleen into oligopeptide powder that is easily absorbed by the human body, solves the taste problem, and can exert its function with a small amount of consumption.

[0042] The prepared porcine spleen oligopeptide powder is rich in essential amino acids, providing rich nutritional support for the body; the length of its peptide chains is mainly concentrated in tripeptides, tetrapeptides and pentapeptides, which is conducive to penetrating the intestinal barrier and being directly absorbed; the N-terminus is mainly valine and leucine, and the C-terminus is rich in proline, leucine and phenylalanine, and these characteristics help to exert its physiological functions; in addition, since it is mainly hydrophobic polypeptide, it can maintain a certain stability during gastrointestinal digestion and maintain its physiological functions.

[0043] This method not only has the advantages of simple operation, low production cost, high raw material utilization rate and high product yield, but also endows the product with the advantages of high purity, safety and non-toxicity and easy absorption by the body.

[0044] Through the above technical solutions, while realizing the high-value utilization of pig spleen, the present invention promotes the development of related industries and demonstrates significant application prospects and economic benefits.

[0045] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0046] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent of the present invention shall be subject to the appended claims.

Claims

1. A preparation method of porcine spleen oligopeptide with enhanced immunity, characterized in that, It includes the following steps: Step 1: Select the pig spleen that has passed inspection and quarantine, wash it, add an appropriate amount of water for homogenization treatment, then heat it up to 90 - 95 °C and keep it warm for 15 minutes; Step 2: Cool the tissue homogenate after enzyme inactivation to an appropriate temperature and carry out enzymatic hydrolysis treatment; Step 3: Adjust the pH value and temperature of the enzymatic hydrolysis system to the optimal reaction conditions and continue enzymatic hydrolysis; Step 4: After enzymatic hydrolysis is completed, let it stand for precipitation, and separate the supernatant enzymatic hydrolysis solution and the residual precipitate; Step 5: Concentrate, dialyze and dry the separated enzymatic hydrolysis solution to finally obtain porcine spleen oligopeptides.

2. The preparation method of porcine spleen oligopeptide with enhanced immunity according to claim 1, characterized in that, In Step 1, the pig spleen and water are mixed for homogenization at a weight ratio of 1:

2.

3. The preparation method of porcine spleen oligopeptide with enhanced immunity according to claim 1, characterized in that, The specific content of Step 2 includes: Step 21: Keep the tissue homogenate at 45 - 50 °C and adjust the pH to 2.5 - 3.5; Step 22: Add acid protease to the system, with the enzyme addition amount being 6200 U / g, and stir and react uniformly for 5 - 6 hours under constant temperature conditions; Step 23: After the reaction is completed, heat the enzymatic hydrolysis system to 90 - 95 °C and maintain it for 15 minutes to inactivate the protease and terminate the reaction.

4. The preparation method of porcine spleen oligopeptide with enhanced immunity according to claim 1, characterized in that, In Step 3, adjust the pH value of the enzymatic hydrolysis system to 2.

5. The preparation method of porcine spleen oligopeptide with enhanced immunity according to claim 1, characterized in that, The specific content of Step 5 includes: Step 51: Carry out vacuum concentration on the enzymatic hydrolysis solution at 65 - 75 °C to make the solid content 30% - 45%; Step 52: Dialyze the concentrated solution using a dialysis membrane with a molecular weight cut-off of 100 Da for 24 - 48 h to remove small molecule impurities; Step 53: Carry out freeze-drying on the dialyzed concentrated solution to obtain the finished oligopeptide powder.

6. The preparation method of porcine spleen oligopeptide with enhanced immunity according to claim 1 or 5, characterized in that, In Step 5, the content of oligopeptides in the obtained porcine spleen oligopeptides accounts for more than 70% of the total peptide components, and the proportion of peptide segments with a molecular weight less than 1100 Da exceeds 80%.

7. Application of the porcine spleen oligopeptides prepared by the preparation method of porcine spleen oligopeptides with enhanced immunity as claimed in any one of claims 1 - 6 in regulating the immune function of the body and as a basic raw material for the development of immune adjuvants.

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