Antibacterial porcine spleen peptide and its preparation method and application
By extracting and optimizing the preparation of MLW active peptides and LGF active peptides from pig spleens, the problem of underutilization of pig spleen resources was solved, and antibacterial pig spleen peptides with excellent antibacterial properties were prepared, opening up its application prospects in the field of antibacterial.
Patent Information
- Application Number
- CN202411516038.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2025-02-11
- Estimated Expiration
- 2044-10-29
AI Technical Summary
In the prior art, the in-depth development and utilization of pig spleens is less, resulting in a large amount of high-quality protein resources being wasted and high-quality pig spleen polypeptide products are lacking.
The MLW active peptide and LGF active peptide were extracted from the pig spleen, and the antibacterial pig spleen peptide was prepared through enzymatic lysis, filtration, ultrafiltration, concentration, vacuum drying, etc., to optimize the pH value, protease type and dosage during the enzymatic lysis process. To improve yield and antibacterial properties.
The prepared antibacterial pig spleen peptide has excellent antibacterial properties for both Gram-negative and positive bacteria, providing broad application prospects, and effectively improving the antibacterial properties of pig spleen peptide.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field of active peptides, and in particular to an antibacterial porcine spleen peptide and a preparation method and application thereof. Background Art
[0002] Bioactive peptides are derived from proteins and are biologically active fragments of proteins that have functions beyond basic nutritional needs.
[0003] Unique biological activity has attracted more and more attention due to its contribution to human health. Since peptides are complex structures hidden in the mother protein, it is difficult for them to effectively exert their biological activity. After external protease hydrolysis, fermentation and other biochemical reactions or gastrointestinal digestion, they can produce a variety of physiological functions and have a positive impact on human health.
[0004] In recent years, many studies have been devoted to the identification of novel egg white protein peptides. The publication of the chicken genome has greatly improved the ability to identify proteins and peptides in complex samples. Studies have shown that different types of bioactive peptides can be obtained by enzymatic hydrolysis of egg white protein with specific proteases.
[0005] Currently, bioactive peptides have been used in prevention and treatment. Pig spleen is rich in protein and contains a large number of bioactive substances, which have physiological functions such as disease prevention and treatment and immune regulation. At present, there is little in-depth development and utilization of pig spleen, resulting in a large amount of high-quality protein resources being wasted. Therefore, using pig spleen as raw material to develop high-quality pig spleen polypeptide products has good prospects and significance. Summary of the invention
[0006] In order to solve the above technical problems, the present application provides an antibacterial porcine spleen peptide and a preparation method and application thereof.
[0007] In a first aspect, the present application provides an antibacterial porcine spleen peptide, which is selected from one or more of MLW active peptides and LGF active peptides; the amino acid sequence of the MLW active peptide is: methionine-leucine-tryptophan; the amino acid sequence of the LGF active peptide is: leucine-glycine-phenylalanine.
[0008] Preferably, the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-10:1-10.
[0009] Preferably, the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-5:6-10.
[0010] In a specific embodiment, the weight ratio of the MLW active peptide to the LGF active peptide can be 1-3:1, 1-5:3, 1-8:5, 1-8:6, 1-10:8, 1:1-10, 3-5:1, 3-5:5, 3-8:6, 3-10:8, 3:3-10, 5-8:1, 5-10:3, 5:3-6, 5:5-8, 5:5-10, 6-8:1, 6-8:3, 6-10:5, 6-10:8, 6:3-10, 8:1-5, 8:3-6, 8:5-10, 8:5-6, 8:8-10, 10:1-6, 10:3-8, 8-10:5, 10:6-10, 10:6-8.
[0011] In some specific embodiments, the weight ratio of the MLW active peptide to the LGF active peptide can also be 1:1, 1:3, 1:5, 1:6, 1:8, 1:10, 3:1, 3:5, 3:6, 3:8, 3:10, 5:1, 5:3, 5:6, 5:8, 5:10, 6:1, 6:3, 6:5, 6:8, 6:10, 8:1, 8:3, 8:5, 8:6, 8:10, 10:1, 10:3, 10:5, 10:6, 10:8.
[0012] Through experimental analysis, it can be known that the present application selects the porcine spleen peptide composed of a mixture of MLW active peptide and LGF active peptide in the above weight ratio to further improve the antibacterial properties of the porcine spleen peptide.
[0013] In a second aspect, the present application provides a method for preparing the above-mentioned antibacterial porcine spleen peptide, which specifically comprises the following steps: taking the porcine spleen for enzymatic hydrolysis, filtering, ultrafiltration, concentration, vacuum drying, and screening and separating to obtain the peptide.
[0014] Preferably, the specific steps of enzymatic hydrolysis of the pig spleen are: taking fresh pig spleen, adding physiological saline, adjusting the pH to 7.5-9.0, adding a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:1-5, and enzymatic hydrolysis at 45-65°C for 2-4h; then adjusting the pH to 6.5-7.5, adding flavor protease, and enzymatic hydrolysis at 45-65°C for 2-4h; boiling to inactivate the enzyme.
[0015] Through experimental analysis, the applicant found that in the process of pig spleen enzymatic hydrolysis, by reasonably selecting a suitable pH environment and reasonably matching the type and dosage of proteases for the two stages of enzymatic hydrolysis, the production of MLW active peptides and LGF active peptides can be effectively increased.
[0016] Preferably, the weight ratio of the mixture of pancreatic enzyme and alkaline protease to the pig spleen is 0.0005-0.0015:1.
[0017] Preferably, the weight ratio of the mixture of pancreatic enzyme and alkaline protease to the pig spleen is 0.0008-0.0012:1.
[0018] Preferably, the weight ratio of the flavor protease to the weight of the pig spleen is 0.0002-0.001:1.
[0019] Preferably, the weight ratio of the flavor protease to the weight of the pig spleen is 0.0004-0.008:1.
[0020] In a third aspect, the present application provides the use of the above-mentioned antibacterial porcine spleen peptide in the preparation of antibacterial drugs.
[0021] In summary, the technical solution of this application has the following effects:
[0022] One or more of the antibacterial porcine spleen peptide MLW active peptide and LGF active peptide prepared by the technical solution of the present application have excellent antibacterial properties against both Gram-negative bacteria and Gram-positive bacteria, and have broad application prospects in the antibacterial field.
[0023] The present application obtains MLW active peptide and LGF active peptide with high yield by optimizing the preparation method of antibacterial porcine spleen peptide. DETAILED DESCRIPTION
[0024] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example Example 1
[0025] Example 1 provides an antibacterial porcine spleen peptide.
[0026] The preparation method of the antibacterial porcine spleen peptide in the embodiment is:
[0027] Enzymatic hydrolysis of pig spleen: take 10kg of fresh pig spleen, wash it with drinking water until there is no blood, grind it with a colloid mill, add 50kg of physiological saline, adjust the pH to 8.0, add 0.01kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:2, and perform enzymatic hydrolysis at 55℃ for 3h; then adjust the pH to 7.0, add 0.006kg of flavor protease, and perform enzymatic hydrolysis at 50℃ for 3h; boil to inactivate the enzyme to obtain an enzymatic solution.
[0028] Centrifugal filtration, ultrafiltration, concentration, vacuum drying: The enzymatic hydrolyzate cooled to room temperature was centrifuged at 6000r / min for 20min to obtain the supernatant, which was first filtered through a 0.45μm filter membrane and the filtrate was collected for the next step. The filtrate was separated with 10kDa, 5kDa, and 3kDa ultrafiltration membranes to collect polypeptides of different molecular weights (>10kDa, 5-10kDa, 3-5kDa, and <3kDa), and the four polypeptide solutions of different molecular weights were vacuum freeze-dried and stored at -80°C.
[0029] Screening and separation: LC-MS / MS liquid mass analysis was performed on the polypeptide components with molecular weight less than 3kDa. LC-MS / MS analysis and database search and comparison were performed to obtain 958μg MLW active peptide and 1547μg LGF active peptide. Embodiment 2-8
[0030] Examples 2-8 each provide an antibacterial porcine spleen peptide.
[0031] The difference between the above embodiment and embodiment 1 is that the method of enzymatic hydrolysis of pig spleen is different, as shown below.
[0032] In Example 2: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.016 kg of a mixture of pancreatin, alkaline protease, and flavor protease in a weight ratio of 5:2:1, and hydrolyzed at 55°C for 3 hours; boiled to inactivate the enzymes, and obtained an enzymatic solution. In this example, 737 μg of MLW active peptide and 1056 μg of LGF active peptide were obtained.
[0033] In Example 3: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and flavor protease in a weight ratio of 5:2, and hydrolyzed at 55°C for 3 h; then adjusted to pH 7.0, added with 0.006 kg of alkaline protease, and hydrolyzed at 50°C for 3 h; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 925 μg of MLW active peptide and 874 μg of LGF active peptide were obtained.
[0034] In Example 4: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to a pH value of 7.0, added with 0.006 kg of flavor protease, and hydrolyzed at 50°C for 3 h; then adjusted to a pH of 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:2, and hydrolyzed at 55°C for 3 h; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 623 μg of MLW active peptide and 1076 μg of LGF active peptide were obtained.
[0035] In Example 5: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:0.5, and hydrolyzed at 55°C for 3 h; then adjusted to pH 7.0, added with 0.006 kg of flavor protease, and hydrolyzed at 50°C for 3 h; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 527 μg of MLW active peptide and 1078 μg of LGF active peptide were obtained.
[0036] In Example 6: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:6, and hydrolyzed at 55°C for 3 h; then adjusted to pH 7.0, added with 0.006 kg of flavor protease, and hydrolyzed at 50°C for 3 h; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 908 μg of MLW active peptide and 779 μg of LGF active peptide were obtained.
[0037] In Example 7: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:1, and hydrolyzed at 55°C for 3 h; then adjusted to pH 7.0, added with 0.006 kg of flavor protease, and hydrolyzed at 50°C for 3 h; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 938 μg of MLW active peptide and 1378 μg of LGF active peptide were obtained.
[0038] In Example 8: Enzymatic hydrolysis of pig spleen: 10 kg of fresh pig spleen was taken, washed with drinking water until there was no blood, crushed with a colloid mill, added with 10 kg of physiological saline, adjusted to pH 8.0, added with 0.01 kg of a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:5 (1:1), and hydrolyzed at 55°C for 3 hours; then adjusted to pH 7.0, added with 0.006 kg of flavor protease, and hydrolyzed at 50°C for 3 hours; boiled to inactivate the enzyme, and obtained an enzymatic solution. In this example, 1027 μg of MLW active peptide and 1239 μg of LGF active peptide were obtained.
[0039] The other process parameters in the above embodiment are the same as those in embodiment 1.
[0040] Through the preparation method of Examples 1-8 to prepare antibacterial porcine spleen peptides and the experimental results, it can be known that the specific steps of porcine spleen enzymatic hydrolysis are as follows: adjusting the pH to 7.5-9.0, adding a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:1-5, and enzymolysis at 45-65°C for 2-4h; then adjusting the pH to 6.5-7.5, adding flavor protease, and enzymolysis at 45-65°C for 2-4h; a higher yield of MLW active peptides and LGF active peptides can be obtained. Examples 9-17
[0041] Examples 9-17 each provide an antibacterial porcine spleen peptide.
[0042] The difference between the above examples is that the weight ratio between the MLW active peptide and the LGF active peptide is as follows: The MLW active peptide and the LGF active peptide in the above examples are derived from Example 1.
[0043] In Example 9: the antibacterial porcine spleen peptide is the MLW active peptide.
[0044] In Example 10: the antibacterial porcine spleen peptide is LGF active peptide.
[0045] In Example 11: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1:10.
[0046] In Example 12: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 3:8.
[0047] In Example 13: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 5:6.
[0048] In Example 14: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 10:1.
[0049] In Example 15: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 6:5.
[0050] In Example 16: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1:15.
[0051] In Example 17: the antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 15:1.
[0052] Performance testing
[0053] Evaluation of the antibacterial performance of antibacterial porcine spleen peptide: The antibacterial porcine spleen peptide powder was dissolved in sterile water to prepare a 20 mg / mL antibacterial porcine spleen peptide sample solution. 100 μL of 5×10 5 CFU / mL bacteria in Mueller HintonBroth medium, and the last well is an empty medium control without bacteria. Add 10μL of antibacterial porcine spleen peptide sample solution to the first well, and dilute it down in sequence by two-fold dilution method, with three replicates for each concentration. Seal the 96-well plate with sealing film and incubate at 37℃ for 20 hours; detect the absorbance value of each well plate at 600nm with an enzyme reader to detect its minimum inhibitory concentration (MIC).
[0054] There are four types of bacteria used for testing, including two Gram-negative bacteria, namely Escherichia coli and Klebsiella pneumoniae; and two Gram-positive bacteria, namely Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.
[0055] Test results: as shown in Table 1.
[0056] Table 1 Performance test results of antibacterial porcine spleen peptides in Examples 9-17
[0057]
[0058] Combined with the test results in Table 1, it can be seen that the porcine spleen peptide prepared by the technical solution of the present application has excellent antibacterial properties against two types of Gram-negative bacteria, Escherichia coli and Klebsiella pneumoniae; and two types of Gram-positive bacteria, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.
[0059] By comparing the test results of Examples 9-17, it can be seen that the present application selects a pig spleen peptide composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-10:1-10, which can effectively improve the antibacterial properties of the pig spleen peptide; further, the present application selects a pig spleen peptide composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-5:6-10.
[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.
Claims
1. An antibacterial porcine spleen peptide, characterized in that: The antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-10:1-10; the amino acid sequence of the MLW active peptide is: methionine-leucine-tryptophan; the amino acid sequence of the LGF active peptide is: leucine-glycine-phenylalanine.
2. The antibacterial porcine spleen peptide according to claim 1, characterized in that The antibacterial porcine spleen peptide is composed of a mixture of MLW active peptide and LGF active peptide in a weight ratio of 1-5:6-10.
3. The method for preparing the antibacterial porcine spleen peptide according to any one of claims 1 to 2, characterized in that: The specific steps include: After enzymatic hydrolysis of pig spleen, it is centrifuged, ultrafiltered, concentrated, vacuum dried, and screened to obtain; The specific steps of enzymolysis of pig spleen are: taking fresh pig spleen, adding physiological saline, adjusting the pH to 7.5-9.0, adding a mixture of pancreatic enzyme and alkaline protease in a weight ratio of 5:1-5, and enzymolysis at 45-65°C for 2-4 hours; then adjusting the pH to 6.5-7.5, adding flavor protease, and enzymolysis at 45-65°C for 2-4 hours; boiling to inactivate the enzyme to obtain an enzymolysis solution; The weight ratio of the mixture of pancreatin and alkaline protease to the pig spleen is 0.005-0.015:10; The weight ratio of the flavor protease to the weight of the pig spleen is 0.002-0.01:10; The specific steps of centrifugal filtration, ultrafiltration, concentration and vacuum drying are as follows: the enzymatic hydrolysate cooled to room temperature is centrifuged at 6000r / min for 20min to obtain the supernatant, firstly filtered through a 0.45μm filter membrane to collect the filtrate; the filtrate is separated by ultrafiltration membranes of 10kDa, 5kDa and 3kDa to collect polypeptides with different molecular weights of >10kDa, 5-10kDa, 3-5kDa and <3kDa, and the polypeptide solutions of the four different molecular weights are vacuum freeze-dried and stored at -80°C; The specific steps of the screening and separation are: performing LC-MS / MS liquid mass analysis and database search and comparison on the polypeptide components with molecular weight less than 3kDa.
4. The method for preparing the antibacterial porcine spleen peptide according to claim 3, characterized in that: The weight ratio of the mixture of pancreatic enzyme and alkaline protease to the pig spleen is 0.008-0.012:
10.
5. The method for preparing the antibacterial porcine spleen peptide according to claim 3, characterized in that: The weight ratio of the flavor protease to the weight of the pig spleen is 0.004-0.008:
10.
6. The use of the antibacterial porcine spleen peptide according to any one of claims 1 to 2 in the preparation of antibacterial drugs, characterized in that: The antibacterial drug is selected from any one of an anti-Escherichia coli drug, an anti-Klebsiella pneumoniae drug, and an anti-Staphylococcus aureus drug.
7. The use of the antibacterial porcine spleen peptide in the preparation of antibacterial drugs according to claim 6, characterized in that: The anti-Staphylococcus aureus drug is an anti-methicillin-resistant Staphylococcus aureus drug.